Virus genomics

The Sanger Institute's Kellam laboratory researches genetic variation of hosts and viruses to determine the molecular and pathogenic outcomes of virus infections. Our research question is 'can whole virus and host genome sequencing reveal genome-wide selective pressures and molecular mechanisms that influence virus and host interactions?'

Virus infections remain one of the major causes of human and animal disease. Geographic locations of current infections of humans and other animals are visible through HealthMap. Determining whole virus genome diversity and the dynamics of viral genome variation is necessary for understanding disease pathogenesis and epidemiology. Similarly, determining the genetic variation in host genes and their expression in the infected cells is necessary to understand virus replication, the antiviral state and disease processes. It is clear that understanding genetic variability across whole virus and host genomes will present new opportunities to inhibit virus replication and provide novel insights into the biology of viral infections.

[Wellcome Images]

Background

Although virus genomes are small they are incredibly diverse and subject to rapid genetic change. In addition, the host organisms viruses infected produce a wide array of defences encoded by genes that are also subjected to accelerated evolution. At the most extreme end of this evolution is the mutation and selection of unique B cells that produce antibodies to counter the diversity of virus proteins and provide immunological memory. The aim of the Virus Genomics team is to understand how genetic changes in viruses and hosts influence the biological properties of viral pathogenesis, persistence and host susceptibility to infection.

Research

Our research spans the breadth of virus and host genetic variation and is underpinned by computational methods and data resources directed towards investigating host and viral genetic variation. Our research is split over three areas of interest, virus genetics, immune repertoire genetics and host genetics. Our approach is to determine such 'infection' genetic variation and understand its consequences through functional molecular virology experiments.

Virus genetics

Through the use of next generation sequencing methods we are able to sequence the full genomes of many different viruses. We are using next-generation sequencing to explore the genetic variation of respiratory virus infections including influenza A and influenza B viruses, human coronaviruses such as MERS-CoV and respiratory syncytial disease virus (RSV). We use the same methods to sequence full virus genomes of blood borne viruses such as HIV and HCV, and gastroenteritis viruses such as noroviruses and rotaviruses. We are engaged in international collaborations to move full virus genome sequencing into national health services, to undertake large-scale virus genome-wide association studies and to use virus genetics to understand generalised virus epidemics. Our research studies into XMRV and MERS-CoV have been featured by the media.

Host genetics

The hosts response to virus infection can range from asymptomatic, typical or a population average response or a severe response to infection, often when significant virus genetic variation is absent. Therefore, intrinsic aspects of how the host organism responds to infection must contribute to the range of pathogenicity. We are using host whole genome and whole exome sequencing together with in vivo and in vitro molecular virology to understand the role of host genes in virus replication. In particular, we are focusing on genes of the Unfolded Protein Response (UPR) and their role in controlling virus replication and interferon stimulated genes such as InterFeron Inducible TransMembrane (IFITM) proteins and their role as virus entry restriction factors. Our research into IFITM3 has been covered widely by the media.

B-cell immune repertoires

Once a virus infection is established cells of the adaptive immune system, namely B and T lymphocytes proliferate in response to virus antigens to both control virus replication and provide immunological memory thereby preventing reinfection. The diversity of the B-cell immune repertoire is conferred by recombination of variable (V), diversity (D) and joining (J) genes of the B-cell locus that form the VDJ rearranged B-cell receptor. We are using NGS to follow this B-cell repertoire diversity, plus extra diversity generated by somatic hypermutation during normal B-cell immunological function and diseases of B-cell such as lymphomas.

We are interested to hear from post-doctoral scientists wishing to pursue career development fellowships in any of our area of interests and undergraduates looking to pursue a PhD at the Wellcome Trust Sanger Institute.

Selected Publications

2013 Publications

  • A systematic review of definitions of extreme phenotypes of HIV control and progression.

    Gurdasani D, Iles L, Dillon DG, Young EH, Olson AD, Naranbhai V, Fidler S, Gkrania-Klotsas E, Post FA, Kellam P, Porter K and Sandhu MS

    aWellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton bStrangeways Research Laboratory, Department of Public Health and Primary Care, University of Cambridge, Wort's Causeway, Cambridge cMedical Research Council, Clinical Trials Unit, Aviation House, London, UK dCentre for the AIDS Programme of Research in South Africa (CAPRISA), Doris Duke Medical Research Institute, Nelson R Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa eWellcome Trust Centre for Human Genetics, Nuffield Department of Medicine, University of Oxford, Oxford fImperial College Healthcare NHS Trust, London gCambridge University Hospitals NHS Foundation Trust, Department of Infectious Diseases, Addenbrooke's Hospital, Cambridge hKing's College London, Weston Education Centre iDivision of Infection and Immunity, University College London, London, UK.

    The study of individuals at opposite ends of the HIV clinical spectrum can provide invaluable insights into HIV biology. Heterogeneity in criteria used to define these individuals can introduce inconsistencies in results from research and make it difficult to identify biological mechanisms underlying these phenotypes. In this systematic review, we formally quantified the heterogeneity in definitions used for terms referring to extreme phenotypes in the literature, and identified common definitions and components used to describe these phenotypes. We assessed 714 definitions of HIV extreme phenotypes in 501 eligible studies published between 1 January 2000 and 15 March 2012, and identified substantial variation among these. This heterogeneity in definitions may represent important differences in biological endophenotypes and clinical progression profiles of individuals selected by these, suggesting the need for harmonized definitions. In this context, we were able to identify common components in existing definitions that may provide a framework for developing consensus definitions for these phenotypes in HIV infection.

    Funded by: Medical Research Council: G0901213; Wellcome Trust

    AIDS (London, England) 2014;28;2;149-62

  • Transmission and evolution of the Middle East respiratory syndrome coronavirus in Saudi Arabia: a descriptive genomic study.

    Cotten M, Watson SJ, Kellam P, Al-Rabeeah AA, Makhdoom HQ, Assiri A, Al-Tawfiq JA, Alhakeem RF, Madani H, AlRabiah FA, Al Hajjar S, Al-nassir WN, Albarrak A, Flemban H, Balkhy HH, Alsubaie S, Palser AL, Gall A, Bashford-Rogers R, Rambaut A, Zumla AI and Memish ZA

    Wellcome Trust Sanger Institute, Hinxton, UK.

    Background: Since June, 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) has, worldwide, caused 104 infections in people including 49 deaths, with 82 cases and 41 deaths reported from Saudi Arabia. In addition to confirming diagnosis, we generated the MERS-CoV genomic sequences obtained directly from patient samples to provide important information on MERS-CoV transmission, evolution, and origin.

    Methods: Full genome deep sequencing was done on nucleic acid extracted directly from PCR-confirmed clinical samples. Viral genomes were obtained from 21 MERS cases of which 13 had 100%, four 85-95%, and four 30-50% genome coverage. Phylogenetic analysis of the 21 sequences, combined with nine published MERS-CoV genomes, was done.

    Findings: Three distinct MERS-CoV genotypes were identified in Riyadh. Phylogeographic analyses suggest the MERS-CoV zoonotic reservoir is geographically disperse. Selection analysis of the MERS-CoV genomes reveals the expected accumulation of genetic diversity including changes in the S protein. The genetic diversity in the Al-Hasa cluster suggests that the hospital outbreak might have had more than one virus introduction.

    Interpretation: We present the largest number of MERS-CoV genomes (21) described so far. MERS-CoV full genome sequences provide greater detail in tracking transmission. Multiple introductions of MERS-CoV are identified and suggest lower R0 values. Transmission within Saudi Arabia is consistent with either movement of an animal reservoir, animal products, or movement of infected people. Further definition of the exposures responsible for the sporadic introductions of MERS-CoV into human populations is urgently needed.

    Funding: Saudi Arabian Ministry of Health, Wellcome Trust, European Community, and National Institute of Health Research University College London Hospitals Biomedical Research Centre.

    Funded by: Wellcome Trust: 093724

    Lancet 2013;382;9909;1993-2002

  • Chicken interferon-inducible transmembrane protein 3 restricts influenza viruses and lyssaviruses in vitro.

    Smith SE, Gibson MS, Wash RS, Ferrara F, Wright E, Temperton N, Kellam P and Fife M

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom.

    Interferon-inducible transmembrane protein 3 (IFITM3) is an effector protein of the innate immune system. It confers potent, cell-intrinsic resistance to infection by diverse enveloped viruses both in vitro and in vivo, including influenza viruses, West Nile virus, and dengue virus. IFITM3 prevents cytosolic entry of these viruses by blocking complete virus envelope fusion with cell endosome membranes. Although the IFITM locus, which includes IFITM1, -2, -3, and -5, is present in mammalian species, this locus has not been unambiguously identified or functionally characterized in avian species. Here, we show that the IFITM locus exists in chickens and is syntenic with the IFITM locus in mammals. The chicken IFITM3 protein restricts cell infection by influenza A viruses and lyssaviruses to a similar level as its human orthologue. Furthermore, we show that chicken IFITM3 is functional in chicken cells and that knockdown of constitutive expression in chicken fibroblasts results in enhanced infection by influenza A virus. Chicken IFITM2 and -3 are constitutively expressed in all tissues examined, whereas IFITM1 is only expressed in the bursa of Fabricius, gastrointestinal tract, cecal tonsil, and trachea. Despite being highly divergent at the amino acid level, IFITM3 proteins of birds and mammals can restrict replication of viruses that are able to infect different host species, suggesting IFITM proteins may provide a crucial barrier for zoonotic infections.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/J004448/1; Medical Research Council: G0600369, G1000413; PHS HHS: 098051

    Journal of virology 2013;87;23;12957-66

  • Network properties derived from deep sequencing of human B-cell receptor repertoires delineate B-cell populations.

    Bashford-Rogers RJ, Palser AL, Huntly BJ, Rance R, Vassiliou GS, Follows GA and Kellam P

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom;

    The adaptive immune response selectively expands B- and T-cell clones following antigen recognition by B- and T-cell receptors (BCR and TCR), respectively. Next-generation sequencing is a powerful tool for dissecting the BCR and TCR populations at high resolution, but robust computational analyses are required to interpret such sequencing. Here, we develop a novel computational approach for BCR repertoire analysis using established next-generation sequencing methods coupled with network construction and population analysis. BCR sequences organize into networks based on sequence diversity, with differences in network connectivity clearly distinguishing between diverse repertoires of healthy individuals and clonally expanded repertoires from individuals with chronic lymphocytic leukemia (CLL) and other clonal blood disorders. Network population measures defined by the Gini Index and cluster sizes quantify the BCR clonality status and are robust to sampling and sequencing depths. BCR network analysis therefore allows the direct and quantifiable comparison of BCR repertoires between samples and intra-individual population changes between temporal or spatially separated samples and over the course of therapy.

    Funded by: Wellcome Trust: 079249, 095663, 100140

    Genome research 2013;23;11;1874-84

  • Hospital outbreak of Middle East respiratory syndrome coronavirus.

    Assiri A, McGeer A, Perl TM, Price CS, Al Rabeeah AA, Cummings DA, Alabdullatif ZN, Assad M, Almulhim A, Makhdoom H, Madani H, Alhakeem R, Al-Tawfiq JA, Cotten M, Watson SJ, Kellam P, Zumla AI, Memish ZA and KSA MERS-CoV Investigation Team

    Global Center for Mass Gatherings Medicine, Ministry of Health, Riyadh, Saudi Arabia.

    Background: In September 2012, the World Health Organization reported the first cases of pneumonia caused by the novel Middle East respiratory syndrome coronavirus (MERS-CoV). We describe a cluster of health care-acquired MERS-CoV infections.

    Methods: Medical records were reviewed for clinical and demographic information and determination of potential contacts and exposures. Case patients and contacts were interviewed. The incubation period and serial interval (the time between the successive onset of symptoms in a chain of transmission) were estimated. Viral RNA was sequenced.

    Results: Between April 1 and May 23, 2013, a total of 23 cases of MERS-CoV infection were reported in the eastern province of Saudi Arabia. Symptoms included fever in 20 patients (87%), cough in 20 (87%), shortness of breath in 11 (48%), and gastrointestinal symptoms in 8 (35%); 20 patients (87%) presented with abnormal chest radiographs. As of June 12, a total of 15 patients (65%) had died, 6 (26%) had recovered, and 2 (9%) remained hospitalized. The median incubation period was 5.2 days (95% confidence interval [CI], 1.9 to 14.7), and the serial interval was 7.6 days (95% CI, 2.5 to 23.1). A total of 21 of the 23 cases were acquired by person-to-person transmission in hemodialysis units, intensive care units, or in-patient units in three different health care facilities. Sequencing data from four isolates revealed a single monophyletic clade. Among 217 household contacts and more than 200 health care worker contacts whom we identified, MERS-CoV infection developed in 5 family members (3 with laboratory-confirmed cases) and in 2 health care workers (both with laboratory-confirmed cases).

    Conclusions: Person-to-person transmission of MERS-CoV can occur in health care settings and may be associated with considerable morbidity. Surveillance and infection-control measures are critical to a global public health response.

    Funded by: NIGMS NIH HHS: U01 GM070708, U54 GM088491; Wellcome Trust: 093724

    The New England journal of medicine 2013;369;5;407-16

  • Metagenomic study of the viruses of African straw-coloured fruit bats: detection of a chiropteran poxvirus and isolation of a novel adenovirus.

    Baker KS, Leggett RM, Bexfield NH, Alston M, Daly G, Todd S, Tachedjian M, Holmes CE, Crameri S, Wang LF, Heeney JL, Suu-Ire R, Kellam P, Cunningham AA, Wood JL, Caccamo M and Murcia PR

    University of Cambridge, Department of Veterinary Medicine, Madingley Rd, Cambridge, Cambridgeshire, CB3 0ES, United Kingdom. kf281@cam.ac.uk

    Viral emergence as a result of zoonotic transmission constitutes a continuous public health threat. Emerging viruses such as SARS coronavirus, hantaviruses and henipaviruses have wildlife reservoirs. Characterising the viruses of candidate reservoir species in geographical hot spots for viral emergence is a sensible approach to develop tools to predict, prevent, or contain emergence events. Here, we explore the viruses of Eidolon helvum, an Old World fruit bat species widely distributed in Africa that lives in close proximity to humans. We identified a great abundance and diversity of novel herpes and papillomaviruses, described the isolation of a novel adenovirus, and detected, for the first time, sequences of a chiropteran poxvirus closely related with Molluscum contagiosum. In sum, E. helvum display a wide variety of mammalian viruses, some of them genetically similar to known human pathogens, highlighting the possibility of zoonotic transmission.

    Funded by: Medical Research Council: G0801822; Wellcome Trust

    Virology 2013;441;2;95-106

  • The CD225 domain of IFITM3 is required for both IFITM protein association and inhibition of influenza A virus and dengue virus replication.

    John SP, Chin CR, Perreira JM, Feeley EM, Aker AM, Savidis G, Smith SE, Elia AE, Everitt AR, Vora M, Pertel T, Elledge SJ, Kellam P and Brass AL

    Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    The interferon-induced transmembrane protein 3 (IFITM3) gene is an interferon-stimulated gene that inhibits the replication of multiple pathogenic viruses in vitro and in vivo. IFITM3 is a member of a large protein superfamily, whose members share a functionally undefined area of high amino acid conservation, the CD225 domain. We performed mutational analyses of IFITM3 and identified multiple residues within the CD225 domain, consisting of the first intramembrane domain (intramembrane domain 1 [IM1]) and a conserved intracellular loop (CIL), that are required for restriction of both influenza A virus (IAV) and dengue virus (DENV) infection in vitro. Two phenylalanines within IM1 (F75 and F78) also mediate a physical association between IFITM proteins, and the loss of this interaction decreases IFITM3-mediated restriction. By extension, similar IM1-mediated associations may contribute to the functions of additional members of the CD225 domain family. IFITM3's distal N-terminal domain is also needed for full antiviral activity, including a tyrosine (Y20), whose alteration results in mislocalization of a portion of IFITM3 to the cell periphery and surface. Comparative analyses demonstrate that similar molecular determinants are needed for IFITM3's restriction of both IAV and DENV. However, a portion of the CIL including Y99 and R87 is preferentially needed for inhibition of the orthomyxovirus. Several IFITM3 proteins engineered with rare single-nucleotide polymorphisms demonstrated reduced expression or mislocalization, and these events were associated with enhanced viral replication in vitro, suggesting that possessing such alleles may impact an individual's risk for viral infection. On the basis of this and other data, we propose a model for IFITM3-mediated restriction.

    Funded by: Howard Hughes Medical Institute; NIAID NIH HHS: 1R01AI091786, R01 AI091786; Wellcome Trust

    Journal of virology 2013;87;14;7837-52

  • Activation of the B cell antigen receptor triggers reactivation of latent Kaposi's sarcoma-associated herpesvirus in B cells.

    Kati S, Tsao EH, Günther T, Weidner-Glunde M, Rothämel T, Grundhoff A, Kellam P and Schulz TF

    Institute of Virology, Hanover Medical School, Hanover, Germany.

    Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus and the cause of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease. Latently infected B cells are the main reservoir of this virus in vivo, but the nature of the stimuli that lead to its reactivation in B cells is only partially understood. We established stable BJAB cell lines harboring latent KSHV by cell-free infection with recombinant virus carrying a puromycin resistance marker. Our latently infected B cell lines, termed BrK.219, can be reactivated by triggering the B cell receptor (BCR) with antibodies to surface IgM, a stimulus imitating antigen recognition. Using this B cell model system we studied the mechanisms that mediate the reactivation of KSHV in B cells following the stimulation of the BCR and could identify phosphatidylinositol 3-kinase (PI3K) and X-box binding protein 1 (XBP-1) as proteins that play an important role in the BCR-mediated reactivation of latent KSHV.

    Journal of virology 2013;87;14;8004-16

  • Reporting and methodological quality of systematic reviews in the orthopaedic literature.

    Gagnier JJ and Kellam PJ

    Department of Orthopaedic Surgery, University of Michigan, 24 Frank Lloyd Wright Drive, MedSport, Domino's Farms, Ann Arbor, MI 48106-0391, USA. jgagnier@umich.edu

    Background: Properly designed and conducted systematic reviews can reliably produce valid pooled treatment-effect estimates and are an important resource for clinical decision-making. The purpose of this report was to assess the reporting and methodological quality of systematic reviews in orthopaedic journals.

    Methods: With use of the 2010 Institute for Scientific Information (ISI) Thomson Reuters Journal Citation Reports, the five orthopaedic surgery journals with the highest impact factors were searched by one individual over a five-year period (from 2006 to 2010) for systematic reviews and meta-analyses. The two authors separately and independently assessed the included studies. The reporting quality was assessed with use of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement, and the methodological quality was assessed with use of the Assessment of Multiple Systematic Reviews (AMSTAR) guidelines, both of which are accepted instruments. We calculated the proportions of each item reported within and across journals.

    Results: Seventy-six systematic reviews and meta-analyses were included. Of the five journals that were examined, articles from The Journal of Bone and Joint Surgery (American Volume) had the best reporting. Articles from The American Journal of Sports Medicine fulfilled the most methodological quality items. The papers from all of the journals reported an average of only 68% of the PRISMA items and only 54% of the AMSTAR quality items.

    Conclusions: Both reporting and methodological quality in the top five orthopaedic journals were poor; the reporting quality was slightly superior to the methodological quality. Although there was a wide range of reporting quality and methodological quality scores across the journals, the included articles demonstrated inadequate adherence to accepted standards of quality. The use of PRISMA and AMSTAR guidelines in designing, implementing, and writing systematic reviews is recommended to improve the quality of systematic reviews and meta-analyses in orthopaedic journals.

    The validity of published systematic reviews in orthopaedics is questionable, and their contribution to clinical decision-making is suboptimal. Clinicians should be careful when interpreting and applying findings of current orthopaedic systematic reviews.

    The Journal of bone and joint surgery. American volume 2013;95;11;e771-7

  • Full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus.

    Cotten M, Lam TT, Watson SJ, Palser AL, Petrova V, Grant P, Pybus OG, Rambaut A, Guan Y, Pillay D, Kellam P and Nastouli E

    Wellcome Trust Sanger Institute, Hinxton, UK.

    A novel betacoronavirus associated with lethal respiratory and renal complications was recently identified in patients from several countries in the Middle East. We report the deep genome sequencing of the virus directly from a patient's sputum sample. Our high-throughput sequencing yielded a substantial depth of genome sequence assembly and showed the minority viral variants in the specimen. Detailed phylogenetic analysis of the virus genome (England/Qatar/2012) revealed its close relationship to European bat coronaviruses circulating among the bat species of the Vespertilionidae family. Molecular clock analysis showed that the 2 human infections of this betacoronavirus in June 2012 (EMC/2012) and September 2012 (England/Qatar/2012) share a common virus ancestor most likely considerably before early 2012, suggesting the human diversity is the result of multiple zoonotic events.

    Funded by: Medical Research Council: MR/K006584/1; Wellcome Trust: 093724, 095831

    Emerging infectious diseases 2013;19;5;736-42B

  • Evolution of equine influenza virus in vaccinated horses.

    Murcia PR, Baillie GJ, Stack JC, Jervis C, Elton D, Mumford JA, Daly J, Kellam P, Grenfell BT, Holmes EC and Wood JL

    Medical Research Council-University of Glasgow Centre for Virus Research, Institute of Infection, Inflammation and Immunity, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

    Influenza A viruses are characterized by their ability to evade host immunity, even in vaccinated individuals. To determine how prior immunity shapes viral diversity in vivo, we studied the intra- and interhost evolution of equine influenza virus in vaccinated horses. Although the level and structure of genetic diversity were similar to those in naïve horses, intrahost bottlenecks may be more stringent in vaccinated animals, and mutations shared among horses often fall close to putative antigenic sites.

    Funded by: Medical Research Council: G0801822; NIGMS NIH HHS: R01 GM080533, R01 GM080533-06; Wellcome Trust

    Journal of virology 2013;87;8;4768-71

  • The evolutionary dynamics of influenza A virus adaptation to mammalian hosts.

    Bhatt S, Lam TT, Lycett SJ, Leigh Brown AJ, Bowden TA, Holmes EC, Guan Y, Wood JL, Brown IH, Kellam P, Combating Swine Influenza Consortium and Pybus OG

    Department of Zoology, University of Oxford, Oxford, UK.

    Few questions on infectious disease are more important than understanding how and why avian influenza A viruses successfully emerge in mammalian populations, yet little is known about the rate and nature of the virus' genetic adaptation in new hosts. Here, we measure, for the first time, the genomic rate of adaptive evolution of swine influenza viruses (SwIV) that originated in birds. By using a curated dataset of more than 24 000 human and swine influenza gene sequences, including 41 newly characterized genomes, we reconstructed the adaptive dynamics of three major SwIV lineages (Eurasian, EA; classical swine, CS; triple reassortant, TR). We found that, following the transfer of the EA lineage from birds to swine in the late 1970s, EA virus genes have undergone substantially faster adaptive evolution than those of the CS lineage, which had circulated among swine for decades. Further, the adaptation rates of the EA lineage antigenic haemagglutinin and neuraminidase genes were unexpectedly high and similar to those observed in human influenza A. We show that the successful establishment of avian influenza viruses in swine is associated with raised adaptive evolution across the entire genome for many years after zoonosis, reflecting the contribution of multiple mutations to the coordinated optimization of viral fitness in a new environment. This dynamics is replicated independently in the polymerase genes of the TR lineage, which established in swine following separate transmission from non-swine hosts.

    Funded by: Biotechnology and Biological Sciences Research Council; Medical Research Council: MC_G0902096; Wellcome Trust

    Philosophical transactions of the Royal Society of London. Series B, Biological sciences 2013;368;1614;20120382

  • Viral population analysis and minority-variant detection using short read next-generation sequencing.

    Watson SJ, Welkers MR, Depledge DP, Coulter E, Breuer JM, de Jong MD and Kellam P

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

    RNA viruses within infected individuals exist as a population of evolutionary-related variants. Owing to evolutionary change affecting the constitution of this population, the frequency and/or occurrence of individual viral variants can show marked or subtle fluctuations. Since the development of massively parallel sequencing platforms, such viral populations can now be investigated to unprecedented resolution. A critical problem with such analyses is the presence of sequencing-related errors that obscure the identification of true biological variants present at low frequency. Here, we report the development and assessment of the Quality Assessment of Short Read (QUASR) Pipeline (http://sourceforge.net/projects/quasr) specific for virus genome short read analysis that minimizes sequencing errors from multiple deep-sequencing platforms, and enables post-mapping analysis of the minority variants within the viral population. QUASR significantly reduces the error-related noise in deep-sequencing datasets, resulting in increased mapping accuracy and reduction of erroneous mutations. Using QUASR, we have determined influenza virus genome dynamics in sequential samples from an in vitro evolution of 2009 pandemic H1N1 (A/H1N1/09) influenza from samples sequenced on both the Roche 454 GSFLX and Illumina GAIIx platforms. Importantly, concordance between the 454 and Illumina sequencing allowed unambiguous minority-variant detection and accurate determination of virus population turnover in vitro.

    Funded by: Medical Research Council: G0700814; Wellcome Trust

    Philosophical transactions of the Royal Society of London. Series B, Biological sciences 2013;368;1614;20120205

  • Different patterns of Epstein-Barr virus latency in endemic Burkitt lymphoma (BL) lead to distinct variants within the BL-associated gene expression signature.

    Kelly GL, Stylianou J, Rasaiyaah J, Wei W, Thomas W, Croom-Carter D, Kohler C, Spang R, Woodman C, Kellam P, Rickinson AB and Bell AI

    School of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.

    Epstein-Barr virus (EBV) is present in all cases of endemic Burkitt lymphoma (BL) but in few European/North American sporadic BLs. Gene expression arrays of sporadic tumors have defined a consensus BL profile within which tumors are classifiable as "molecular BL" (mBL). Where endemic BLs fall relative to this profile remains unclear, since they not only carry EBV but also display one of two different forms of virus latency. Here, we use early-passage BL cell lines from different tumors, and BL subclones from a single tumor, to compare EBV-negative cells with EBV-positive cells displaying either classical latency I EBV infection (where EBNA1 is the only EBV antigen expressed from the wild-type EBV genome) or Wp-restricted latency (where an EBNA2 gene-deleted virus genome broadens antigen expression to include the EBNA3A, -3B, and -3C proteins and BHRF1). Expression arrays show that both types of endemic BL fall within the mBL classification. However, while EBV-negative and latency I BLs show overlapping profiles, Wp-restricted BLs form a distinct subgroup, characterized by a detectable downregulation of the germinal center (GC)-associated marker Bcl6 and upregulation of genes marking early plasmacytoid differentiation, notably IRF4 and BLIMP1. Importantly, these same changes can be induced in EBV-negative or latency I BL cells by infection with an EBNA2-knockout virus. Thus, we infer that the distinct gene profile of Wp-restricted BLs does not reflect differences in the identity of the tumor progenitor cell per se but differences imposed on a common progenitor by broadened EBV gene expression.

    Funded by: Cancer Research UK: C910/A8829

    Journal of virology 2013;87;5;2882-94

  • Interferon-induced transmembrane protein-3 genetic variant rs12252-C is associated with severe influenza in Chinese individuals.

    Zhang YH, Zhao Y, Li N, Peng YC, Giannoulatou E, Jin RH, Yan HP, Wu H, Liu JH, Liu N, Wang DY, Shu YL, Ho LP, Kellam P, McMichael A and Dong T

    Beijing You'an Hospital, Capital Medical University, Beijing PO 100069, China.

    The SNP rs12252-C allele alters the function of interferon-induced transmembrane protein-3 increasing the disease severity of influenza virus infection in Caucasians, but the allele is rare. However, rs12252-C is much more common in Han Chinese. Here we report that the CC genotype is found in 69% of Chinese patients with severe pandemic influenza A H1N1/09 virus infection compared with 25% in those with mild infection. Specifically, the CC genotype was estimated to confer a sixfold greater risk for severe infection than the CT and TT genotypes. More importantly, because the risk genotype occurs with such a high frequency, its effect translates to a large population-attributable risk of 54.3% for severe infection in the Chinese population studied compared with 5.4% in Northern Europeans. Interferon-induced transmembrane protein-3 genetic variants could, therefore, have a strong effect of the epidemiology of influenza in China and in people of Chinese descent.

    Funded by: Medical Research Council: G0600520, G1001046; Wellcome Trust

    Nature communications 2013;4;1418

  • Restriction of V3 region sequence divergence in the HIV-1 envelope gene during antiretroviral treatment in a cohort of recent seroconverters.

    Gall A, Kaye S, Hué S, Bonsall D, Rance R, Baillie GJ, Fidler SJ, Weber JN, McClure MO, Kellam P and SPARTAC Trial Investigators

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

    Background: Dynamic changes in Human Immunodeficiency Virus 1 (HIV-1) sequence diversity and divergence are associated with immune control during primary infection and progression to AIDS. Consensus sequencing or single genome amplification sequencing of the HIV-1 envelope (env) gene, in particular the variable (V) regions, is used as a marker for HIV-1 genome diversity, but population diversity is only minimally, or semi-quantitatively sampled using these methods.

    Results: Here we use second generation deep sequencing to determine inter-and intra-patient sequence heterogeneity and to quantify minor variants in a cohort of individuals either receiving or not receiving antiretroviral treatment following seroconversion; the SPARTAC trial. We show, through a cross-sectional study of sequence diversity of the env V3 in 30 antiretroviral-naive patients during primary infection that considerable population structure diversity exists, with some individuals exhibiting highly constrained plasma virus diversity. Diversity was independent of clinical markers (viral load, time from seroconversion, CD4 cell count) of infection. Serial sampling over 60 weeks of non-treated individuals that define three initially different diversity profiles showed that complex patterns of continuing HIV-1 sequence diversification and divergence could be readily detected. Evidence for minor sequence turnover, emergence of new variants and re-emergence of archived variants could be inferred from this analysis. Analysis of viral divergence over the same time period in patients who received short (12 weeks, ART12) or long course antiretroviral therapy (48 weeks, ART48) and a non-treated control group revealed that ART48 successfully suppressed viral divergence while ART12 did not have a significant effect.

    Conclusions: Deep sequencing is a sensitive and reliable method for investigating the diversity of the env V3 as an important component of HIV-1 genome diversity. Detailed insights into the complex early intra-patient dynamics of env V3 diversity and divergence were explored in antiretroviral-naïve recent seroconverters. Long course antiretroviral therapy, initiated soon after seroconversion and administered for 48 weeks, restricts HIV-1 divergence significantly. The effect of ART12 and ART48 on clinical markers of HIV infection and progression is currently investigated in the SPARTAC trial.

    Funded by: NIAID NIH HHS: R01 AI046995; Wellcome Trust

    Retrovirology 2013;10;8

2012 Publications

  • Transmission of equine influenza virus during an outbreak is characterized by frequent mixed infections and loose transmission bottlenecks.

    Hughes J, Allen RC, Baguelin M, Hampson K, Baillie GJ, Elton D, Newton JR, Kellam P, Wood JL, Holmes EC and Murcia PR

    Medical Research Council-University of Glasgow Centre for Virus Research, Institute of Infection, Inflammation and Immunity, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

    The ability of influenza A viruses (IAVs) to cross species barriers and evade host immunity is a major public health concern. Studies on the phylodynamics of IAVs across different scales - from the individual to the population - are essential for devising effective measures to predict, prevent or contain influenza emergence. Understanding how IAVs spread and evolve during outbreaks is critical for the management of epidemics. Reconstructing the transmission network during a single outbreak by sampling viral genetic data in time and space can generate insights about these processes. Here, we obtained intra-host viral sequence data from horses infected with equine influenza virus (EIV) to reconstruct the spread of EIV during a large outbreak. To this end, we analyzed within-host viral populations from sequences covering 90% of the infected yards. By combining gene sequence analyses with epidemiological data, we inferred a plausible transmission network, in turn enabling the comparison of transmission patterns during the course of the outbreak and revealing important epidemiological features that were not apparent using either approach alone. The EIV populations displayed high levels of genetic diversity, and in many cases we observed distinct viral populations containing a dominant variant and a number of related minor variants that were transmitted between infectious horses. In addition, we found evidence of frequent mixed infections and loose transmission bottlenecks in these naturally occurring populations. These frequent mixed infections likely influence the size of epidemics.

    Funded by: Medical Research Council: G0801822, G0901135; NIGMS NIH HHS: 2 R01 GM080533-06; Wellcome Trust

    PLoS pathogens 2012;8;12;e1003081

  • Universal amplification, next-generation sequencing, and assembly of HIV-1 genomes.

    Gall A, Ferns B, Morris C, Watson S, Cotten M, Robinson M, Berry N, Pillay D and Kellam P

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom.

    Whole HIV-1 genome sequences are pivotal for large-scale studies of inter- and intrahost evolution, including the acquisition of drug resistance mutations. The ability to rapidly and cost-effectively generate large numbers of HIV-1 genome sequences from different populations and geographical locations and determine the effect of minority genetic variants is, however, a limiting factor. Next-generation sequencing promises to bridge this gap but is hindered by the lack of methods for the enrichment of virus genomes across the phylogenetic breadth of HIV-1 and methods for the robust assembly of the virus genomes from short-read data. Here we report a method for the amplification, next-generation sequencing, and unbiased de novo assembly of HIV-1 genomes of groups M, N, and O, as well as recombinants, that does not require prior knowledge of the sequence or subtype. A sensitivity of at least 3,000 copies/ml was determined by using plasma virus samples of known copy numbers. We applied our novel method to compare the genome diversities of HIV-1 groups, subtypes, and genes. The highest level of diversity was found in the env, nef, vpr, tat, and rev genes and parts of the gag gene. Furthermore, we used our method to investigate mutations associated with HIV-1 drug resistance in clinical samples at the level of the complete genome. Drug resistance mutations were detected as both major variant and minor species. In conclusion, we demonstrate the feasibility of our method for large-scale HIV-1 genome sequencing. This will enable the phylogenetic and phylodynamic resolution of the ongoing pandemic and efficient monitoring of complex HIV-1 drug resistance genotypes.

    Funded by: Wellcome Trust: S0753

    Journal of clinical microbiology 2012;50;12;3838-44

  • Estimating reassortment rates in co-circulating Eurasian swine influenza viruses.

    Lycett SJ, Baillie G, Coulter E, Bhatt S, Kellam P, McCauley JW, Wood JL, Brown IH, Pybus OG, Leigh Brown AJ and Combating Swine Influenza Initiative-COSI Consortium

    Institute of Evolutionary Biology, University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh EH9 3JT, UK. samantha.lycett@ed.ac.uk

    Swine have often been considered as a mixing vessel for different influenza strains. In order to assess their role in more detail, we undertook a retrospective sequencing study to detect and characterize the reassortants present in European swine and to estimate the rate of reassortment between H1N1, H1N2 and H3N2 subtypes with Eurasian (avian-like) internal protein-coding segments. We analysed 69 newly obtained whole genome sequences of subtypes H1N1-H3N2 from swine influenza viruses sampled between 1982 and 2008, using Illumina and 454 platforms. Analyses of these genomes, together with previously published genomes, revealed a large monophyletic clade of Eurasian swine-lineage polymerase segments containing H1N1, H1N2 and H3N2 subtypes. We subsequently examined reassortments between the haemagglutinin and neuraminidase segments and estimated the reassortment rates between lineages using a recently developed evolutionary analysis method. High rates of reassortment between H1N2 and H1N1 Eurasian swine lineages were detected in European strains, with an average of one reassortment every 2-3 years. This rapid reassortment results from co-circulating lineages in swine, and in consequence we should expect further reassortments between currently circulating swine strains and the recent swine-origin H1N1v pandemic strain.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/H014306/1; Medical Research Council: MC_G0902096, MC_U117512723; Wellcome Trust

    The Journal of general virology 2012;93;Pt 11;2326-36

  • Permissive and restricted virus infection of murine embryonic stem cells.

    Wash R, Calabressi S, Franz S, Griffiths SJ, Goulding D, Tan EP, Wise H, Digard P, Haas J, Efstathiou S and Kellam P

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

    Recent RNA interference (RNAi) studies have identified many host proteins that modulate virus infection, but small interfering RNA 'off-target' effects and the use of transformed cell lines limit their conclusiveness. As murine embryonic stem (mES) cells can be genetically modified and resources exist where many and eventually all known mouse genes are insertionally inactivated, it was reasoned that mES cells would provide a useful alternative to RNAi screens. Beyond allowing investigation of host-pathogen interactions in vitro, mES cells have the potential to differentiate into other primary cell types, as well as being used to generate knockout mice for in vivo studies. However, mES cells are poorly characterized for virus infection. To investigate whether ES cells can be used to explore host-virus interactions, this study characterized the responses of mES cells following infection by herpes simplex virus type 1 (HSV-1) and influenza A virus. HSV-1 replicated lytically in mES cells, although mES cells were less permissive than most other cell types tested. Influenza virus was able to enter mES cells and express some viral proteins, but the replication cycle was incomplete and no infectious virus was produced. Knockdown of the host protein AHCYL1 in mES cells reduced HSV-1 replication, showing the potential for using mES cells to study host-virus interactions. Transcriptional profiling, however, indicated the lack of an efficient innate immune response in these cells. mES cells may thus be useful to identify host proteins that play a role in virus replication, but they are not suitable to determine factors that are involved in innate host defence.

    Funded by: Medical Research Council: G9800943, MR/J002232/1; Wellcome Trust

    The Journal of general virology 2012;93;Pt 10;2118-30

  • IFITM3 restricts the morbidity and mortality associated with influenza.

    Everitt AR, Clare S, Pertel T, John SP, Wash RS, Smith SE, Chin CR, Feeley EM, Sims JS, Adams DJ, Wise HM, Kane L, Goulding D, Digard P, Anttila V, Baillie JK, Walsh TS, Hume DA, Palotie A, Xue Y, Colonna V, Tyler-Smith C, Dunning J, Gordon SB, GenISIS Investigators, MOSAIC Investigators, Smyth RL, Openshaw PJ, Dougan G, Brass AL and Kellam P

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton CB10 1SA, UK.

    The 2009 H1N1 influenza pandemic showed the speed with which a novel respiratory virus can spread and the ability of a generally mild infection to induce severe morbidity and mortality in a subset of the population. Recent in vitro studies show that the interferon-inducible transmembrane (IFITM) protein family members potently restrict the replication of multiple pathogenic viruses. Both the magnitude and breadth of the IFITM proteins' in vitro effects suggest that they are critical for intrinsic resistance to such viruses, including influenza viruses. Using a knockout mouse model, we now test this hypothesis directly and find that IFITM3 is essential for defending the host against influenza A virus in vivo. Mice lacking Ifitm3 display fulminant viral pneumonia when challenged with a normally low-pathogenicity influenza virus, mirroring the destruction inflicted by the highly pathogenic 1918 'Spanish' influenza. Similar increased viral replication is seen in vitro, with protection rescued by the re-introduction of Ifitm3. To test the role of IFITM3 in human influenza virus infection, we assessed the IFITM3 alleles of individuals hospitalized with seasonal or pandemic influenza H1N1/09 viruses. We find that a statistically significant number of hospitalized subjects show enrichment for a minor IFITM3 allele (SNP rs12252-C) that alters a splice acceptor site, and functional assays show the minor CC genotype IFITM3 has reduced influenza virus restriction in vitro. Together these data reveal that the action of a single intrinsic immune effector, IFITM3, profoundly alters the course of influenza virus infection in mouse and humans.

    Funded by: Cancer Research UK: 13031; Chief Scientist Office; Medical Research Council: G0600511, G0800767, G0800777, G0802752, G0901697, MC_G1001212, MC_U122785833; NIAID NIH HHS: R01 AI091786, R01AI091786; Wellcome Trust: 090382, 090382/Z/09/Z, 090385/Z/09/Z, 098051

    Nature 2012;484;7395;519-23

  • Evolutionary dynamics of local pandemic H1N1/2009 influenza virus lineages revealed by whole-genome analysis.

    Baillie GJ, Galiano M, Agapow PM, Myers R, Chiam R, Gall A, Palser AL, Watson SJ, Hedge J, Underwood A, Platt S, McLean E, Pebody RG, Rambaut A, Green J, Daniels R, Pybus OG, Kellam P and Zambon M

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom.

    Virus gene sequencing and phylogenetics can be used to study the epidemiological dynamics of rapidly evolving viruses. With complete genome data, it becomes possible to identify and trace individual transmission chains of viruses such as influenza virus during the course of an epidemic. Here we sequenced 153 pandemic influenza H1N1/09 virus genomes from United Kingdom isolates from the first (127 isolates) and second (26 isolates) waves of the 2009 pandemic and used their sequences, dates of isolation, and geographical locations to infer the genetic epidemiology of the epidemic in the United Kingdom. We demonstrate that the epidemic in the United Kingdom was composed of many cocirculating lineages, among which at least 13 were exclusively or predominantly United Kingdom clusters. The estimated divergence times of two of the clusters predate the detection of pandemic H1N1/09 virus in the United Kingdom, suggesting that the pandemic H1N1/09 virus was already circulating in the United Kingdom before the first clinical case. Crucially, three clusters contain isolates from the second wave of infections in the United Kingdom, two of which represent chains of transmission that appear to have persisted within the United Kingdom between the first and second waves. This demonstrates that whole-genome analysis can track in fine detail the behavior of individual influenza virus lineages during the course of a single epidemic or pandemic.

    Funded by: Medical Research Council: MC_U117512723; Wellcome Trust: 095831

    Journal of virology 2012;86;1;11-8

  • Marked endotheliotropism of highly pathogenic avian influenza virus H5N1 following intestinal inoculation in cats.

    Reperant LA, van de Bildt MW, van Amerongen G, Leijten LM, Watson S, Palser A, Kellam P, Eissens AC, Frijlink HW, Osterhaus AD and Kuiken T

    Department of Ecology and Evolutionary Biology, Princeton University, Princeton, New Jersey, USA.

    Highly pathogenic avian influenza virus (HPAIV) H5N1 can infect mammals via the intestine; this is unusual since influenza viruses typically infect mammals via the respiratory tract. The dissemination of HPAIV H5N1 following intestinal entry and associated pathogenesis are largely unknown. To assess the route of spread of HPAIV H5N1 to other organs and to determine its associated pathogenesis, we inoculated infected chicken liver homogenate directly into the intestine of cats by use of enteric-coated capsules. Intestinal inoculation of HPAIV H5N1 resulted in fatal systemic disease. The spread of HPAIV H5N1 from the lumen of the intestine to other organs took place via the blood and lymphatic vascular systems but not via neuronal transmission. Remarkably, the systemic spread of the virus via the vascular system was associated with massive infection of endothelial and lymphendothelial cells, resulting in widespread hemorrhages. This is unique for influenza in mammals and resembles the pathogenesis of HPAIV infection in terrestrial poultry. It contrasts with the pathogenesis of systemic disease from the same virus following entry via the respiratory tract, where lesions are characterized mainly by necrosis and inflammation and are associated with the presence of influenza virus antigen in parenchymal, not endothelial cells. The marked endotheliotropism of the virus following intestinal inoculation indicates that the pathogenesis of systemic influenza virus infection in mammals may differ according to the portal of entry.

    Journal of virology 2012;86;2;1158-65

  • Analysis of high-depth sequence data for studying viral diversity: a comparison of next generation sequencing platforms using Segminator II.

    Archer J, Baillie G, Watson SJ, Kellam P, Rambaut A and Robertson DL

    Computational and Evolutionary Biology, Faculty of Life Sciences, University of Manchester, Manchester, UK. john.archer@manchester.ac.uk

    Background: Next generation sequencing provides detailed insight into the variation present within viral populations, introducing the possibility of treatment strategies that are both reactive and predictive. Current software tools, however, need to be scaled up to accommodate for high-depth viral data sets, which are often temporally or spatially linked. In addition, due to the development of novel sequencing platforms and chemistries, each with implicit strengths and weaknesses, it will be helpful for researchers to be able to routinely compare and combine data sets from different platforms/chemistries. In particular, error associated with a specific sequencing process must be quantified so that true biological variation may be identified.

    Results: Segminator II was developed to allow for the efficient comparison of data sets derived from different sources. We demonstrate its usage by comparing large data sets from 12 influenza H1N1 samples sequenced on both the 454 Life Sciences and Illumina platforms, permitting quantification of platform error. For mismatches median error rates at 0.10 and 0.12%, respectively, suggested that both platforms performed similarly. For insertions and deletions median error rates within the 454 data (at 0.3 and 0.2%, respectively) were significantly higher than those within the Illumina data (0.004 and 0.006%, respectively). In agreement with previous observations these higher rates were strongly associated with homopolymeric stretches on the 454 platform. Outside of such regions both platforms had similar indel error profiles. Additionally, we apply our software to the identification of low frequency variants.

    Conclusion: We have demonstrated, using Segminator II, that it is possible to distinguish platform specific error from biological variation using data derived from two different platforms. We have used this approach to quantify the amount of error present within the 454 and Illumina platforms in relation to genomic location as well as location on the read. Given that next generation data is increasingly important in the analysis of drug-resistance and vaccine trials, this software will be useful to the pathogen research community. A zip file containing the source code and jar file is freely available for download from http://www.bioinf.manchester.ac.uk/segminator/.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/H012419/1; Wellcome Trust: 095831

    BMC bioinformatics 2012;13;47

  • Evolution of an Eurasian avian-like influenza virus in naïve and vaccinated pigs.

    Murcia PR, Hughes J, Battista P, Lloyd L, Baillie GJ, Ramirez-Gonzalez RH, Ormond D, Oliver K, Elton D, Mumford JA, Caccamo M, Kellam P, Grenfell BT, Holmes EC and Wood JL

    Cambridge Infectious Diseases Consortium, Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.

    Influenza viruses are characterized by an ability to cross species boundaries and evade host immunity, sometimes with devastating consequences. The 2009 pandemic of H1N1 influenza A virus highlights the importance of pigs in influenza emergence, particularly as intermediate hosts by which avian viruses adapt to mammals before emerging in humans. Although segment reassortment has commonly been associated with influenza emergence, an expanded host-range is also likely to be associated with the accumulation of specific beneficial point mutations. To better understand the mechanisms that shape the genetic diversity of avian-like viruses in pigs, we studied the evolutionary dynamics of an Eurasian Avian-like swine influenza virus (EA-SIV) in naïve and vaccinated pigs linked by natural transmission. We analyzed multiple clones of the hemagglutinin 1 (HA1) gene derived from consecutive daily viral populations. Strikingly, we observed both transient and fixed changes in the consensus sequence along the transmission chain. Hence, the mutational spectrum of intra-host EA-SIV populations is highly dynamic and allele fixation can occur with extreme rapidity. In addition, mutations that could potentially alter host-range and antigenicity were transmitted between animals and mixed infections were commonplace, even in vaccinated pigs. Finally, we repeatedly detected distinct stop codons in virus samples from co-housed pigs, suggesting that they persisted within hosts and were transmitted among them. This implies that mutations that reduce viral fitness in one host, but which could lead to fitness benefits in a novel host, can circulate at low frequencies.

    Funded by: Medical Research Council: G0801822; NICHD NIH HHS: R24 HD047879; NIGMS NIH HHS: R01 GM080533-05; Wellcome Trust

    PLoS pathogens 2012;8;5;e1002730

2011 Publications

  • Determinants of bluetongue virus virulence in murine models of disease.

    Caporale M, Wash R, Pini A, Savini G, Franchi P, Golder M, Patterson-Kane J, Mertens P, Di Gialleonardo L, Armillotta G, Lelli R, Kellam P and Palmarini M

    Medical Research Council-University of Glasgow Centre for Virus Research, Institute of Infection, Inflammation and Immunity, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

    Bluetongue is a major infectious disease of ruminants that is caused by bluetongue virus (BTV). In this study, we analyzed virulence and genetic differences of (i) three BTV field strains from Italy maintained at either a low (L strains) or high (H strains) passage number in cell culture and (ii) three South African "reference" wild-type strains and their corresponding live attenuated vaccine strains. The Italian BTV L strains, in general, were lethal for both newborn NIH-Swiss mice inoculated intracerebrally and adult type I interferon receptor-deficient (IFNAR(-/-)) mice, while the virulence of the H strains was attenuated significantly in both experimental models. Similarly, the South African vaccine strains were not pathogenic for IFNAR(-/-) mice, while the corresponding wild-type strains were virulent. Thus, attenuation of the virulence of the BTV strains used in this study is not mediated by the presence of an intact interferon system. No clear distinction in virulence was observed for the South African BTV strains in newborn NIH-Swiss mice. Full genomic sequencing revealed relatively few amino acid substitutions, scattered in several different viral proteins, for the strains found to be attenuated in mice compared to the pathogenic related strains. However, only the genome segments encoding VP1, VP2, and NS2 consistently showed nonsynonymous changes between all virulent and attenuated strain pairs. This study established an experimental platform for investigating the determinants of BTV virulence. Future studies using reverse genetics will allow researchers to precisely map and "weight" the relative influences of the various genome segments and viral proteins on BTV virulence.

    Funded by: Wellcome Trust

    Journal of virology 2011;85;21;11479-89

  • Metagenomics and the molecular identification of novel viruses.

    Bexfield N and Kellam P

    Department of Veterinary Medicine, University of Cambridge, Cambridge, UK. nb289@cam.ac.uk

    There have been rapid recent developments in establishing methods for identifying and characterising viruses associated with animal and human diseases. These methodologies, commonly based on hybridisation or PCR techniques, are combined with advanced sequencing techniques termed 'next generation sequencing'. Allied advances in data analysis, including the use of computational transcriptome subtraction, have also impacted the field of viral pathogen discovery. This review details these molecular detection techniques, discusses their application in viral discovery, and provides an overview of some of the novel viruses discovered. The problems encountered in attributing disease causality to a newly identified virus are also considered.

    Veterinary journal (London, England : 1997) 2011;190;2;191-8

  • Phylogenetic analysis of murine leukemia virus sequences from longitudinally sampled chronic fatigue syndrome patients suggests PCR contamination rather than viral evolution.

    Katzourakis A, Hué S, Kellam P and Towers GJ

    Department of Zoology, University of Oxford, South Parks Road, Oxford OX13PS, United Kingdom.

    Xenotropic murine leukemia virus (MLV)-related virus (XMRV) has been amplified from human prostate cancer and chronic fatigue syndrome (CFS) patient samples. Other studies failed to replicate these findings and suggested PCR contamination with a prostate cancer cell line, 22Rv1, as a likely source. MLV-like sequences have also been detected in CFS patients in longitudinal samples 15 years apart. Here, we tested whether sequence data from these samples are consistent with viral evolution. Our phylogenetic analyses strongly reject a model of within-patient evolution and demonstrate that the sequences from the first and second time points represent distinct endogenous murine retroviruses, suggesting contamination.

    Funded by: Medical Research Council: G0801172, G9721629; Wellcome Trust: 090940, WT090940

    Journal of virology 2011;85;20;10909-13

  • X-box binding protein 1 induces the expression of the lytic cycle transactivator of Kaposi's sarcoma-associated herpesvirus but not Epstein-Barr virus in co-infected primary effusion lymphoma.

    Lai IY, Farrell PJ and Kellam P

    University College London, MRC Centre for Molecular Virology, Department of Infection, Division of Infection and Immunity, Windeyer Institute of Medical Science, 46 Cleveland Street, London W1T 4JF, UK.

    Cells of primary effusion lymphoma (PEL), a B-cell non-Hodgkin's lymphoma, are latently infected by Kaposi's sarcoma-associated herpesvirus (KSHV), with about 80 % of PEL also co-infected with Epstein-Barr virus (EBV). Both viruses can be reactivated into their lytic replication cycle in PEL by chemical inducers. However, simultaneous activation of both lytic cascades leads to mutual lytic cycle co-repression. The plasma cell-differentiation factor X-box binding protein 1 (XBP-1) transactivates the KSHV immediate-early promoter leading to the production of the replication and transcription activator protein (RTA), and reactivation of KSHV from latency. XBP-1 has been reported to act similarly on the EBV immediate-early promoter Zp, leading to the production of the lytic-cycle transactivator protein BZLF1. Here we show that activated B-cell terminal-differentiation transcription factor X-box binding protein 1 (XBP-1s) does not induce EBV BZLF1 and BRLF1 expression in PEL and BL cell lines, despite inducing lytic reactivation of KSHV in PEL. We show that XBP-1s transactivates the KSHV RTA promoter but does not transactivate the EBV BZLF1 promoter in non-B-cells by using a luciferase assay. Co-expression of activated protein kinase D, which can phosphorylate and inactivate class II histone deacetylases (HDACs), does not rescue XBP-1 activity on Zp nor does it induce BZLF1 and BRLF1 expression in PEL. Finally, chemical inducers of KSHV and EBV lytic replication in PEL, including HDAC inhibitors, do not lead to XBP-1 activation. We conclude that XBP-1 specifically reactivates the KSHV lytic cycle in dually infected PELs.

    Funded by: Cancer Research UK; Wellcome Trust

    The Journal of general virology 2011;92;Pt 2;421-31

  • A viral discovery methodology for clinical biopsy samples utilising massively parallel next generation sequencing.

    Daly GM, Bexfield N, Heaney J, Stubbs S, Mayer AP, Palser A, Kellam P, Drou N, Caccamo M, Tiley L, Alexander GJ, Bernal W and Heeney JL

    Department of Veterinary Medicine, The University of Cambridge, Cambridge, United Kingdom.

    Here we describe a virus discovery protocol for a range of different virus genera, that can be applied to biopsy-sized tissue samples. Our viral enrichment procedure, validated using canine and human liver samples, significantly improves viral read copy number and increases the length of viral contigs that can be generated by de novo assembly. This in turn enables the Illumina next generation sequencing (NGS) platform to be used as an effective tool for viral discovery from tissue samples.

    Funded by: Wellcome Trust

    PloS one 2011;6;12;e28879

  • Analysis of XMRV integration sites from human prostate cancer tissues suggests PCR contamination rather than genuine human infection.

    Garson JA, Kellam P and Towers GJ

    MRC Centre for Medical Molecular Virology, Division of Infection and Immunity, University College London, 46 Cleveland St, London W1T 4JF, UK.

    XMRV is a gammaretrovirus associated in some studies with human prostate cancer and chronic fatigue syndrome. Central to the hypothesis of XMRV as a human pathogen is the description of integration sites in DNA from prostate tumour tissues. Here we demonstrate that 2 of 14 patient-derived sites are identical to sites cloned in the same laboratory from experimentally infected DU145 cells. Identical integration sites have never previously been described in any retrovirus infection. We propose that the patient-derived sites are the result of PCR contamination. This observation further undermines the notion that XMRV is a genuine human pathogen.

    Funded by: Medical Research Council: G0801172, G9721629; Wellcome Trust: 090940, WT076608, WT090940

    Retrovirology 2011;8;13

  • Assessment of a 44 gene classifier for the evaluation of chronic fatigue syndrome from peripheral blood mononuclear cell gene expression.

    Frampton D, Kerr J, Harrison TJ and Kellam P

    Department of Infection, Division of Infection and Immunity, University College London, London, United Kingdom.

    Chronic fatigue syndrome (CFS) is a clinically defined illness estimated to affect millions of people worldwide causing significant morbidity and an annual cost of billions of dollars. Currently there are no laboratory-based diagnostic methods for CFS. However, differences in gene expression profiles between CFS patients and healthy persons have been reported in the literature. Using mRNA relative quantities for 44 previously identified reporter genes taken from a large dataset comprising both CFS patients and healthy volunteers, we derived a gene profile scoring metric to accurately classify CFS and healthy samples. This metric out-performed any of the reporter genes used individually as a classifier of CFS.To determine whether the reporter genes were robust across populations, we applied this metric to classify a separate blind dataset of mRNA relative quantities from a new population of CFS patients and healthy persons with limited success. Although the metric was able to successfully classify roughly two-thirds of both CFS and healthy samples correctly, the level of misclassification was high. We conclude many of the previously identified reporter genes are study-specific and thus cannot be used as a broad CFS diagnostic.

    PloS one 2011;6;3;e16872

  • No evidence of XMRV or related retroviruses in a London HIV-1-positive patient cohort.

    Gray ER, Garson JA, Breuer J, Edwards S, Kellam P, Pillay D and Towers GJ

    Department of Infection and Immunity, University College London, London, United Kingdom. e.gray@ucl.ac.uk

    Background: Several studies have implicated a recently discovered gammaretrovirus, XMRV (Xenotropic murine leukaemia virus-related virus), in chronic fatigue syndrome and prostate cancer, though whether as causative agent or opportunistic infection is unclear. It has also been suggested that the virus can be found circulating amongst the general population. The discovery has been controversial, with conflicting results from attempts to reproduce the original studies.

    We extracted peripheral blood DNA from a cohort of 540 HIV-1-positive patients (approximately 20% of whom have never been on anti-retroviral treatment) and determined the presence of XMRV and related viruses using TaqMan PCR. While we were able to amplify as few as 5 copies of positive control DNA, we did not find any positive samples in the patient cohort.

    In view of these negative findings in this highly susceptible group, we conclude that it is unlikely that XMRV or related viruses are circulating at a significant level, if at all, in HIV-1-positive patients in London or in the general population.

    Funded by: Department of Health; Medical Research Council: G0801172, G9721629; Wellcome Trust: 090940, WT090940

    PloS one 2011;6;3;e18096

  • Specific capture and whole-genome sequencing of viruses from clinical samples.

    Depledge DP, Palser AL, Watson SJ, Lai IY, Gray ER, Grant P, Kanda RK, Leproust E, Kellam P and Breuer J

    Division of Infection and Immunity, University College London, London, United Kingdom. d.depledge@ucl.ac.uk

    Whole genome sequencing of viruses directly from clinical samples is integral for understanding the genetics of host-virus interactions. Here, we report the use of sample sparing target enrichment (by hybridisation) for viral nucleic acid separation and deep-sequencing of herpesvirus genomes directly from a range of clinical samples including saliva, blood, virus vesicles, cerebrospinal fluid, and tumour cell lines. We demonstrate the effectiveness of the method by deep-sequencing 13 highly cell-associated human herpesvirus genomes and generating full length genome alignments at high read depth. Moreover, we show the specificity of the method enables the study of viral population structures and their diversity within a range of clinical samples types.

    Funded by: Department of Health; Medical Research Council: G07008, G0700814, G0900950; Wellcome Trust: 081703MA

    PloS one 2011;6;11;e27805

  • Disease-associated XMRV sequences are consistent with laboratory contamination.

    Hué S, Gray ER, Gall A, Katzourakis A, Tan CP, Houldcroft CJ, McLaren S, Pillay D, Futreal A, Garson JA, Pybus OG, Kellam P and Towers GJ

    MRC Centre for Medical Molecular Virology, Division of Infection and Immunity, University College London, 46 Cleveland St, London W1T 4JF, UK.

    Background: Xenotropic murine leukaemia viruses (MLV-X) are endogenous gammaretroviruses that infect cells from many species, including humans. Xenotropic murine leukaemia virus-related virus (XMRV) is a retrovirus that has been the subject of intense debate since its detection in samples from humans with prostate cancer (PC) and chronic fatigue syndrome (CFS). Controversy has arisen from the failure of some studies to detect XMRV in PC or CFS patients and from inconsistent detection of XMRV in healthy controls.

    Results: Here we demonstrate that Taqman PCR primers previously described as XMRV-specific can amplify common murine endogenous viral sequences from mouse suggesting that mouse DNA can contaminate patient samples and confound specific XMRV detection. To consider the provenance of XMRV we sequenced XMRV from the cell line 22Rv1, which is infected with an MLV-X that is indistinguishable from patient derived XMRV. Bayesian phylogenies clearly show that XMRV sequences reportedly derived from unlinked patients form a monophyletic clade with interspersed 22Rv1 clones (posterior probability >0.99). The cell line-derived sequences are ancestral to the patient-derived sequences (posterior probability >0.99). Furthermore, pol sequences apparently amplified from PC patient material (VP29 and VP184) are recombinants of XMRV and Moloney MLV (MoMLV) a virus with an envelope that lacks tropism for human cells. Considering the diversity of XMRV we show that the mean pairwise genetic distance among env and pol 22Rv1-derived sequences exceeds that of patient-associated sequences (Wilcoxon rank sum test: p = 0.005 and p < 0.001 for pol and env, respectively). Thus XMRV sequences acquire diversity in a cell line but not in patient samples. These observations are difficult to reconcile with the hypothesis that published XMRV sequences are related by a process of infectious transmission.

    Conclusions: We provide several independent lines of evidence that XMRV detected by sensitive PCR methods in patient samples is the likely result of PCR contamination with mouse DNA and that the described clones of XMRV arose from the tumour cell line 22Rv1, which was probably infected with XMRV during xenografting in mice. We propose that XMRV might not be a genuine human pathogen.

    Funded by: Medical Research Council: G0801172, G0801172(87743), G9721629; Wellcome Trust: 090940, WT076608, WT090940

    Retrovirology 2010;7;1;111

2010 Publications

  • The RING-CH ligase K5 antagonizes restriction of KSHV and HIV-1 particle release by mediating ubiquitin-dependent endosomal degradation of tetherin.

    Pardieu C, Vigan R, Wilson SJ, Calvi A, Zang T, Bieniasz P, Kellam P, Towers GJ and Neil SJ

    MRC Centre for Medical Molecular Virology, University College London, London, United Kingdom.

    Tetherin (CD317/BST2) is an interferon-induced membrane protein that inhibits the release of diverse enveloped viral particles. Several mammalian viruses have evolved countermeasures that inactivate tetherin, with the prototype being the HIV-1 Vpu protein. Here we show that the human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) is sensitive to tetherin restriction and its activity is counteracted by the KSHV encoded RING-CH E3 ubiquitin ligase K5. Tetherin expression in KSHV-infected cells inhibits viral particle release, as does depletion of K5 protein using RNA interference. K5 induces a species-specific downregulation of human tetherin from the cell surface followed by its endosomal degradation. We show that K5 targets a single lysine (K18) in the cytoplasmic tail of tetherin for ubiquitination, leading to relocalization of tetherin to CD63-positive endosomal compartments. Tetherin degradation is dependent on ESCRT-mediated endosomal sorting, but does not require a tyrosine-based sorting signal in the tetherin cytoplasmic tail. Importantly, we also show that the ability of K5 to substitute for Vpu in HIV-1 release is entirely dependent on K18 and the RING-CH domain of K5. By contrast, while Vpu induces ubiquitination of tetherin cytoplasmic tail lysine residues, mutation of these positions has no effect on its antagonism of tetherin function, and residual tetherin is associated with the trans-Golgi network (TGN) in Vpu-expressing cells. Taken together our results demonstrate that K5 is a mechanistically distinct viral countermeasure to tetherin-mediated restriction, and that herpesvirus particle release is sensitive to this mode of antiviral inhibition.

    Funded by: Medical Research Council: G0801172, G0801172(87743), G0801937, G9721629; Wellcome Trust: 076608, WT082274MA

    PLoS pathogens 2010;6;4;e1000843

  • Regulation of the Epstein-Barr virus Zp promoter in B lymphocytes during reactivation from latency.

    McDonald C, Karstegl CE, Kellam P and Farrell PJ

    Department of Virology, Imperial College Faculty of Medicine, St Mary's Campus, London W2 1PG, UK.

    Ten novel mutations were introduced into the Zp promoter to test the role of sequences outside the established transcription factor-binding sites in Epstein-Barr virus (EBV) reactivation. Most of these had only small effects, but mutations in the ZID site were shown to reduce Zp activity strongly at early times after induction by anti-immunoglobulin (anti-Ig). The binding of MEF2 transcription factor to ZID was characterized in detail and linked functionally to Zp promoter activity. The presence of XBP-1s, the active form of XBP-1, after administration of anti-Ig to Akata Burkitt's lymphoma cells is consistent with a role for this factor in reactivation of the EBV lytic cycle, although signalling through MEF2D was quantitatively much more significant in activation of Zp. Silencing of Zp during latency is thought to be primarily a consequence of a repressive chromatin structure on Zp, and this aspect of Zp regulation can be observed in the Akata genome through protection of Zp from activation by BZLF1 in the absence of signalling from the B-cell receptor.

    The Journal of general virology 2010;91;Pt 3;622-9

  • KSHV-encoded miRNAs target MAF to induce endothelial cell reprogramming.

    Hansen A, Henderson S, Lagos D, Nikitenko L, Coulter E, Roberts S, Gratrix F, Plaisance K, Renne R, Bower M, Kellam P and Boshoff C

    Cancer Research UK Viral Oncology Group, University College London Cancer Institute, University College London, London WC1E 6BT, United Kingdom.

    Kaposi sarcoma herpesvirus (KSHV) induces transcriptional reprogramming of endothelial cells. In particular, KSHV-infected lymphatic endothelial cells (LECs) show an up-regulation of genes associated with blood vessel endothelial cells (BECs). Consequently, KSHV-infected tumor cells in Kaposi sarcoma are poorly differentiated endothelial cells, expressing markers of both LECs and BECs. MicroRNAs (miRNAs) are short noncoding RNA molecules that act post-transcriptionally to negatively regulate gene expression. Here we validate expression of the KSHV-encoded miRNAs in Kaposi sarcoma lesions and demonstrate that these miRNAs contribute to viral-induced reprogramming by silencing the cellular transcription factor MAF (musculoaponeurotic fibrosarcoma oncogene homolog). MAF is expressed in LECs but not in BECs. We identify a novel role for MAF as a transcriptional repressor, preventing expression of BEC-specific genes, thereby maintaining the differentiation status of LECs. These findings demonstrate that viral miRNAs could influence the differentiation status of infected cells, and thereby contribute to KSHV-induced oncogenesis.

    Funded by: Cancer Research UK; Medical Research Council: G0800168

    Genes & development 2010;24;2;195-205

2009 Publications

  • Microarray-based determination of the lytic cascade of human herpesvirus 6B.

    Tsao EH, Kellam P, Sin CS, Rasaiyaah J, Griffiths PD and Clark DA

    Department of Infection, Division of Infection and Immunity, Royal Free and University College Medical School of UCL, London, UK.

    The lytic gene expression of several members of the human herpesvirus family has been profiled by using gene-expression microarrays; however, the lytic cascade of roseoloviruses has not been studied in similar depth. Based on the complete DNA genome sequences of human herpesvirus 6 variant A (HHV-6A) and variant B (HHV-6B), we constructed a cDNA microarray containing DNA probes to their predicted open reading frames, plus 914 human genes. Gene-expression profiling of HHV-6B strain Z29 in SupT1 cells over a 60 h time-course post-infection, together with kinetic classification of the HHV-6B genes in the presence of either cycloheximide or phosphonoacetic acid, allowed the placement of HHV-6B genes into defined kinetic classes. Eighty-nine HHV-6B genes were divided into four different expression kinetic classes: eight immediate-early, 44 early, 33 late and four biphasic. Clustering of genes with similar expression profiles implied a shared function, thus revealing possible roles of previously uncharacterized HHV-6B genes.

    Funded by: Biotechnology and Biological Sciences Research Council: EGM17738

    The Journal of general virology 2009;90;Pt 11;2581-91

  • Transcriptional and functional defects of dendritic cells derived from the MUTZ-3 leukaemia line.

    Rasaiyaah J, Noursadeghi M, Kellam P and Chain B

    Division of Infection and Immunity, University College London, London, UK.

    Dendritic cells (DC) generated from MUTZ-3, an immortalized acute myeloid leukaemia-derived cell line, have potential application as a model for the study of human DC, and as a tool with which to stimulate immunotherapeutic responses to cancer. However, the relationship of MUTZ-3 DC to their non-transformed counterparts remains incompletely understood. Immunoselected CD14+ MUTZ-3 cells were used to generate a homogeneous population of DC (M3DC). These cells had a cell surface phentoype and morphology characteristic of conventional monocyte-derived DC (MDDC). Whole genome transcriptome comparison of M3DC and MDDC however, revealed extensive differences between these two cell types. Functional ontology-based data analysis revealed three enriched clusters of genes downregulated in M3DC, with functions in pathogen recognition, DC maturation and cytokine/chemokine signalling. Downregulation of protein expression was confirmed for several of these genes. The molecular differences were accompanied by a profoundly impaired phenotypic and functional response of M3DC to microbial stimulation. The immortalized phenotype of MUTZ-3 therefore reflects not only deregulated proliferative capacity, but substantial perturbation of normal antigen-presenting cell function. These results have important implications for studies using MUTZ-3 as a model of MDDC or for cancer immunotherapy.

    Funded by: Biotechnology and Biological Sciences Research Council; Wellcome Trust: 077161

    Immunology 2009;127;3;429-41

  • X-box binding protein 1 contributes to induction of the Kaposi's sarcoma-associated herpesvirus lytic cycle under hypoxic conditions.

    Dalton-Griffin L, Wilson SJ and Kellam P

    Department of Infection, UCL, London, United Kingdom.

    Kaposi's sarcoma-associated herpesvirus (KSHV), like other herpesviruses, has two stages to its life cycle: latency and lytic replication. KSHV is required for development of Kaposi's sarcoma, a tumor of endothelial origin, and is associated with the B-cell tumor primary effusion lymphoma (PEL) and the plasmablastic variant of multicentric Castleman's disease, all of which are characterized by predominantly latent KSHV infection. Recently, we and others have shown that the activated form of transcription factor X-box binding protein 1 (XBP-1) is a physiological trigger of KSHV lytic reactivation in PEL. Here, we show that XBP-1s transactivates the ORF50/RTA promoter though an ACGT core containing the XBP-1 response element, an element previously identified as a weakly active hypoxia response element (HRE). Hypoxia induces the KSHV lytic cycle, and active HREs that respond to hypoxia-inducible factor 1alpha are present in the ORF50/RTA promoter. Hypoxia also induces active XBP-1s, and here, we show that both transcription factors contribute to the induction of RTA expression, leading to the production of infectious KSHV under hypoxic conditions.

    Funded by: Cancer Research UK; Medical Research Council; Wellcome Trust

    Journal of virology 2009;83;14;7202-9

  • Genome-wide innate immune responses in HIV-1-infected macrophages are preserved despite attenuation of the NF-kappa B activation pathway.

    Noursadeghi M, Tsang J, Miller RF, Straschewski S, Kellam P, Chain BM and Katz DR

    Infection and Immunity, University College London, London, United Kingdom. m.noursadeghi@ucl.ac.uk

    Macrophages contribute to HIV-1 infection at many levels. They provide permissive cells at the site of inoculation, augment virus transfer to T cells, generate long-lived viral reservoirs, and cause bystander cell apoptosis. A body of evidence suggests that the role of macrophages in cellular host defense is also compromised by HIV-1 infection. In this respect, macrophages are potent cells of the innate immune system that initiate and regulate wide-ranging immunological responses. This study focuses on the effect of HIV-1 infection on innate immune responses by macrophages at the level of signal transduction, whole genome transcriptional profiling, and cytokine secretion. We show that in an ex vivo model, M-CSF-differentiated monocyte-derived macrophages uniformly infected with replicating CCR5-tropic HIV-1, without cytopathic effect, exhibit selective attenuation of the NF-kappaB activation pathway in response to TLR4 and TLR2 stimulation. However, functional annotation clustering analysis of genome-wide transcriptional responses to LPS stimulation suggests substantial preservation of gene expression changes at the systems level, with modest attenuation of a subset of up-regulated LPS-responsive genes, and no effect on a selection of inflammatory cytokine responses at the protein level. These results extend existing reports of inhibitory interactions between HIV-1 accessory proteins and NF-kappaB signaling pathways, and whole genome expression profiling provides comprehensive assessment of the consequent effects on immune response gene expression. Unexpectedly, our data suggest innate immune responses are broadly preserved with limited exceptions, and pave the way for further study of the complex relationship between HIV-1 and immunological pathways within macrophages.

    Funded by: Wellcome Trust: 077161, WT077161

    Journal of immunology (Baltimore, Md. : 1950) 2009;182;1;319-28

  • Identification of genes differentially expressed as result of adenovirus type 5- and adenovirus type 12-transformation.

    Strath J, Georgopoulos LJ, Kellam P and Blair GE

    Institute of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK. janet_strath@hotmail.com

    Background: Cells transformed by human adenoviruses (Ad) exhibit differential capacities to induce tumours in immunocompetent rodents; for example, Ad12-transformed rodent cells are oncogenic whereas Ad5-transformed cells are not. The E1A gene determines oncogenic phenotype, is a transcriptional regulator and dysregulates host cell gene expression, a key factor in both cellular transformation and oncogenesis. To reveal differences in gene expression between cells transformed with oncogenic and non-oncogenic adenoviruses we have performed comparative analysis of transcript profiles with the aim of identifying candidate genes involved in the process of neoplastic transformation.

    Results: Analysis of microarray data revealed that a total of 232 genes were differentially expressed in Ad12 E1- or Ad5 E1-transformed BRK cells compared to untransformed baby rat kidney (BRK) cells. Gene information was available for 193 transcripts and using gene ontology (GO) classifications and literature searches it was possible to assign known or suggested functions to 166 of these identified genes. A subset of differentially-expressed genes from the microarray was further examined by real-time PCR and Western blotting using BRK cells immortalised by Ad12 E1A or Ad5 E1A in addition to Ad12 E1- or Ad5 E1-transformed BRK cells. Up-regulation of RelA and significant dysregulation of collagen type I mRNA transcripts and proteins were found in Ad-transformed cells.

    Conclusion: These results suggest that a complex web of cellular pathways become altered in Ad-transformed cells and that Ad E1A is sufficient for the observed dysregulation. Further work will focus on investigating which splice variant of Ad E1A is responsible for the observed dysregulation at the pathway level, and the mechanisms of E1A-mediated transcriptional regulation.

    Funded by: Biotechnology and Biological Sciences Research Council

    BMC genomics 2009;10;67

  • Infectious causes of cancer and their detection.

    Dalton-Griffin L and Kellam P

    Department of Infection, University College London, Cleveland Street, London W1T 4JF, UK.

    Molecular techniques for identifying pathogens associated with cancer continue to be developed, including one reported recently in BMC Medical Genomics. Identifying a causal infectious agent helps in understanding the biology of these cancers and can lead ultimately to the development of antimicrobial drugs and vaccines for their treatment and prevention.

    Journal of biology 2009;8;7;67

2008 Publications

  • Computational inference of replication and transcription activator regulator activity in herpesvirus from gene expression data.

    Recchia A, Wit E, Vinciotti V and Kellam P

    University of Groningen (RUG), Institute of Mathematics and Computing Science, Groningen, Netherlands.

    One of the main aims of system biology is to understand the structure and dynamics of genomic systems. A computational approach, facilitated by new technologies for high-throughput quantitative experimental data, is put forward to investigate the regulatory system of dynamic interaction among genes in Kaposi's sarcoma-associated herpesvirus network after induction of lytic replication. A reconstruction of transcription factor activity and gene-regulatory kinetics using data from a time-course microarray experiment is proposed. The computational approach uses nonlinear differential equations. In particular, the quantitative Michaelis-Menten model of gene-regulatory kinetics is extended to allow for post-transcriptional modifications and synergic interactions between target genes and the Rta transcription factor. The kinetic method is developed within a Bayesian inferential framework using Markov chain Monte Carlo. The profile of the Rta transcriptional regulator, other post-transcriptional regulatory genes and gene-specific kinetic parameters are inferred from the gene expression data of the target genes. The method described here provides an example of a principled approach to handle a wide range of transcriptional network architectures and regulatory activation mechanisms to reconstruct the activity of several transcription factors and activation kinetic parameters in a single regulatory network.

    IET systems biology 2008;2;6;385-96

  • Patient-specific simulation as a basis for clinical decision-making.

    Sadiq SK, Mazzeo MD, Zasada SJ, Manos S, Stoica I, Gale CV, Watson SJ, Kellam P, Brew S and Coveney PV

    Centre for Computational Science, Department of Chemistry, University College London, London WC1H 0AJ, UK.

    Patient-specific medical simulation holds the promise of determining tailored medical treatment based on the characteristics of an individual patient (for example, using a genotypic assay of a sequence of DNA). Decision-support systems based on patient-specific simulation can potentially revolutionize the way that clinicians plan courses of treatment for various conditions, ranging from viral infections to arterial abnormalities. Basing medical decisions on the results of simulations that use models derived from data specific to the patient in question means that the effectiveness of a range of potential treatments can be assessed before they are actually administered, preventing the patient from experiencing unnecessary or ineffective treatments. We illustrate the potential for patient-specific simulation by first discussing the scale of the evolving international grid infrastructure that is now available to underpin such applications. We then consider two case studies, one concerned with the treatment of patients with HIV/AIDS and the other addressing neuropathologies associated with the intracranial vasculature. Such patient-specific medical simulations require access to both appropriate patient data and the computational resources on which to perform potentially very large simulations. Computational infrastructure providers need to furnish access to a wide range of different types of resource, typically made available through heterogeneous computational grids, and to institute policies that facilitate the performance of patient-specific simulations on those resources. To support these kinds of simulations, where life and death decisions are being made, computational resource providers must give urgent priority to such jobs, for example by allowing them to pre-empt the queue on a machine and run straight away. We describe systems that enable such priority computing.

    Philosophical transactions. Series A, Mathematical, physical, and engineering sciences 2008;366;1878;3199-219

  • Bim-mediated deletion of antigen-specific CD8 T cells in patients unable to control HBV infection.

    Lopes AR, Kellam P, Das A, Dunn C, Kwan A, Turner J, Peppa D, Gilson RJ, Gehring A, Bertoletti A and Maini MK

    Division of Infection and Immunity and Centre for Sexual Health and HIV Research, University College London, London, United Kingdom.

    HBV-specific CD8(+) T cells are critical for a successful immune response to HBV infection. They are markedly diminished in number in patients who fail to control the virus, but the mechanisms resulting in their depletion remain ill defined. Here, we dissected the defective HBV-specific CD8(+) T cell response associated with chronic HBV infection by gene expression profiling. We found that HBV-specific CD8(+) T cells from patients with different clinical outcomes could be distinguished by their patterns of gene expression. Microarray analysis revealed that overlapping clusters of functionally related apoptotic genes were upregulated in HBV-specific CD8(+) T cells from patients with chronic compared with resolved infection. Further analysis confirmed that levels of the proapoptotic protein Bcl2-interacting mediator (Bim) were upregulated in HBV-specific CD8(+) T cells from patients with chronic HBV infection. Blocking Bim-mediated apoptosis enhanced recovery of HBV-specific CD8(+) T cells both in culture and directly ex vivo. Consistent with evidence that Bim mediates apoptosis of CD8(+) T cells expressing low levels of CD127 (IL-7R), the few surviving HBV-specific CD8(+) T cells were CD127(hi )and had elevated levels of the antiapoptotic protein Mcl1, suggesting they were amenable to IL-7-mediated rescue from apoptosis. We therefore postulate that Bim-mediated attrition of HBV-specific CD8(+) T cells contributes to the inability of these cell populations to persist and control viral replication.

    Funded by: Medical Research Council: G108/515

    The Journal of clinical investigation 2008;118;5;1835-45

  • Gene expression subtypes in patients with chronic fatigue syndrome/myalgic encephalomyelitis.

    Kerr JR, Petty R, Burke B, Gough J, Fear D, Sinclair LI, Mattey DL, Richards SC, Montgomery J, Baldwin DA, Kellam P, Harrison TJ, Griffin GE, Main J, Enlander D, Nutt DJ and Holgate ST

    Department of Cellular & Molecular Medicine, St. George's University of London, London. jkerr@sgul.ac.uk

    Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a multisystem disease, the pathogenesis of which remains undetermined. We set out to determine the precise abnormalities of gene expression in the blood of patients with CFS/ME. We analyzed gene expression in peripheral blood from 25 patients with CFS/ME diagnosed according to the Centers for Disease Control and Prevention diagnostic criteria and 50 healthy blood donors, using a microarray with a cutoff fold difference of expression of >or=2.5. Genes showing differential expression were further analyzed in 55 patients with CFS/ME and 75 healthy blood donors, using quantitative polymerase chain reaction. Differential expression was confirmed for 88 genes; 85 were upregulated, and 3 were downregulated. Highly represented functions were hematological disease and function, immunological disease and function, cancer, cell death, immune response, and infection. Clustering of quantitative polymerase chain reaction data from patients with CFS/ME revealed 7 subtypes with distinct differences in Medical Outcomes Survey Short Form-36 scores, clinical phenotypes, and severity.

    Funded by: Medical Research Council: G0800766

    The Journal of infectious diseases 2008;197;8;1171-84

  • Gene3D: comprehensive structural and functional annotation of genomes.

    Yeats C, Lees J, Reid A, Kellam P, Martin N, Liu X and Orengo C

    UCL, Department of Molecular Biology & Biochemistry, Darwin Building, Gower St, London, UK. yeats@biochem.ucl.ac.uk

    Gene3D provides comprehensive structural and functional annotation of most available protein sequences, including the UniProt, RefSeq and Integr8 resources. The main structural annotation is generated through scanning these sequences against the CATH structural domain database profile-HMM library. CATH is a database of manually derived PDB-based structural domains, placed within a hierarchy reflecting topology, homology and conservation and is able to infer more ancient and divergent homology relationships than sequence-based approaches. This data is supplemented with Pfam-A, other non-domain structural predictions (i.e. coiled coils) and experimental data from UniProt. In order to enhance the investigations possible with this data, we have also incorporated a variety of protein annotation resources, including protein-protein interaction data, GO functional assignments, KEGG pathways, FUNCAT functional descriptions and links to microarray expression data. All of this data can be accessed through a newly re-designed website that has a focus on flexibility and clarity, with searches that can be restricted to a single genome or across the entire sequence database. Currently Gene3D contains over 3.5 million domain assignments for nearly 5 million proteins including 527 completed genomes. This is available at: http://gene3d.biochem.ucl.ac.uk/

    Nucleic acids research 2008;36;Database issue;D414-8

  • X box binding protein XBP-1s transactivates the Kaposi's sarcoma-associated herpesvirus (KSHV) ORF50 promoter, linking plasma cell differentiation to KSHV reactivation from latency.

    Wilson SJ, Tsao EH, Webb BL, Ye H, Dalton-Griffin L, Tsantoulas C, Gale CV, Du MQ, Whitehouse A and Kellam P

    Department of Infection, UCL, 46 Cleveland Street, London W1T 4JF, United Kingdom.

    Reactivation of lytic replication from viral latency is a defining property of all herpesviruses. Despite this, the authentic physiological cues for the latent-lytic switch are unclear. Such cues should ensure that viral lytic replication occurs under physiological conditions, predominantly in sites which facilitate transmission to permissive uninfected cells and new susceptible hosts. Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with the B-cell neoplasm primary effusion lymphoma (PEL), in which the virus remains latent. We have previously shown that PEL cells have the gene expression profile and immunophenotype of cycling preplasma cells (plasmablasts). Here, we show that the highly active spliced isoform of plasma cell transcription factor X box binding protein 1 (XBP-1s) is a lytic switch for KSHV. XBP-1s is normally absent in PEL, but the induction of endoplasmic reticulum stress leads to XBP-1s generation, plasma cell-like differentiation, and lytic reactivation of KSHV. XBP-1s binds to and activates the KSHV immediate-early gene ORF50 and synergizes with the ORF50 gene product RTA to induce a full lytic cycle. These data suggest that KSHV remains latent until B-cell terminal differentiation into plasma cells, the transcriptional environment of which provides the physiological "lytic switch" through XBP-1s. This links B-cell terminal differentiation to KSHV lytic reactivation.

    Funded by: Wellcome Trust

    Journal of virology 2007;81;24;13578-86

2007 Publications

  • Phylogenetic surveillance of viral genetic diversity and the evolving molecular epidemiology of human immunodeficiency virus type 1.

    Gifford RJ, de Oliveira T, Rambaut A, Pybus OG, Dunn D, Vandamme AM, Kellam P, Pillay D and UK Collaborative Group on HIV Drug Resistance

    Department of Infection, University College London, London, United Kingdom. rjmg@stanford.edu

    With ongoing generation of viral genetic diversity and increasing levels of migration, the global human immunodeficiency virus type 1 (HIV-1) epidemic is becoming increasingly heterogeneous. In this study, we investigate the epidemiological characteristics of 5,675 HIV-1 pol gene sequences sampled from distinct infections in the United Kingdom. These sequences were phylogenetically analyzed in conjunction with 976 complete-genome and 3,201 pol gene reference sequences sampled globally and representing the broad range of HIV-1 genetic diversity, allowing us to estimate the probable geographic origins of the various strains present in the United Kingdom. A statistical analysis of phylogenetic clustering in this data set identified several independent transmission chains within the United Kingdom involving recently introduced strains and indicated that strains more commonly associated with infections acquired heterosexually in East Africa are spreading among men who have sex with men. Coalescent approaches were also used and indicated that the transmission chains that we identify originated in the late 1980s to early 1990s. Similar changes in the epidemiological structuring of HIV epidemics are likely to be taking in place in other industrialized nations with large immigrant populations. The framework implemented here takes advantage of the vast amount of routinely generated HIV-1 sequence data and can provide epidemiological insights not readily obtainable through standard surveillance methods.

    Funded by: Medical Research Council: MC_U122886351

    Journal of virology 2007;81;23;13050-6

  • Dendritic cells and myeloid leukaemias: plasticity and commitment in cell differentiation.

    Rasaiyaah J, Yong K, Katz DR, Kellam P and Chain BM

    Department of Immunology and Molecular Pathology, UCL, London, UK.

    Dendritic cells (DCs) are key antigen-presenting cells (APCs), which link innate and adaptive immunity, ultimately activating antigen-specific T cells. This review examines the relationship between the acute and chronic myeloid leukaemias and cells with DC properties. DCs are non-dividing terminally differentiated cells, and ex vivo leukaemic cells or cell lines show little similarity to DCs. However, many leukaemias differentiate further in response to defined stimuli, and retain a degree of lineage plasticity. Therefore, several studies have explored the response of leukaemic cells to the in vitro regimens used to differentiate ex vivo primary DCs. Recent data suggest that the most 'dendritic-like' cells can be derived from more undifferentiated myeloid leukaemias, such as the myelomonocytic Mutz-3 cell line. These findings have important implications for understanding the developmental origins of DCs, for harnessing the APC properties of this class of tumour to stimulate the therapeutic anti-tumour immunity, and for developing useful models for the study of human DC physiology and pathology. There is a strong rationale for further exploration of this class of tumour and its relationship to the normal DC.

    British journal of haematology 2007;138;3;281-90

  • LEDGF/p75 functions downstream from preintegration complex formation to effect gene-specific HIV-1 integration.

    Shun MC, Raghavendra NK, Vandegraaff N, Daigle JE, Hughes S, Kellam P, Cherepanov P and Engelman A

    Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Division of AIDS, Harvard Medical School, Boston, Massachusetts 02115, USA.

    LEDGF/p75 directly interacts with lentiviral integrase proteins and can modulate their enzymatic activities and chromosomal association. A novel genetic knockout model was established that allowed us for the first time to analyze HIV-1 integration in the absence of LEDGF/p75 protein. Supporting a crucial role for the cofactor in viral replication, HIV-1 vector integration and reporter gene expression were significantly reduced in LEDGF-null cells. Yet, integrase processed the viral cDNA termini normally and maintained its local target DNA sequence preference during integration. Preintegration complexes extracted from knockout cells moreover supported normal levels of DNA strand transfer activity in vitro. In contrast, HIV-1 lost its strong bias toward integrating into transcription units, displaying instead increased affinity for promoter regions and CpG islands. Our results reveal LEDGF/p75 as a critical targeting factor, commandeering lentiviruses from promoter- and/or CpG island-proximal pathways that are favored by other members of Retroviridae. Akin to yeast retrotransposons, disrupting the lentiviral targeting mechanism significantly perturbs overall integration.

    Funded by: Medical Research Council: G0600009; NIAID NIH HHS: AI39394

    Genes & development 2007;21;14;1767-78

  • Specific gene expression profiles in systemic juvenile idiopathic arthritis.

    Ogilvie EM, Khan A, Hubank M, Kellam P and Woo P

    University College London, London, UK.

    Objective: Patients with systemic juvenile idiopathic arthritis (JIA) have arthritis, quotidian fevers, and other extraarticular features. This disease often remains severe and debilitating. The purpose of this study was to compare gene expression profiles in peripheral blood mononuclear cells (PBMCs) from patients with active and inactive systemic JIA to define and better understand the cause of active disease.

    Methods: Gene expression profiles of PBMCs were determined in cells from 9 patients with active systemic JIA and 8 patients with inactive systemic JIA. Unsupervised clustering and significance analysis were performed. We compared the systemic JIA profile with data from patients with polyarticular JIA, chronic infantile neurologic, cutaneous, articular syndrome, Kawasaki disease, and systemic lupus erythematosus to identify disease-specific genes. Quantitative reverse transcription-polymerase chain reaction of selected genes was performed on negatively selected B cells, T cells, and monocytes.

    Results: Unsupervised clustering of expressed genes resulted in 2 groups that corresponded to the clinical status of the patients (active and inactive disease) and was independent of their medications. A total of 286 genes were identified as significantly up-regulated in patients with active disease and 86% of them were specific to systemic JIA. Interleukin-6 (IL-6) was expressed in monocytes and B cells, IL-10 in monocytes, and suppressor of cytokine signaling 3 in monocytes and T cells from patients with active disease.

    Conclusion: Gene expression profiles in PBMCs identified disease-specific genes in patients with systemic JIA. Cell type analyses should allow further insight into the mechanisms of the disease.

    Funded by: Arthritis Research UK: 17287

    Arthritis and rheumatism 2007;56;6;1954-65

  • Current research priorities in chronic fatigue syndrome/myalgic encephalomyelitis: disease mechanisms, a diagnostic test and specific treatments.

    Kerr JR, Christian P, Hodgetts A, Langford PR, Devanur LD, Petty R, Burke B, Sinclair LI, Richards SC, Montgomery J, McDermott CR, Harrison TJ, Kellam P, Nutt DJ, Holgate ST and Collaborative Clinical Study Group

    Department of Cellular & Molecular Medicine, St George's University of London, London, UK. jkerr@sgul.ac.uk

    Chronic fatigue syndrome (CFS) is an illness characterised by disabling fatigue of at least 6 months duration, which is accompanied by various rheumatological, infectious and neuropsychiatric symptoms. A collaborative study group has been formed to deal with the current areas for development in CFS research--namely, to develop an understanding of the molecular pathogenesis of CFS, to develop a diagnostic test and to develop specific and curative treatments. Various groups have studied the gene expression in peripheral blood of patients with CFS, and from those studies that have been confirmed using polymerase chain reaction (PCR), clearly, the most predominant functional theme is that of immunity and defence. However, we do not yet know the precise gene signature and metabolic pathways involved. Currently, this is being dealt with using a microarray representing 47,000 human genes and variants, massive parallel signature sequencing and real-time PCR. It will be important to ensure that once a gene signature has been identified, it is specific to CFS and does not occur in other diseases and infections. A diagnostic test is being developed using surface-enhanced, laser-desorption and ionisation-time-of-flight mass spectrometry based on a pilot study in which putative biomarkers were identified. Finally, clinical trials are being planned; novel treatments that we believe are important to trial in patients with CFS are interferon-beta and one of the anti-tumour necrosis factor-alpha drugs.

    Funded by: Medical Research Council: G0800766

    Journal of clinical pathology 2007;60;2;113-6

2006 Publications

  • Assessment of automated genotyping protocols as tools for surveillance of HIV-1 genetic diversity.

    Gifford R, de Oliveira T, Rambaut A, Myers RE, Gale CV, Dunn D, Shafer R, Vandamme AM, Kellam P, Pillay D and UK Collaborative Group on HIV Drug Resistance

    Department of Infection, University College London, UK. r.gifford@ucl.ac.uk

    Background: The routine use of drug resistance testing provides an abundant source of HIV-1 sequence data. However, it is not clear how reliable standard genotyping of these sequences is for describing HIV-1 genetic variation and for detecting novel genetic variants and epidemiological trends.

    Objectives: To compare assignment of HIV-1 resistance test sequences to reference strains across commonly used genotyping protocols.

    Methods: Subtype assignments were compared across three standard genotyping protocols for 10 537 resistance test sequences, representing approximately one-fifth of all reported infections in the United Kingdom. Sequences that were inconsistently genotyped across methods, or that were unassigned by at least one method, were examined for evidence of recombination using sliding-window-based approaches.

    Results: Although agreement across methods was high for subtypes B, C and H, it was generally much lower (< 50%) for other subtypes. Disagreement between methods typically involved closely related, but epidemiologically distinct, groups or involved a significant proportion ( approximately 12%) of divergent sequences in which analysis revealed widespread evidence of recombination and a remarkable diversity of unusual recombinant forms.

    Conclusions: With frequent long-distance transfer of viral strains and widespread recombination between them, genetic and epidemiological relationships within HIV-1 are becoming increasingly complex. Current methods of subtype assignment vary in their ability to identify novel genetic variants and to distinguish epidemiologically distinct strains. Capturing meaningful epidemiological information from resistance test data will require a critical understanding of the methodologies used in order to appreciate the possible sources of error and misclassification.

    Funded by: Medical Research Council: MC_U122886351

    AIDS (London, England) 2006;20;11;1521-9

  • Genotyping Hepatitis B virus from whole- and sub-genomic fragments using position-specific scoring matrices in HBV STAR.

    Myers R, Clark C, Khan A, Kellam P and Tedder R

    Division of Infection and Immunity, Royal Free and University College Medical School, The Windeyer Building, 46 Cleveland Street, London W1T 4JF, UK.

    Hepatitis B virus (HBV) genomes have been classified into eight genotypes based on phylogenetic analysis of sequence variation. Identifying and tracking the movement of HBV genotypes is important in terms of both monitoring infection rates and predicting disease and treatment. An HBV genotyping tool has been developed that compares query sequences with position-specific scoring matrices representing the eight HBV genotypes. This tool (hbv star) is rapid, robust and accurate and assigns genotype based on a statistically defined scoring model. hbv star confidently assigned 90% of 590 full-length HBV genomes to an HBV genotype (Z score >2.0). Thirty-two of the residual 48 sequences were identified as non-human primate viruses and 16 sequences were identified as recombinant or putative recombinants. Receiver-Operated Characteristic (ROC) analysis was used to compare the accuracy of genotype prediction using basal core promoter sequences and surface and core genes with the accuracy achieved by using full-length sequences. A web interface to hbv star is available at http://www.vgb.ucl.ac.uk/starn.shtml.

    The Journal of general virology 2006;87;Pt 6;1459-64

  • Infectogenomics: insights from the host genome into infectious diseases.

    Kellam P and Weiss RA

    MRC/UCL Centre for Medical Molecular Virology, Division of Infection & Immunity, University College London, London W1T 4JF, UK.

    Five years into the human postgenomic era, we are gaining considerable knowledge about host-pathogen interactions through host genomes. This "infectogenomics" approach should yield further insights into both diagnostic and therapeutic advances, as well as normal cellular function.

    Cell 2006;124;4;695-7

  • A low temperature method of isolating normal human articular chondrocytes.

    Hidvegi NC, Sales KM, Izadi D, Ong J, Kellam P, Eastwood D and Butler PE

    Department of Plastic Surgery, Royal Free Hospital School of Medicine, London, UK. nhidvegi@hotmail.com

    Objective: Numerous methods for isolation of human chondrocytes are reported in the literature, most based on isolation from animal cartilage. Normal human articular cartilage (NHAC) poses particular problems for isolating chondrocytes when compared to animal or other types of human cartilage: a hardy matrix, combined with few and friable chondrocytes makes isolation difficult. Our objective was to develop an efficient method of isolating chondrocytes from NHAC without jeopardising the viability of these cells.

    Design: In this study we demonstrate that lowering the enzymatic digestion temperature to 27 degrees C increases cell yield and chondrocyte viability. We then optimised this low temperature isolation of chondrocytes from NHAC by comparing the relative efficacies of trypsin and protease and hyaluronidase in combination with different types of collagenase (I, II and XI) at releasing chondrocytes from their surrounding cartilaginous matrix. Enzymes were tested at different concentrations and for differing times. Outcome measures included determining the amount of cartilage digested, the number of viable chondrocytes isolated per gram of cartilage and cell adherence rates.

    Conclusions: From these set of experiments, the method that maximised cell yield without jeopardising cell viability proved to be a two stage process: pre-digestion step using trypsin for 15 min; followed by overnight digestion with a combination of two types of collagenase (types I and II) and at a lower temperature of 27 degrees C. This has resulted in an efficient and robust method of releasing chondrocytes from cartilage, without jeopardising the viability of these cells.

    Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society 2006;14;1;89-93

  • Attacking pathogens through their hosts.

    Kellam P

    Virus Genomics and Bioinformatics Group, Division of Infection and Immunity, University College London, London W1T 4JF, UK. p.kellam@ucl.ac.uk

    Through understanding the intricacies of host-pathogen interactions, it is now possible to inhibit the growth of microbes, especially viruses, by targeting host-cell proteins and functions. This new antimicrobial strategy has proved effective in the laboratory and in the clinic, and it has great potential for the future.

    Genome biology 2006;7;1;201

  • Robust Selection of Predictive Genes via a Simple Classifier.

    Vinciotti V, Tucker A, Kellam P and Liu X

    School of Information Systems, Computing and Mathematics, Brunel University, Uxbridge, UK. veronica.vinciotti@brunel.ac.uk

    Identifying genes that direct the mechanism of a disease from expression data is extremely useful in understanding how that mechanism works. This in turn may lead to better diagnoses and potentially could lead to a cure for that disease. This task becomes extremely challenging when the data are characterised by only a small number of samples and a high number of dimensions, as is often the case with gene expression data. Motivated by this challenge, we present a general framework that focuses on simplicity and data perturbation. These are the keys for robust identification of the most predictive features in such data. Within this framework, we propose a simple selective naive Bayes classifier discovered using a global search technique, and combine it with data perturbation to increase its robustness for small sample sizes. An extensive validation of the method was carried out using two applied datasets from the field of microarrays and a simulated dataset, all confounded by small sample sizes and high dimensionality. The method has been shown to be capable of selecting genes known to be associated with prostate cancer and viral infections.

    Applied bioinformatics 2006;5;1;1-11

2005 Publications

  • A statistical model for HIV-1 sequence classification using the subtype analyser (STAR).

    Myers RE, Gale CV, Harrison A, Takeuchi Y and Kellam P

    Department of Immunology and Molecular Pathology, University College London, UK.

    Motivation: HIV-1 antiretroviral drug resistance testing produces large amounts of HIV-1 protease and reverse transcriptase sequences. These provide an excellent resource to study the incidence, spread and clinical significance of HIV-1 subtypes. We have produced a program, Subtype Analyser (STAR) that rapidly and accurately subtypes HIV-1. Here we have determined a robust and statistically validated model for subtype assignment.

    Results: We have significantly extended our HIV-1 subtyping tool (STAR), such that each query sequence when evaluated against subtype profile alignments, returns a discriminating score based on the ratio of subtype positive to negative amino acid positions. These scores were transformed into a Z-score distribution and evaluated. Of the 141 sequences used to define the subtype alignments, 98% were correctly reclassified. Inclusion of additional recombination detection within STAR increased the detection of known recombinant sequences to 95%.

    Availability: STAR is available as compiled (Linux Fedora 3) or source code from http://pgv19.virol.ucl.ac.uk/download/star_linux.tar

    Contact: p.kellam@ucl.ac.uk

    http://pgv19.virol.ucl.ac.uk/download/star_supplement

    Bioinformatics (Oxford, England) 2005;21;17;3535-40

  • Gene expression in peripheral blood mononuclear cells from patients with chronic fatigue syndrome.

    Kaushik N, Fear D, Richards SC, McDermott CR, Nuwaysir EF, Kellam P, Harrison TJ, Wilkinson RJ, Tyrrell DA, Holgate ST and Kerr JR

    Department of Paediatric Infectious Diseases, St Marys Campus, Imperial College, Norfolk Place, London W2 1PG, UK.

    Background: Chronic fatigue syndrome (CFS) is a multisystem disease, the pathogenesis of which remains undetermined.

    Aims: To test the hypothesis that there are reproducible abnormalities of gene expression in patients with CFS compared with normal healthy persons.

    Methods: To gain further insight into the pathogenesis of this disease, gene expression was analysed in peripheral blood mononuclear cells from 25 patients with CFS diagnosed according to the Centers for Disease Control criteria and 25 normal blood donors matched for age, sex, and geographical location, using a single colour microarray representing 9522 human genes. After normalisation, average difference values for each gene were compared between test and control groups using a cutoff fold difference of expression > or = 1.5 and a p value of 0.001. Genes showing differential expression were further analysed using Taqman real time polymerase chain reaction (PCR) in fresh samples.

    Results: Analysis of microarray data revealed differential expression of 35 genes. Real time PCR confirmed differential expression in the same direction as array results for 16 of these genes, 15 of which were upregulated (ABCD4, PRKCL1, MRPL23, CD2BP2, GSN, NTE, POLR2G, PEX16, EIF2B4, EIF4G1, ANAPC11, PDCD2, KHSRP, BRMS1, and GABARAPL1) and one of which was downregulated (IL-10RA). This profile suggests T cell activation and perturbation of neuronal and mitochondrial function. Upregulation of neuropathy target esterase and eukaryotic translation initiation factor 4G1 may suggest links with organophosphate exposure and virus infection, respectively.

    Conclusion: These results suggest that patients with CFS have reproducible alterations in gene regulation.

    Funded by: Medical Research Council: MC_U117588499

    Journal of clinical pathology 2005;58;8;826-32

  • An experimental evaluation of a loop versus a reference design for two-channel microarrays.

    Vinciotti V, Khanin R, D'Alimonte D, Liu X, Cattini N, Hotchkiss G, Bucca G, de Jesus O, Rasaiyaah J, Smith CP, Kellam P and Wit E

    Department of Information Systems and Computing, Brunel University Uxbridge UB8 3PH, UK. veronica.vinciotti@brunel.ac.uk

    Motivation: Despite theoretical arguments that so-called 'loop designs' for two-channel DNA microarray experiments are more efficient, biologists continue to use 'reference designs'. We describe two sets of microarray experiments with RNA from two different biological systems (TPA-stimulated mammalian cells and Streptomyces coelicolor). In each case, both a loop and a reference design were used with the same RNA preparations with the aim of studying their relative efficiency.

    Results: The results of these experiments show that (1) the loop design attains a much higher precision than the reference design, (2) multiplicative spot effects are a large source of variability, and if they are not accounted for in the mathematical model, for example, by taking log-ratios or including spot effects, then the model will perform poorly. The first result is reinforced by a simulation study. Practical recommendations are given on how simple loop designs can be extended to more realistic experimental designs and how standard statistical methods allow the experimentalist to use and interpret the results from loop designs in practice.

    Availability: The data and R code are available at http://exgen.ma.umist.ac.uk

    Contact: veronica.vinciotti@brunel.ac.uk.

    Bioinformatics (Oxford, England) 2005;21;4;492-501

2004 Publications

  • Development of a novel human immunodeficiency virus type 1 subtyping tool, Subtype Analyzer (STAR): analysis of subtype distribution in London.

    Gale CV, Myers R, Tedder RS, Williams IG and Kellam P

    Department of Infection, University College London, London W1T 4JF, UK.

    We have developed a high throughput computational tool for assigning subtype to HIV-1, based solely on protease and reverse transcriptase (PR-RT) amino acid sequence, generated routinely for clinical assessment of genotypic drug resistance. Subtype-specific profiles were created by generation of position-specific scoring matrices (PSSMs) from multiple amino acids alignments of HIV-1 sequence data from GenBank, phylogenetically divided into subtypes A, AG, B, C, D, F/K, G, H, and J and the separate groups N and O. Query sequences of unknown subtype are aligned with these profiles and a score is derived by comparing each amino acid position in the unknown sequence to the normalized frequency distribution of amino acids at the corresponding positions in the subtype alignments. The highest score is used to assign subtype to the query sequence. Leave one out cross-validation analysis showed the Subtype Analyzer (STAR) was 99% accurate in subtype assignation. STAR can be updated with additional subtype-specific sequence data from sequence databases. STAR was used to classify HIV-1 PR-RT sequences from 843 HIV-1 clinical isolates submitted for drug resistance profiling in London. Within this dataset 26.9% of sequences were classified by STAR as non-B subtypes.

    AIDS research and human retroviruses 2004;20;5;457-64

  • Poxvirus genomes: a phylogenetic analysis.

    Gubser C, Hué S, Kellam P and Smith GL

    Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, UK.

    The evolutionary relationships of 26 sequenced members of the poxvirus family have been investigated by comparing their genome organization and gene content and by using DNA and protein sequences for phylogenetic analyses. The central region of the genome of chordopoxviruses (ChPVs) is highly conserved in gene content and arrangement, except for some gene inversions in Fowlpox virus (FPV) and species-specific gene insertions in FPV and Molluscum contagiosum virus (MCV). In the central region 90 genes are conserved in all ChPVs, but no gene from near the termini is conserved throughout the subfamily. Inclusion of two entomopoxvirus (EnPV) sequences reduces the number of conserved genes to 49. The EnPVs are divergent from ChPVs and between themselves. Relationships between ChPV genera were evaluated by comparing the genome size, number of unique genes, gene arrangement and phylogenetic analyses of protein sequences. Overall, genus Avipoxvirus is the most divergent. The next most divergent ChPV genus is Molluscipoxvirus, whose sole member, MCV, infects only man. The Suipoxvirus, Capripoxvirus, Leporipoxvirus and Yatapoxvirus genera cluster together, with Suipoxvirus and Capripoxvirus sharing a common ancestor, and are distinct from the genus Orthopoxvirus (OPV). Within the OPV genus, Monkeypox virus, Ectromelia virus and Cowpox virus strain Brighton Red (BR) do not group closely with any other OPV, Variola virus and Camelpox virus form a subgroup, and Vaccinia virus is most closely related to CPV-GRI-90. This suggests that CPV-BR and GRI-90 should be separate species.

    The Journal of general virology 2004;85;Pt 1;105-17

  • Consensus clustering and functional interpretation of gene-expression data.

    Swift S, Tucker A, Vinciotti V, Martin N, Orengo C, Liu X and Kellam P

    Department of Information Systems and Computing, Brunel University, Uxbridge UB8 3PH, UK.

    Microarray analysis using clustering algorithms can suffer from lack of inter-method consistency in assigning related gene-expression profiles to clusters. Obtaining a consensus set of clusters from a number of clustering methods should improve confidence in gene-expression analysis. Here we introduce consensus clustering, which provides such an advantage. When coupled with a statistically based gene functional analysis, our method allowed the identification of novel genes regulated by NFkappaB and the unfolded protein response in certain B-cell lymphomas.

    Genome biology 2004;5;11;R94

2003 Publications

  • Kaposi's sarcoma-associated herpesvirus-infected primary effusion lymphoma has a plasma cell gene expression profile.

    Jenner RG, Maillard K, Cattini N, Weiss RA, Boshoff C, Wooster R and Kellam P

    Wohl Virion Centre, Department of Immunology and Molecular Pathology, Windeyer Institute, University College London, London W1T 4JF, United Kingdom.

    Kaposi's sarcoma-associated herpesvirus is associated with three human tumors: Kaposi's sarcoma, and the B cell lymphomas, plasmablastic lymphoma associated with multicentric Castleman's disease, and primary effusion lymphoma (PEL). Epstein-Barr virus, the closest human relative of Kaposi's sarcoma-associated herpesvirus, mimics host B cell signaling pathways to direct B cell development toward a memory B cell phenotype. Epstein-Barr virus-associated B cell tumors are presumed to arise as a consequence of this virus-mediated B cell activation. The stage of B cell development represented by PEL, how this stage relates to tumor pathology, and how this information may be used to treat the disease are largely unknown. In this study we used gene expression profiling to order a range of B cell tumors by stage of development. PEL gene expression closely resembles that of malignant plasma cells, including the low expression of mature B cell genes. The unfolded protein response is partially activated in PEL, but is fully activated in plasma cell tumors, linking endoplasmic reticulum stress to plasma cell development through XBP-1. PEL cells can be defined by the overexpression of genes involved in inflammation, cell adhesion, and invasion, which may be responsible for their presentation in body cavities. Similar to malignant plasma cells, all PEL samples tested express the vitamin D receptor and are sensitive to the vitamin D analogue drug EB 1089 (Seocalcitol).

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;18;10399-404

  • Rabbit endogenous retrovirus-H encodes a functional protease.

    Voisset C, Myers RE, Carne A, Kellam P and Griffiths DJ

    Wohl Virion Centre, Windeyer Institute of Medical Sciences, University College London, 46 Cleveland Street, London W1T 4JF, UK.

    Recent studies have revealed that 'human retrovirus-5' sequences found in human samples belong to a rabbit endogenous retrovirus family named RERV-H. A part of the gag-pro region of the RERV-H genome was amplified by PCR from DNA in human samples and several forms of RERV-H protease were expressed in bacteria. The RERV-H protease was able to cleave itself from a precursor protein and was also able to cleave the RERV-H Gag polyprotein precursor in vitro whereas a form of the protease with a mutation engineered into the active site was inactive. Potential N- and C-terminal autocleavage sites were characterized. The RERV-H protease was sensitive to pepstatin A, showing it to be an aspartic protease. Moreover, it was strongly inhibited by PYVPheStaAMT, a pseudopeptide inhibitor specific for Mason-Pfizer monkey virus and avian myeloblastosis-associated virus. A structural model of the RERV-H protease was constructed that, together with the activity data, confirms that this is a retroviral aspartic protease.

    The Journal of general virology 2003;84;Pt 1;215-25

  • Viral bioinformatics: computational views of host and pathogen.

    Kellam P, Holzerlandt R, Gramoustianou E, Jenner R and Kwan A

    Virus Genomics and Bioinformatics Group, Department of Immunology and Molecular Pathology, University College, Windeyer Institute of Medical Sciences, 46 Cleveland Street, London W1T4JF, UK.

    Wherever cellular life occurs, viruses are also found. As a result, complex organism and cellular antiviral responses co-evolve with virally encoded countermeasures. Since viruses co-opt or interfere with specific cellular pathways during their replication, knowledge of viral genome sequences has helped fundamental understanding of host biology. During viral infection, shifts in the balance between host and viral biological processes result in acute or chronic viral disease pathology accompanied with either active viral replication, viral containment/persistence or viral clearance. Studying host-virus interactions at the level of single gene effects, however, fails to produce a global systems-level understanding. This should now be achievable in the context of complete host and pathogen genome sequences. New experimental methods and computational approaches are rapidly developing, allowing global views of dynamic viral and cellular molecular mechanisms. Systems level virology using DNA microarrays and specific viral data resources will reveal the detailed cellular context in which viruses replicate, highlighting common and distinct antiviral mechanisms, the effect of different host cell gene expression programs, and the response of cells to similar or diverse virus types. Ultimately, microbiology and immunology will tend towards a systems-level view of how host and pathogen interact.

    Novartis Foundation symposium 2003;254;234-47; discussion 247-52

2002 Publications

  • PFDB: a generic protein family database integrating the CATH domain structure database with sequence based protein family resources.

    Shepherd AJ, Martin NJ, Johnson RG, Kellam P and Orengo CA

    Department of Biochemistry and Molecular Biology, University College, London, Gower Street, London WC1E 6BT.

    Motivation: The PFDB (Protein Family Database) is a new database designed to integrate protein family-related data with relevant functional and genomic data. It currently manages biological data for three projects-the CATH protein domain database (Orengo et al., 1997; Pearl et al., 2001), the VIDA virus domains database (Albà et al., 2001) and the Gene3D database (Buchan et al., 2001). The PFDB has been designed to accommodate protein families identified by a variety of sequence based or structure based protocols and provides a generic resource for biological research by enabling mapping between different protein families and diverse biochemical and genetic data, including complete genomes.

    Results: A characteristic feature of the PFDB is that it has a number of meta-level entities (for example aggregation, collection and inclusion) represented as base tables in the final design. The explicit representation of relationships at the meta-level has a number of advantages, including flexibility-both in terms of the range of queries that can be formulated and the ability to integrate new biological entities within the existing design. A potential drawback with this approach-poor performance caused by the number of joins across meta-level tables-is avoided by implementing the PFDB with materialized views using the mature relational database technology of Oracle 8i. The resultant database is both fast and flexible. This paper presents the principles on which the database has been designed and implemented, and describes the current status of the database and query facilities supported.

    Bioinformatics (Oxford, England) 2002;18;12;1666-72

  • Identification of new herpesvirus gene homologs in the human genome.

    Holzerlandt R, Orengo C, Kellam P and Albà MM

    Wohl Virion Centre, Department of Immunology and Molecular Pathology, University College London, London W1T 4JF, United Kingdom.

    Viruses are intracellular parasites that use many cellular pathways during their replication. Large DNA viruses, such as herpesviruses, have captured a repertoire of cellular genes to block or mimic host immune responses, apoptosis regulation, and cell-cycle control mechanisms. We have conducted a systematic search for all homologs of herpesvirus proteins in the human genome using position-specific scoring matrices representing herpesvirus protein sequence domains, and pair-wise sequence comparisons. The analysis shows that approximately 13% of the herpesvirus proteins have clear sequence similarity to products of the human genome. Different human herpesviruses vary in their numbers of human homologs, indicating distinct rates of gene acquisition in different lineages. Our analysis has identified new families of herpesvirus/human homologs from viruses including human herpesvirus 5 (human cytomegalovirus; HCMV) and human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus; KSHV), which may play important roles in host-virus interactions.

    Genome research 2002;12;11;1739-48

  • Gammaherpesvirus lytic gene expression as characterized by DNA array.

    Ahn JW, Powell KL, Kellam P and Alber DG

    Wolfson Institute for Biomedical Research, University College London, London WC1E 6BT, United Kingdom.

    Gammaherpesviruses are associated with a number of diseases including lymphomas and other malignancies. Murine gammaherpesvirus 68 (MHV-68) constitutes the most amenable animal model for this family of pathogens. However experimental characterization of gammaherpesvirus gene expression, at either the protein or RNA level, lags behind that of other, better-studied alpha- and beta-herpesviruses. We have developed a cDNA array to globally characterize MHV-68 gene expression profiles, thus providing an experimental supplement to a genome that is chiefly annotated by homology. Viral genes started to be transcribed as early as 3 h postinfection (p.i.), and this was followed by a rapid escalation of gene expression that could be seen at 5 h p.i. Individual genes showed their own transcription profiles, and most genes were still being expressed at 18 h p.i. Open reading frames (ORFs) M3 (chemokine-binding protein), 52, and M9 (capsid protein) were particularly noticeable due to their very high levels of expression. Hierarchical cluster analysis of transcription profiles revealed four main groups of genes and allowed functional predictions to be made by comparing expression profiles of uncharacterized genes to those of genes of known function. Each gene was also categorized according to kinetic class by blocking de novo protein synthesis and viral DNA replication in vitro. One gene, ORF 73, was found to be expressed with alpha-kinetics, 30 genes were found to be expressed with beta-kinetics, and 42 genes were found to be expressed with gamma-kinetics. This fundamental characterization furthers the development of this model and provides an experimental basis for continued investigation of gammaherpesvirus pathology.

    Journal of virology 2002;76;12;6244-56

  • Virus bioinformatics: databases and recent applications.

    Kellam P and Albà MM

    Wohl Virion Centre, Department of Immunology and Molecular Pathology, Windeyer Institute of Medical Sciences, University College London, London, UK. p.kellam@ucl.ac.uk

    Bioinformatics is now used as an umbrella term for almost all aspects of computational biology. Bioinformatics research will have an impact on all of biology, and virology is not immune from these research methods. Although virology has been slower to embrace bioinformatics this is now changing, particularly in the areas of viral sequences databasing and the systematic identification of viral and host homologous proteins. Here we will review some of these recent advances focusing mainly on the herpesvirus.

    Applied bioinformatics 2002;1;1;37-42

2001 Publications

  • Post-genomic virology: the impact of bioinformatics, microarrays and proteomics on investigating host and pathogen interactions.

    Kellam P

    Wohl Virion Centre, Department of Immunology and Molecular Pathology, Windeyer Institute of Medical Sciences, University College London, 46 Cleveland Street, London W1T 4JF, UK. p.kellam@ucl.ac.uk

    Post-genomic research encompasses many diverse aspects of modern science. These include the two broad subject areas of computational biology (bioinformatics) and functional genomics. Laboratory based functional genomics aims to measure and assess either the messenger RNA (mRNA) levels (transcriptome studies) or the protein content (proteome studies) of cells and tissues. All of these methods have been applied recently to the study of host and pathogen interactions for both bacteria and viruses. A basic overview of the technology is given in this review together with approaches to data analysis. The wealth of information produced from even these preliminary studies has shown the generalities, subtleties and specificities of host-pathogen interactions. Such research should ultimately result in new methods for diagnosing and treating infectious diseases.

    Reviews in medical virology 2001;11;5;313-29

  • VIDA: a virus database system for the organization of animal virus genome open reading frames.

    Albà MM, Lee D, Pearl FM, Shepherd AJ, Martin N, Orengo CA and Kellam P

    Wohl Virion Centre, Department of Immunology and Molecular Pathology, Windeyer Institute of Medical Sciences, University College London, London, UK.

    VIDA is a new virus database that organizes open reading frames (ORFs) from partial and complete genomic sequences from animal viruses. Currently VIDA includes all sequences from GenBank for Herpesviridae, Coronaviridae and Arteriviridae. The ORFs are organized into homologous protein families, which are identified on the basis of sequence similarity relationships. Conserved sequence regions of potential functional importance are identified and can be retrieved as sequence alignments. We use a controlled taxonomical and functional classification for all the proteins and protein families in the database. When available, protein structures that are related to the families have also been included. The database is available for online search and sequence information retrieval at http://www.biochem.ucl.ac.uk/bsm/virus_database/ VIDA.html.

    Nucleic acids research 2001;29;1;133-6

  • Genomewide function conservation and phylogeny in the Herpesviridae.

    Albà MM, Das R, Orengo CA and Kellam P

    Wohl Virion Centre, Department of Immunology and Molecular Pathology, University College London, London W1T 4JF, UK.

    The Herpesviridae are a large group of well-characterized double-stranded DNA viruses for which many complete genome sequences have been determined. We have extracted protein sequences from all predicted open reading frames of 19 herpesvirus genomes. Sequence comparison and protein sequence clustering methods have been used to construct herpesvirus protein homologous families. This resulted in 1692 proteins being clustered into 243 multiprotein families and 196 singleton proteins. Predicted functions were assigned to each homologous family based on genome annotation and published data and each family classified into seven broad functional groups. Phylogenetic profiles were constructed for each herpesvirus from the homologous protein families and used to determine conserved functions and genomewide phylogenetic trees. These trees agreed with molecular-sequence-derived trees and allowed greater insight into the phylogeny of ungulate and murine gammaherpesviruses.

    Genome research 2001;11;1;43-54

  • Kaposi's sarcoma-associated herpesvirus latent and lytic gene expression as revealed by DNA arrays.

    Jenner RG, Albà MM, Boshoff C and Kellam P

    Wohl Virion Centre, Department of Immunology and Molecular Pathology, Windeyer Institute, University College London, London W1T 4JF, United Kingdom.

    Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is associated with three human tumors, Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. KSHV encodes a number of homologs of cellular proteins involved in the cell cycle, signal transduction, and modulation of the host immune response. Of the virus complement of over 85 open reading frames (ORFs), the expression of only a minority has been characterized individually. We have constructed a nylon membrane-based DNA array which allows the expression of almost every ORF of KSHV to be measured simultaneously. A PEL-derived cell line, BC-3, was used to study the expression of KSHV during latency and after the induction of lytic replication. Cluster analysis, which arranges genes according to their expression profile, revealed a correlation between expression and assigned gene function that is consistent with the known stages of the herpesvirus life cycle. Furthermore, latent and lytic genes thought to be functionally related cluster into groups. The correlation between gene expression and function also infers possible roles for KSHV genes yet to be characterized.

    Journal of virology 2001;75;2;891-902

  • Microarray gene expression database: progress towards an international repository of gene expression data.

    Kellam P

    Wohl Virion Centre, Department of Immunology and Molecular Pathology, Windeyer Institute, University College London, London W1T 4JF, UK. p.kellam@ucl.ac.uk

    Genome biology 2001;2;5;REPORTS4011

2000 Publications

  • The latent nuclear antigen of Kaposi sarcoma-associated herpesvirus targets the retinoblastoma-E2F pathway and with the oncogene Hras transforms primary rat cells.

    Radkov SA, Kellam P and Boshoff C

    The CRC Viral Oncology Group, The Wolfson Institute for Biomedical Research, Cruciform Building and The Wohl Virion Centre, Departments of Oncology and Molecular Pathology, University College London, London WC1E 6AE, UK.

    Kaposi sarcoma-associated herpesvirus (KSHV) is involved in the etiopathogenesis of Kaposi sar-coma and certain lymphoproliferative disorders. Open reading frame (ORF) 73 encodes the main immunogenic latent nuclear antigen (LNA-1) of KSHV. LNA-1 maintains the KSHV episome and tethers the viral genome to chromatin during mitosis. In addition, LNA-1 interacts with p53 and represses its transcriptional activity. Here we show that LNA-1 also interacts with the retinoblastoma protein. LNA-1 transactivated an artificial promoter carrying the cell cycle transcription factor E2F DNA-binding sequences and also upregulated the cyclin E (CCNEI) promoter, but not the B-myb (MYBL2) promoter. LNA-1 overcame the flat-cell phenotype induced by retinoblastoma protein in Saos2 cells. In cooperation with the cellular oncogene Harvey rat sarcoma viral oncogene homolog (Hras), LNA-1 transformed primary rat embryo fibroblasts and rendered them tumorigenic. These findings indicate that LNA-1 acts as a transcription co-factor and may contribute to KSHV-induced oncogenesis by targeting the retinoblastoma protein-E2F transcriptional regulatory pathway.

    Nature medicine 2000;6;10;1121-7

  • HHV-8 is associated with a plasmablastic variant of Castleman disease that is linked to HHV-8-positive plasmablastic lymphoma.

    Dupin N, Diss TL, Kellam P, Tulliez M, Du MQ, Sicard D, Weiss RA, Isaacson PG and Boshoff C

    Departments of Oncology, Molecular Pathology and Histopathology, University College London, UK.

    Castleman disease (CD) is a lymphoproliferative disorder of unknown etiology that is associated with the development of secondary tumors, including B-cell lymphoma. Human herpesvirus 8 (HHV-8) (Kaposi's sarcoma-associated herpesvirus) sequences have been described in some cases of multicentric Castleman disease (MCD). Using a monoclonal antibody against an HHV-8-latent nuclear antigen, we show that HHV-8 is specifically associated with a variant of MCD in which HHV-8-positive plasmablasts that show lambda light-chain restriction localize in the mantle zone of B-cell follicles and coalesce to form microscopic lymphomas in some cases. Furthermore, we show that the frank plasmablastic lymphoma that develops in patients with this plasmablastic variant of MCD is also positive for HHV-8 and lambda light chain. Plasmablastic lymphoma associated with MCD is a new disease entity associated with HHV-8 infection. (Blood. 2000;95:1406-1412)

    Blood 2000;95;4;1406-12

  • Host-pathogen studies in the post-genomic era.

    Kellam P

    Wohl Virion Centre, Department of Molecular Pathology, Windeyer Institute, University College London, London, W1P 6DB, UK. p.kellam@ucl.ac.uk

    Several studies are starting to show the power of DNA microarrays to identify interactions between animal hosts and their pathogens, and have revealed interesting correlations between host responses to different infectious agents.

    Genome biology 2000;1;2;REVIEWS1009

1999 Publications

  • Characterization of monoclonal antibodies raised against the latent nuclear antigen of human herpesvirus 8.

    Kellam P, Bourboulia D, Dupin N, Shotton C, Fisher C, Talbot S, Boshoff C and Weiss RA

    Institute of Cancer Research, Chester Beatty Laboratories, London SW3 6JB, United Kingdom. p.Kellam@ucl.ac.uk

    Human herpesvirus 8 (HHV-8; also designated Kaposi's sarcoma-associated herpesvirus) is the likely etiological agent of Kaposi's sarcoma (KS). HHV-8 encodes a latent nuclear antigen (LNA) which is the product of the viral gene orf 73. LNA is recognized by most infected patient sera and is the basis of current immunofluorescence assays used in epidemiological studies of HHV-8 infection. Here we describe the characterization of four monoclonal antibodies raised to the C-terminal third of LNA-glutathione S-transferase fusion proteins. These monoclonal antibodies recognized discrete linear epitopes within the C terminus and repetitive region of LNA, detected antigen in primary effusion lymphoma (PEL) cells, and precipitated a 220- to 230-kDa protein doublet corresponding to LNA from HHV-8-infected PEL cell lines. In situ immunocytochemistry of KS lesions with these antibodies show that LNA is extensively expressed in KS spindle cells.

    Journal of virology 1999;73;6;5149-55

  • Transcriptional analysis of human herpesvirus-8 open reading frames 71, 72, 73, K14, and 74 in a primary effusion lymphoma cell line.

    Talbot SJ, Weiss RA, Kellam P and Boshoff C

    Department of Medical Microbiology, Medical School, The University of Edinburgh, Teviot Place, Edinburgh, EH8 9AG, United Kingdom. s.talbot@ed.ac.uk

    We examined the transcription and splicing of open reading frames (ORFs) 71 (K13)-74 of human herpesvirus-8 (HHV-8) in the primary effusion lymphoma cell line BCP-1 (latently infected with HHV-8), using a combination of NORTHERN blot analysis, RT-PCR, and rapid amplification of cDNA ends (PCR-RACE). The three genes encoded by ORFs 71, 72, and 73 [viral FLICE inhibitory protein (v-FLIP), v-cyclin, latent nuclear antigen (LNA)] are transcribed from a common transcription start site in BCP-1 cells uninduced (latent) or induced (lytic) with n-butyrate. The resulting transcript is spliced to yield a 5.32-kb message encoding LNA, v-cyclin, and v-FLIP and a 1.7-kb bicistronic message encoding v-cyclin and v-FLIP. The two genes encoded by ORFs K14 and 74 (v-Ox2 and v-GPCR) are transcribed as a 2.7-kb bicistronic transcript that is induced with n-butyrate. A small (149-bp) intron is spliced from the intragenic noncoding region immediately before the v-GPCR initiating codon. Examination of sequence elements in the promoter of the LNA/v-cyclin/v-FLIP operon revealed TAATGARAT and Octamer binding motifs characteristic of herpesvirus immediate-early genes. Sequence elements in the v-Ox2/v-GPCR promoter included AP1 and Zta-like (EBV Zebra transactivator) binding motifs consistent with the n-butyrate induction of this operon.

    Virology 1999;257;1;84-94

  • Distribution of human herpesvirus-8 latently infected cells in Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma.

    Dupin N, Fisher C, Kellam P, Ariad S, Tulliez M, Franck N, van Marck E, Salmon D, Gorin I, Escande JP, Weiss RA, Alitalo K and Boshoff C

    Departments of Oncology and Molecular Pathology, Royal Free and University College Medical School, UCL, London, United Kingdom W1P 6BT.

    Human herpesvirus 8 (HHV-8, also called KSHV) is linked to the etiopathogenesis of Kaposi's sarcoma (KS), multicentric Castleman's disease (MCD), and primary effusion lymphoma (PEL). The universal presence of HHV-8 in early KS has not yet been shown. We used a mAb (LN53) against latent nuclear antigen-1 (LNA-1) of HHV-8 encoded by ORF73 to study the distribution of the cell types latently infected by HHV-8 in patch, plaque, and nodular KS, MCD, and PEL. In early KS, HHV-8 is present in <10% of cells forming the walls of ectatic vessels. In nodular KS, HHV-8 is present in cells surrounding slit-like vessels and in >90% of spindle cells, but not in normal vascular endothelium. In addition, HHV-8 colocalizes with vascular endothelial growth factor receptor-3 (VEGFR-3), a marker of lymphatic and precursor endothelium. In early KS lesions, VEGFR-3 is more extensively expressed than LNA-1, indicating that HHV-8 is not inducing the proliferation of VEGFR-3-positive endothelium directly. In MCD, HHV-8 is present in mantle zone large immunoblastic B cells. No staining for LNA-1 is seen in samples from multiple myeloma, prostate cancer, and angiosarcoma, supporting the absence of any etiological link between these diseases and HHV-8.

    Proceedings of the National Academy of Sciences of the United States of America 1999;96;8;4546-51

1998 Publications

  • Single amino acid substitutions disrupt tetramer formation in the dihydroneopterin aldolase enzyme of Pneumocystis carinii.

    Thomas MC, Ballantine SP, Bethell SS, Bains S, Kellam P and Delves CJ

    Glaxo Wellcome Research and Development, Medicines Research Centre, Stevenage, Hertfordshire, UK.

    In the opportunistic pathogen Pneumocystis carinii, dihydroneopterin aldolase function is expressed as the N-terminal portion of the multifunctional folic acid synthesis protein (Fas). This region encompasses two domains, FasA and FasB, which are 27% amino acid identical. FasA and FasB also share significant amino acid sequence similarity with bacterial dihydroneopterin aldolases. In the present study, this enzyme function has been overproduced as an independent monofunctional activity in Escherichia coli. Recombinant FasAB-Met23 (amino acids 23-290 of the predicted open reading frame) was purified and shown to contain dihydroneopterin aldolase activity. The native FasAB-Met23 is a tetramer of the 30-kDa subunit, demonstrating characteristics of an associating-dissociating equilibrium system in which only the multimeric form of the enzyme is active. Multiple sequence alignment of FasA and FasB with other dihydroneopterin aldolases highlights only three positions where the amino acid is invariable between all the predicted proteins. The role of these conserved amino acid residues in enzyme function was investigated using site-directed mutagenesis. Mutant FasAB-Met23 species were overproduced and purified to near homogeneity. Three FasA domain mutants and two FasB domain mutants had little or no detectable dihydroneopterin aldolase activity, implicating both FasA and FasB in the catalytic mechanism. We show that each mutant protein containing an inactivating amino acid substitution has lost its ability to form stable tetramers.

    Biochemistry 1998;37;33;11629-36

  • Molecular identification of novel viruses.

    Kellam P

    Dept of Virology, Chester Beatty Laboratories, London, UK paulk@icr.ac.uk

    Viruses are responsible for many of the diseases caused by microbial infection. During the past two decades, approximately 20 new human viruses have been discovered. Many of these new viruses were initially identified using molecular biology techniques, a major advantage of which is the ability to search rapidly for new viruses, known viruses or related, but previously unidentified, members of established virus families in disease samples.

    Trends in microbiology 1998;6;4;160-5

  • Human herpesvirus type 8 and Kaposi's sarcoma.

    Weiss RA, Whitby D, Talbot S, Kellam P and Boshoff C

    Institute of Cancer Research, Chester Beatty Laboratories, London, U.K.

    Kaposi's sarcoma-associated herpesvirus or human herpesvirus type 8 (HHV-8) is present in all forms of Kaposi's sarcoma (KS) as well as in primary effusion lymphomas and some cases of Castleman's disease. In KS tissues, HHV-8 is present in endothelial and spindle cells. Current serologic tests suggest that HHV-8 is predominantly found in those at risk of KS and is not as widespread as most other human herpesviruses. HHV-8 encodes various proteins that may play a role in promotion of cellular growth, including cyclin- and G-coupled protein receptor homologues, and anti-apoptotic proteins, including Bcl-2, IL-6 (i.e., interleukin 6), and FLIP (i.e., FLICE inhibitory protein) homologues. In addition, HHV-8 encodes two macrophage inflammatory-like proteins with anti-human immunodeficiency virus and angiogenic potential.

    Journal of the National Cancer Institute. Monographs 1998;23;51-4

1997 Publications

  • Illicit viral DNA.

    Weiss RA and Kellam P

    Nature 1997;390;6657;235-6

  • Identification of a major latent nuclear antigen, LNA-1, in the human herpesvirus 8 genome.

    Kellam P, Boshoff C, Whitby D, Matthews S, Weiss RA and Talbot SJ

    Institute of Cancer Research, Chester Beatty Laboratories, London, United Kingdom.

    Objectives: Human herpesvirus 8 (HHV-8) is strongly associated with all forms of Kaposi's sarcoma (KS) and with primary effusion lymphomas (PEL). KS patients' sera are immunoreactive against discrete nuclear localizing antigens in PEL cell lines. This study sought to identify and characterize these nuclear localizing proteins.

    KS patients' sera were used to screen a cDNA expression library derived from a PEL cell line (BCP-1) latently infected with HHV-8.

    Results: An HHV-8-specific cDNA clone was isolated. It encoded one partial and two complete open reading frames (ORFs): ORF 73, ORF 72 (v-cyclin), and K13, respectively. The immunodominant epitope was mapped to the C-terminal domain of ORF 73. Analysis with the KS patients' sera of HEK 293 cells transfected with a clone encompassing the complete coding region of ORF 73, ORF 72, and K13 gave a nuclear immunofluorescence pattern similar to that observed in BCP-1 cells. Western blot analysis with KS patients' sera of transfected HEK 293 cells revealed an immunoreactive protein of 220 to 230 kD that was similar to that observed previously in PEL cell lines. After induction of lytic replication of HHV-8 in BCP-1 cells with n-butyrate, we observed a major reduction in the expression of an ORF 73-specific 6.6-kb mRNA, indicating that this region is under latent control.

    Conclusions: These data identify a region of HHV-8 encoding for a major immunoreactive latent nuclear antigen (LNA-1), analogous to the Epstein-Barr virus latent nuclear antigens.

    Journal of human virology 1997;1;1;19-29

1995 Publications

  • Functional cloning of the dihydropteroate synthase gene of Staphylococcus haemolyticus.

    Kellam P, Dallas WS, Ballantine SP and Delves CJ

    Structural Biology Group, GlaxoWellcome Research Laboratories, Beckenham, Kent, UK.

    A 1,7-kilobase fragment of the Staphylococcus haemolyticus chromosome containing the dihydropteroate synthase gene has been cloned by complementation in a temperature-sensitive mutant of Escherichia coli. The gene, designated folP, predicts a gene product of 29613 Da which shares significant amino acid sequence identity with other known bacterial dihydropteroate synthases. Analysis of the DNA sequence upstream and downstream of folP identified two further, incomplete open reading frames, one of which shows predicted amino acid sequence similarity to a second bacterial folic acid synthesis enzyme, dihydroneopterin aldolase.

    FEMS microbiology letters 1995;134;2-3;165-9

  • Retroviral recombination can lead to linkage of reverse transcriptase mutations that confer increased zidovudine resistance.

    Kellam P and Larder BA

    Antiviral Therapeutic Research Unit, Wellcome Research Laboratories, Beckenham, Kent, United Kingdom.

    Genetic recombination between viral genomes has been shown to contribute to the generation of genetic diversity during retrovirus infections. The role of recombination in the development of human immunodeficiency virus type 1 (HIV-1) zidovudine resistance was investigated as a possible cause of the formation of the linked Leu-41/Tyr-215 resistance genotype. Zidovudine resistance is conferred by the presence of subsets of four or five amino acid substitutions in the HIV-1 reverse transcriptase. Zidovudine therapy of asymptomatic HIV-1-infected individuals results in the selection of drug-resistant variants that posses defined combinations of the five zidovudine resistance mutations. The linked Leu-41/Tyr-215 resistance genotype appears central to the continued development of high-level zidovudine resistance. By using genetically tagged mutant viruses, it was possible readily to select recombinant viruses from mixed infections of Leu-41 and Tyr-215 single mutants in the presence of zidovudine drup pressure. After three passages of a mixed infection in the presence of drug, 38% of clones screened were recombinant double mutants. In the absence of zidovudine selection, little change in the mixed virus populations was noted. No evidence of de novo generation of mutations at codons 41 and 215 was seen during any in vitro passage. This provides the first example of the role of retroviral recombination in the development of HIV-1 variants with increased drug resistance.

    Journal of virology 1995;69;2;669-74

1994 Publications

  • Zidovudine treatment results in the selection of human immunodeficiency virus type 1 variants whose genotypes confer increasing levels of drug resistance.

    Kellam P, Boucher CA, Tijnagel JM and Larder BA

    Antiviral Therapeutic Research Unit, Wellcome Research Laboratories, Beckenham, Kent, U.K.

    High level resistance to 3'-azido-3'-deoxythymidine (AZT, zidovudine or Retrovir) is conferred by the presence of four or five mutations (Met-41-->Leu; Asp-67-->Asn; Lys-70-->Arg; Thr-215-->Tyr or Phe; Lys-219-->Gln) in the human immunodeficiency virus (HIV) reverse transcriptase. The order of appearance of these five mutations in asymptomatic patients during therapy has been studied. This has enabled us to propose a model for the acquisition of zidovudine resistance mutations during the treatment of high-risk asymptomatic HIV-infected individuals. A consistent acquisition pattern of mutations at codons 41, 70 and 215 was observed in 17 individuals. Complex mixtures of HIV species containing different combinations of single and linked double resistance mutations were present early in zidovudine therapy in isolates from two patients studied in detail. From these mixtures the linked Leu-41/Tyr-215 genotype outgrew all others initially. The development of each new virus population is likely to be mediated primarily by the increase in the level of drug resistance rather than changes in the growth kinetics of the virus. This leads us to conclude that one major driving force in the outgrowth of different mutant viruses is the selective advantage conferred by higher levels of drug resistance.

    The Journal of general virology 1994;75 ( Pt 2);341-51

  • Recombinant virus assay: a rapid, phenotypic assay for assessment of drug susceptibility of human immunodeficiency virus type 1 isolates.

    Kellam P and Larder BA

    Antiviral Therapeutic Research Unit, Wellcome Research Laboratories, Beckenham, Kent, United Kingdom.

    Antiviral drug susceptibility assays for clinical human immunodeficiency virus type 1 (HIV-1) isolates are required to monitor the development of drug resistance during clinical trials and antiretroviral drug therapy. First-generation phenotypic assays possess a number of drawbacks, not least the selection of unrepresentative virus populations during cocultivation. Here we describe a rapid phenotypic assay for the assessment of the susceptibility of clinical isolates to reverse transcriptase (RT) inhibitors. This procedure, called the recombinant virus assay, allows the generation of viable virus by homologous recombination of a PCR-derived pool of RT coding sequences into an RT-deleted, noninfectious proviral clone, pHIV delta BstEII. A nested PCR procedure has been optimized to allow the amplification of an RT pool from both uncultured and cocultured infected patient peripheral blood lymphocyte (PBL) DNA for subsequent use in the creation of recombinant viruses. Analysis of two patients during the course of zidovudine therapy showed that this approach produced viruses which accurately exhibited the same genotype and phenotype as that of the original infected PBL DNA. The recombinant virus assay can be performed in approximately 3 weeks without the use of donor PBLs and therefore represents a rapid, nonselective procedure for the assay of clinical isolates.

    Antimicrobial agents and chemotherapy 1994;38;1;23-30

1993 Publications

  • Quantitative detection of HIV-1 drug resistance mutations by automated DNA sequencing.

    Larder BA, Kohli A, Kellam P, Kemp SD, Kronick M and Henfrey RD

    Antiviral Therapeutic Research Unit, Wellcome Foundation Limited, Beckenham, Kent, UK.

    A comparison has been made between manual and automated DNA sequencing procedures to evaluate the ability to distinguish mixtures of wild-type and mutant sequences. Quantitative detection of such mixtures of HIV-1 drug resistance mutations was best achieved using an automated system that uses fluorescent-labelled sequencing primers. This procedure has a wide range of applications in clinical research, including heterozygote analysis. Software that automatically reports mixed-base positions is presented.

    Nature 1993;365;6447;671-3

  • Convergent combination therapy can select viable multidrug-resistant HIV-1 in vitro.

    Larder BA, Kellam P and Kemp SD

    Antiviral Therapeutic Research Unit, Wellcome Research Laboratories, Beckenham, Kent, UK.

    The reverse transcriptase enzyme of human immunodeficiency virus type 1 (HIV-1) is the target for many inhibitors. Amino-acid substitutions in functional regions of the enzyme that abolish reverse transcriptase activity also prevent HIV-1 replication. But selection pressure by drugs such as AZT (3'-azido-3'deoxythymidine, zidovudine), ddI (2',3'-dideoxyinosine) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) causes outgrowth of resistant variants due to non-lethal mutations in the enzyme. Reports of synergy and lack of cross-resistance between reverse transcriptase inhibitors (refs 7, 9, 10, 12-14, 17, 18, 20, 21), plus the reversal of AZT resistance by mutations induced by ddI and NNRTIs, have indicated that specific drug combinations directed at reverse transcriptase might curtail resistance. Chow et al. extended this concept in a report that specific multiple combinations of resistance mutations in the reverse transcriptase can significantly impair HIV-1 replication. They concluded that evolutionary limitations may exist to prevent the emergence of multidrug resistance to inhibitors of reverse transcriptase. We report here that HIV-1 co-resistant to AZT, ddI and the NNRTI nevirapine can be readily selected in cell culture starting with dual AZT- and ddI-resistant virus. We found no evidence for 'replication incompatible' combinations of resistance mutations, although a mutation (M184-->V) conferring oxathiolane-cytosine nucleoside resistance in reverse transcriptase completely suppressed AZT resistance in a triple-resistant background. These in vitro observations suggest that triple drug combination therapy might ultimately result in co-resistant HIV-1, although they do not preclude assessment of such combinations for treatment of HIV-1 disease.

    Nature 1993;365;6445;451-3

  • Effects of discontinuation of zidovudine treatment on zidovudine sensitivity of human immunodeficiency virus type 1 isolates.

    Boucher CA, van Leeuwen R, Kellam P, Schipper P, Tijnagel J, Lange JM and Larder BA

    Department of Virology, University of Amsterdam, Amsterdam, The Netherlands.

    Zidovudine treatment of individuals infected with human immunodeficiency virus type 1 (HIV-1) results in HIV-1 isolates with a reduced zidovudine sensitivity in vitro. This reduction is due to mutations causing amino acid substitutions at five codons (41, 67, 70, 215, and 219) on the reverse transcriptase enzyme of HIV. HIV-1 isolates were obtained 8 to 69 weeks after therapy discontinuation from 10 patients at different stages of disease. Zidovudine sensitivity was determined by the HeLa CD4+ plaque assay. The presence of the resistance-conferring mutations was determined by using a selective polymerase chain reaction. Sensitivity could be determined for six isolate pairs: one showed a decline in the 50% inhibitory zidovudine concentration after therapy discontinuation; four pairs did not show a change. The majority of changes in the five codons in isolates from all 10 patients were the result of a relative increase in the wild-type sequence. Complete changes from mutant to the wild type were seen for only two codons in isolates from two patients. This study of isolates from a small group of individuals at different stages of disease, who had been taking zidovudine for 1 to 2 years, shows that a period of 1 year without zidovudine may be required to achieve a change from a mutant or mixed virus population to a wild-type virus population.

    Antimicrobial agents and chemotherapy 1993;37;7;1525-30

1992 Publications

  • HIV-1 biological phenotype and the development of zidovudine resistance in relation to disease progression in asymptomatic individuals during treatment.

    Boucher CA, Lange JM, Miedema FF, Weverling GJ, Koot M, Mulder JW, Goudsmit J, Kellam P, Larder BA and Tersmette M

    Department of Virology, University of Amsterdam, The Netherlands.

    Objective: To determine which parameters are associated with clinical progression during zidovudine treatment of asymptomatic HIV-1-infected individuals.

    Methods: Twenty-four initially asymptomatic HIV-1-infected individuals were treated with zidovudine and followed until the development of AIDS or for approximately 3 years. HIV-1 phenotype was determined by cocultivation of patient cells with donor lymphocytes, and by a new assay of direct cocultivation with MT-2 cells. Specific mutations in the HIV-1 reverse transcriptase (RT) gene conferring resistance to zidovudine were detected using a selective polymerase chain reaction.

    Results: Progression to AIDS was more rapid in individuals harbouring syncytium-inducing (SI) viral isolates or showing a conversion from non-syncytium-inducing (NSI) to SI viral isolates. One out of 20 patients who spent a total of 559 months harbouring an NSI phenotype progressed to AIDS, whereas eight out of 12 patients who spent a total of 223 months harbouring an SI phenotype progressed to AIDS (P < 0.001). There was no significant difference between SI and non-SI isolates in the frequency of five mutations causing zidovudine resistance. However, all SI isolates obtained after 2 years of treatment contained mutations in codons 41 and 215 of the RT gene, whereas only five out of 11 (45%) NSI isolates obtained at that time had this combination of mutations.

    Conclusions: Conversion to the SI phenotype cannot be prevented by zidovudine treatment. The presence or appearance of an SI virus heralded disease progression in zidovudine-treated individuals. Further research is required to investigate the relationship between virus phenotype and development of zidovudine resistance.

    AIDS (London, England) 1992;6;11;1259-64

  • Fifth mutation in human immunodeficiency virus type 1 reverse transcriptase contributes to the development of high-level resistance to zidovudine.

    Kellam P, Boucher CA and Larder BA

    Department of Molecular Sciences, Wellcome Research Laboratories, Beckenham Kent, United Kingdom.

    It is recognized that high-level resistance to 3'-azido-3'-deoxythymidine (AZT, zidovudine, or Retrovir) is conferred by the presence of four mutations in the human immunodeficiency virus (HIV) reverse transcriptase [RT; deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase (RNA-directed), EC 2.7.7.49] coding sequence. However, a number of clinical isolates have been observed that exhibit high-level resistance but contain only three of the four identified mutations (Asn-67, Arg-70, and Tyr-215). Construction of a molecular clone with this genotype gave rise to only a partially resistant virus, raising the possibility that an additional mutation existed in some clinical isolates. Using an HIV marker rescue system, we have mapped and identified a fifth mutation conferring resistance to zidovudine, namely, methionine to leucine at codon 41 of HIV RT. An infectious molecular clone containing this mutation together with three previously identified mutations in the RT coding sequence yielded highly resistant HIV after transfection of T cells. Direct detection of the fifth mutation in DNA samples from cocultured peripheral blood lymphocytes by the PCR revealed that it occurred relatively early in the development of zidovudine resistance. However, this mutation was only detected after the appearance of the codon 215 change in the RT coding sequence. Identification of this mutation in addition to the other known mutations conferring resistance enables rapid and direct correlation between an RT genotype and sensitivity of the virus.

    Proceedings of the National Academy of Sciences of the United States of America 1992;89;5;1934-8

  • Ordered appearance of zidovudine resistance mutations during treatment of 18 human immunodeficiency virus-positive subjects.

    Boucher CA, O'Sullivan E, Mulder JW, Ramautarsing C, Kellam P, Darby G, Lange JM, Goudsmit J and Larder BA

    Department of Virology, University of Amsterdam, Netherlands.

    The order of appearance in the reverse transcriptase gene of four mutations implicated in the development of resistance to zidovudine was investigated by selective polymerase chain reaction. Serial human immunodeficiency virus isolates were studied from 18 initially asymptomatic individuals who had been treated with zidovudine for 2 years. Most subjects had similar patterns. The first mutation occurred transiently at codon 70; its disappearance was paralleled by the appearance of a mutation at codon 215. Subsequently, in some individuals, the mutation at codon 70 reappeared. During the 2 years of treatment, no mutations developed at codon 219 and only one at codon 67, suggesting that most individuals developed only partly resistant virus. This was confirmed by plaque-reduction assay. Six subjects progressed to AIDS within the 2-year study period, confirming that the development of highly resistant isolates is not required for progression in treated individuals. No clear temporal relationship was found between the development of partial resistance and progression.

    The Journal of infectious diseases 1992;165;1;105-10

1991 Publications

  • Resistance to ddI and sensitivity to AZT induced by a mutation in HIV-1 reverse transcriptase.

    St Clair MH, Martin JL, Tudor-Williams G, Bach MC, Vavro CL, King DM, Kellam P, Kemp SD and Larder BA

    Division of Virology, Burroughs Wellcome Co., Research Triangle Park, NC 27709.

    Serial human immunodeficiency virus type-1 (HIV-1) isolates were obtained from five individuals with acquired immunodeficiency syndrome (AIDS) who changed therapy to 2',3'-dideoxyinosine (ddI) after at least 12 months of treatment with 3'-azido-3'-deoxythymidine (zidovudine, AZT). The in vitro sensitivity to ddI decreased during the 12 months following ddI initiation, whereas AZT sensitivity increased. Analysis of the reverse transcriptase coding region revealed a mutation associated with reduced sensitivity to ddI. When this mutation was present in the same genome as a mutation known to confer AZT resistance, the isolates showed increased sensitivity to AZT. Analysis of HIV-1 variants confirmed that the ddI resistance mutation alone conferred ddI and 2',3'-dideoxycytidine resistance, and suppressed the effect of the AZT resistance mutation. The use of combination therapy for HIV-1 disease may prevent drug-resistant isolates from emerging.

    Science (New York, N.Y.) 1991;253;5027;1557-9

  • Zidovudine resistance predicted by direct detection of mutations in DNA from HIV-infected lymphocytes.

    Larder BA, Kellam P and Kemp SD

    Molecular Sciences Department, Wellcome Research Laboratories, Beckenham, Kent, UK.

    Zidovudine-resistant strains of HIV have recently been isolated from individuals during prolonged treatment. Analysis of the HIV reverse transcriptase (RT) gene from clinical isolates revealed that resistance was due to multiple nucleotide changes conferring specific amino acid substitutions in this enzyme. In order to correlate the degree of resistance with these amino acid changes, we constructed a series of infectious HIV variants with specific combinations of mutations in the RT gene and assessed their sensitivity to zidovudine. The reproducible nature of the mutations seen in clinical isolates has enabled the polymerase chain reaction to be used to identify lesions associated with resistance. This procedure was validated by analysis of sensitive and resistant clinical isolates with RT genes of known DNA sequence. Using a 'double' amplification procedure, zidovudine sensitivity was assessed by direct detection of specific mutations in DNA from peripheral-blood lymphocyte samples. This should make it possible to test large numbers of individuals receiving zidovudine therapy, with the aim of establishing the clinical significance of the resistant isolates.

    AIDS (London, England) 1991;5;2;137-44

  • The isolation and characterisation of plant sequences homologous to human hypervariable minisatellites.

    Daly A, Kellam P, Berry ST, Chojecki AJ and Barnes SR

    I.C.I Seeds, Jealott's Hill Research Station, Bracknell, Berkshire, Great Britain.

    We have isolated DNA probes, homologous to the human hypervariable minisatellite sequence 33.15, from the genome of rice (Oryza sativa). These probes are capable of producing a multilocus rice DNA fingerprint. The rice sequence has a tandem repeating structure based on a 12 bp GC-rich repeat which shows homology to its human counterpart. This probe detects up to 30 loci which are at a number of unlinked chromosomal sites. The GC-rich sequence is invariably associated with an open reading frame (ORF) of unknown function. The ORF is probably a member of a small multigene family.

    EXS 1991;58;330-41

1990 Publications

  • Zidovudine sensitivity of human immunodeficiency viruses from high-risk, symptom-free individuals during therapy.

    Boucher CA, Tersmette M, Lange JM, Kellam P, de Goede RE, Mulder JW, Darby G, Goudsmit J and Larder BA

    Human Retrovirus Laboratory, University of Amsterdam, Netherlands.

    Human immunodeficiency type 1 isolates from 18 initially symptom-free men who were treated with zidovudine for 2 years were investigated for drug sensitivity. At the start all the men had persistent core antigenaemia; the acquired immunodeficiency syndrome developed in 6 during the study. The polymerase chain reaction was used to detect mutations at residue 215 of reverse transcriptase, a mutation associated with reduced drug sensitivity. After 2 years 16/18 isolates were mutant. However, after about 6 months of treatment the mutation was detected in only 7 isolates, 4 from individuals who subsequently had AIDS. Limited direct virus sensitivity data correlated with the genetic data. The rate of appearance of the 215 mutation seemed to correlate with CD4 counts and viral virulence.

    Lancet 1990;336;8715;585-90

Reviews, invited commentaries and book chapters

Team

Team members

Eva Archer
PhD Student
Rachael Bashford Rogers
PhD Student
Matthew Cotten
Senior Staff Scientist
Carmen Diaz Soria
cds1@sanger.ac.ukPhD Student
Astrid Gall
Staff Scientist
Saad Idris
Wellcome Trust Clinical PhD Fellow
Pinky Langat
PhD Student
Swee Hoe Ong
Senior Computational Biologist
Anne Palser
ap10@sanger.ac.ukStaff Scientist
My Phan
Postdoctoral Fellow
Neneh Sallah
PhD Student
Sarah Smith
PhD Student
Rachael Wash
Staff Scientist
Simon Watson
Senior Computational Biologist

Eva Archer

- PhD Student

I graduated from the University of North Carolina at Chapel Hill in 2012 with a B.Sc. in Chemistry and a B.A. in International Studies. At UNC, I worked in the laboratory of Dr. Kevin Weeks researching RNA structural biology. I am a second year graduate student in the NIH-Cambridge Scholars PhD programme and split my time between the Wellcome Trust Sanger Institute and the National Institutes of Health in Washington, DC.

Research

I work in the Virus Genomics group at the Sanger Institute and the Immunology Laboratory at the Vaccine Research Center with Rick Koup and Danny Douek. My PhD research is focused on two aspects of the early immune response to Simian Immunodeficiency Virus (SIV) in macaques: understanding which subsets of CD4+ T cells are actively infected and produce viral RNAs, and sequencing antibody repertoires throughout infection to study the evolution of the humoral response.

References

  • Long-range architecture in a viral RNA genome.

    Archer EJ, Simpson MA, Watts NJ, O'Kane R, Wang B, Erie DA, McPherson A and Weeks KM

    Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290, USA.

    We have developed a model for the secondary structure of the 1058-nucleotide plus-strand RNA genome of the icosahedral satellite tobacco mosaic virus (STMV) using nucleotide-resolution SHAPE chemical probing of the viral RNA isolated from virions and within the virion, perturbation of interactions distant in the primary sequence, and atomic force microscopy. These data are consistent with long-range base pairing interactions and a three-domain genome architecture. The compact domains of the STMV RNA have dimensions of 10-45 nm. Each of the three domains corresponds to a specific functional component of the virus: The central domain corresponds to the coding sequence of the single (capsid) protein encoded by the virus, whereas the 5' and 3' untranslated domains span signals essential for translation and replication, respectively. This three-domain architecture is compatible with interactions between the capsid protein and short RNA helices previously visualized by crystallography. STMV is among the simplest of the icosahedral viruses but, nonetheless, has an RNA genome with a complex higher-order structure that likely reflects high information content and an evolutionary relationship between RNA domain structure and essential replicative functions.

    Funded by: NIGMS NIH HHS: GM079480, GM080412, R01 GM079480, R01 GM080412

    Biochemistry 2013;52;18;3182-90

Rachael Bashford Rogers

- PhD Student

I am a final year PhD student working under Prof. Paul Kellam and Prof. Allan Bradley. I graduated from Oxford University in 2010 with degree in Chemistry (MChem). My research year, under Prof. Chris Schofield, studied structure and mechanism of the hypoxic response. I also worked under Dr Laura Itzhaki (University of Cambridge) characterising degradation processes of proteins from stalled ribosomes, and the interactions between two breast cancer related genes (2008-2009). I have been involved in other projects such as large-scale analysis of malaria phospho-proteome, an in vitro interaction screen between putative malaria gamete fertility proteins, and RNA expression analysis.

Research

My PhD has been focused on developing highly sensitive molecular frameworks for understanding the dynamics of B- and T-cell populations during infection and malignancy, achieved by high-throughput sequencing, and the development of novel computational methods. Projects range from understanding immune responses in human immunodeficiency virus (HIV), Simian immunodeficiency virus (SIV), Hepatitis B and Hepatitis C in humans and model organisms, to understanding leukemogenesis in chronic lymphocytic leukemia (CLL), and development of diagnostic tools for determining B-cell and T-cell clonality. The work involves multiple national and international collaborations.

References

  • Transmission and evolution of the Middle East respiratory syndrome coronavirus in Saudi Arabia: a descriptive genomic study.

    Cotten M, Watson SJ, Kellam P, Al-Rabeeah AA, Makhdoom HQ, Assiri A, Al-Tawfiq JA, Alhakeem RF, Madani H, AlRabiah FA, Al Hajjar S, Al-nassir WN, Albarrak A, Flemban H, Balkhy HH, Alsubaie S, Palser AL, Gall A, Bashford-Rogers R, Rambaut A, Zumla AI and Memish ZA

    Wellcome Trust Sanger Institute, Hinxton, UK.

    Background: Since June, 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) has, worldwide, caused 104 infections in people including 49 deaths, with 82 cases and 41 deaths reported from Saudi Arabia. In addition to confirming diagnosis, we generated the MERS-CoV genomic sequences obtained directly from patient samples to provide important information on MERS-CoV transmission, evolution, and origin.

    Methods: Full genome deep sequencing was done on nucleic acid extracted directly from PCR-confirmed clinical samples. Viral genomes were obtained from 21 MERS cases of which 13 had 100%, four 85-95%, and four 30-50% genome coverage. Phylogenetic analysis of the 21 sequences, combined with nine published MERS-CoV genomes, was done.

    Findings: Three distinct MERS-CoV genotypes were identified in Riyadh. Phylogeographic analyses suggest the MERS-CoV zoonotic reservoir is geographically disperse. Selection analysis of the MERS-CoV genomes reveals the expected accumulation of genetic diversity including changes in the S protein. The genetic diversity in the Al-Hasa cluster suggests that the hospital outbreak might have had more than one virus introduction.

    Interpretation: We present the largest number of MERS-CoV genomes (21) described so far. MERS-CoV full genome sequences provide greater detail in tracking transmission. Multiple introductions of MERS-CoV are identified and suggest lower R0 values. Transmission within Saudi Arabia is consistent with either movement of an animal reservoir, animal products, or movement of infected people. Further definition of the exposures responsible for the sporadic introductions of MERS-CoV into human populations is urgently needed.

    Funding: Saudi Arabian Ministry of Health, Wellcome Trust, European Community, and National Institute of Health Research University College London Hospitals Biomedical Research Centre.

    Funded by: Wellcome Trust: 093724

    Lancet 2013;382;9909;1993-2002

  • Network properties derived from deep sequencing of human B-cell receptor repertoires delineate B-cell populations.

    Bashford-Rogers RJ, Palser AL, Huntly BJ, Rance R, Vassiliou GS, Follows GA and Kellam P

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom;

    The adaptive immune response selectively expands B- and T-cell clones following antigen recognition by B- and T-cell receptors (BCR and TCR), respectively. Next-generation sequencing is a powerful tool for dissecting the BCR and TCR populations at high resolution, but robust computational analyses are required to interpret such sequencing. Here, we develop a novel computational approach for BCR repertoire analysis using established next-generation sequencing methods coupled with network construction and population analysis. BCR sequences organize into networks based on sequence diversity, with differences in network connectivity clearly distinguishing between diverse repertoires of healthy individuals and clonally expanded repertoires from individuals with chronic lymphocytic leukemia (CLL) and other clonal blood disorders. Network population measures defined by the Gini Index and cluster sizes quantify the BCR clonality status and are robust to sampling and sequencing depths. BCR network analysis therefore allows the direct and quantifiable comparison of BCR repertoires between samples and intra-individual population changes between temporal or spatially separated samples and over the course of therapy.

    Funded by: Wellcome Trust: 079249, 095663, 100140

    Genome research 2013;23;11;1874-84

Matthew Cotten

- Senior Staff Scientist

I have a Ph.D. (biochemistry, University of Iowa, Iowa, USA), and did postdoctoral work at Vanderbilt University (Nashville, Tennessee, USA) and the Institute for Molecular Pathology (IMP, Vienna, Austria) studying chromatin assembly and histone gene regulation. I was a group leader at the IMP, developing artificial viruses for gene transfer, at a biotechnology firm (Axxima, Munich, Germany) developing anti-viral kinase inhibitors and at the MRC Laboratories in Gambia studying the evolution of HIV-2 and managing viral diagnostics. I have peer-reviewed publications in molecular biology, basic virology and viral deep sequencing methods (http://scholar.google.co.uk/citations?user=6eepOOYAAAAJ).

Research

My current research efforts are to help develop high-throughput deep sequencing methods for full RNA viral genomes including norovirus, hepatitis C virus, rotavirus, respiratory syncytial virus, MERS coronavirus and to devise more sensitive methods for virus detection and virus discovery. I work as both a wet-lab scientist as well as writing algorithms for primer design, sequence analysis and data visualization.

References

  • Spread, circulation, and evolution of the Middle East respiratory syndrome coronavirus.

    Cotten M, Watson SJ, Zumla AI, Makhdoom HQ, Palser AL, Ong SH, Al Rabeeah AA, Alhakeem RF, Assiri A, Al-Tawfiq JA, Albarrak A, Barry M, Shibl A, Alrabiah FA, Hajjar S, Balkhy HH, Flemban H, Rambaut A, Kellam P and Memish ZA

    Unlabelled: The Middle East respiratory syndrome coronavirus (MERS-CoV) was first documented in the Kingdom of Saudi Arabia (KSA) in 2012 and, to date, has been identified in 180 cases with 43% mortality. In this study, we have determined the MERS-CoV evolutionary rate, documented genetic variants of the virus and their distribution throughout the Arabian peninsula, and identified the genome positions under positive selection, important features for monitoring adaptation of MERS-CoV to human transmission and for identifying the source of infections. Respiratory samples from confirmed KSA MERS cases from May to September 2013 were subjected to whole-genome deep sequencing, and 32 complete or partial sequences (20 were ≥ 99% complete, 7 were 50 to 94% complete, and 5 were 27 to 50% complete) were obtained, bringing the total available MERS-CoV genomic sequences to 65. An evolutionary rate of 1.12 × 10(-3) substitutions per site per year (95% credible interval [95% CI], 8.76 × 10(-4); 1.37 × 10(-3)) was estimated, bringing the time to most recent common ancestor to March 2012 (95% CI, December 2011; June 2012). Only one MERS-CoV codon, spike 1020, located in a domain required for cell entry, is under strong positive selection. Four KSA MERS-CoV phylogenetic clades were found, with 3 clades apparently no longer contributing to current cases. The size of the population infected with MERS-CoV showed a gradual increase to June 2013, followed by a decline, possibly due to increased surveillance and infection control measures combined with a basic reproduction number (R0) for the virus that is less than 1.

    Importance: MERS-CoV adaptation toward higher rates of sustained human-to-human transmission appears not to have occurred yet. While MERS-CoV transmission currently appears weak, careful monitoring of changes in MERS-CoV genomes and of the MERS epidemic should be maintained. The observation of phylogenetically related MERS-CoV in geographically diverse locations must be taken into account in efforts to identify the animal source and transmission of the virus.

    Funded by: Wellcome Trust

    mBio 2014;5;1

  • Transmission and evolution of the Middle East respiratory syndrome coronavirus in Saudi Arabia: a descriptive genomic study.

    Cotten M, Watson SJ, Kellam P, Al-Rabeeah AA, Makhdoom HQ, Assiri A, Al-Tawfiq JA, Alhakeem RF, Madani H, AlRabiah FA, Al Hajjar S, Al-nassir WN, Albarrak A, Flemban H, Balkhy HH, Alsubaie S, Palser AL, Gall A, Bashford-Rogers R, Rambaut A, Zumla AI and Memish ZA

    Wellcome Trust Sanger Institute, Hinxton, UK.

    Background: Since June, 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) has, worldwide, caused 104 infections in people including 49 deaths, with 82 cases and 41 deaths reported from Saudi Arabia. In addition to confirming diagnosis, we generated the MERS-CoV genomic sequences obtained directly from patient samples to provide important information on MERS-CoV transmission, evolution, and origin.

    Methods: Full genome deep sequencing was done on nucleic acid extracted directly from PCR-confirmed clinical samples. Viral genomes were obtained from 21 MERS cases of which 13 had 100%, four 85-95%, and four 30-50% genome coverage. Phylogenetic analysis of the 21 sequences, combined with nine published MERS-CoV genomes, was done.

    Findings: Three distinct MERS-CoV genotypes were identified in Riyadh. Phylogeographic analyses suggest the MERS-CoV zoonotic reservoir is geographically disperse. Selection analysis of the MERS-CoV genomes reveals the expected accumulation of genetic diversity including changes in the S protein. The genetic diversity in the Al-Hasa cluster suggests that the hospital outbreak might have had more than one virus introduction.

    Interpretation: We present the largest number of MERS-CoV genomes (21) described so far. MERS-CoV full genome sequences provide greater detail in tracking transmission. Multiple introductions of MERS-CoV are identified and suggest lower R0 values. Transmission within Saudi Arabia is consistent with either movement of an animal reservoir, animal products, or movement of infected people. Further definition of the exposures responsible for the sporadic introductions of MERS-CoV into human populations is urgently needed.

    Funding: Saudi Arabian Ministry of Health, Wellcome Trust, European Community, and National Institute of Health Research University College London Hospitals Biomedical Research Centre.

    Funded by: Wellcome Trust: 093724

    Lancet 2013;382;9909;1993-2002

  • Hospital outbreak of Middle East respiratory syndrome coronavirus.

    Assiri A, McGeer A, Perl TM, Price CS, Al Rabeeah AA, Cummings DA, Alabdullatif ZN, Assad M, Almulhim A, Makhdoom H, Madani H, Alhakeem R, Al-Tawfiq JA, Cotten M, Watson SJ, Kellam P, Zumla AI, Memish ZA and KSA MERS-CoV Investigation Team

    Global Center for Mass Gatherings Medicine, Ministry of Health, Riyadh, Saudi Arabia.

    Background: In September 2012, the World Health Organization reported the first cases of pneumonia caused by the novel Middle East respiratory syndrome coronavirus (MERS-CoV). We describe a cluster of health care-acquired MERS-CoV infections.

    Methods: Medical records were reviewed for clinical and demographic information and determination of potential contacts and exposures. Case patients and contacts were interviewed. The incubation period and serial interval (the time between the successive onset of symptoms in a chain of transmission) were estimated. Viral RNA was sequenced.

    Results: Between April 1 and May 23, 2013, a total of 23 cases of MERS-CoV infection were reported in the eastern province of Saudi Arabia. Symptoms included fever in 20 patients (87%), cough in 20 (87%), shortness of breath in 11 (48%), and gastrointestinal symptoms in 8 (35%); 20 patients (87%) presented with abnormal chest radiographs. As of June 12, a total of 15 patients (65%) had died, 6 (26%) had recovered, and 2 (9%) remained hospitalized. The median incubation period was 5.2 days (95% confidence interval [CI], 1.9 to 14.7), and the serial interval was 7.6 days (95% CI, 2.5 to 23.1). A total of 21 of the 23 cases were acquired by person-to-person transmission in hemodialysis units, intensive care units, or in-patient units in three different health care facilities. Sequencing data from four isolates revealed a single monophyletic clade. Among 217 household contacts and more than 200 health care worker contacts whom we identified, MERS-CoV infection developed in 5 family members (3 with laboratory-confirmed cases) and in 2 health care workers (both with laboratory-confirmed cases).

    Conclusions: Person-to-person transmission of MERS-CoV can occur in health care settings and may be associated with considerable morbidity. Surveillance and infection-control measures are critical to a global public health response.

    Funded by: NIGMS NIH HHS: U01 GM070708, U54 GM088491; Wellcome Trust: 093724

    The New England journal of medicine 2013;369;5;407-16

  • Full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus.

    Cotten M, Lam TT, Watson SJ, Palser AL, Petrova V, Grant P, Pybus OG, Rambaut A, Guan Y, Pillay D, Kellam P and Nastouli E

    Wellcome Trust Sanger Institute, Hinxton, UK.

    A novel betacoronavirus associated with lethal respiratory and renal complications was recently identified in patients from several countries in the Middle East. We report the deep genome sequencing of the virus directly from a patient's sputum sample. Our high-throughput sequencing yielded a substantial depth of genome sequence assembly and showed the minority viral variants in the specimen. Detailed phylogenetic analysis of the virus genome (England/Qatar/2012) revealed its close relationship to European bat coronaviruses circulating among the bat species of the Vespertilionidae family. Molecular clock analysis showed that the 2 human infections of this betacoronavirus in June 2012 (EMC/2012) and September 2012 (England/Qatar/2012) share a common virus ancestor most likely considerably before early 2012, suggesting the human diversity is the result of multiple zoonotic events.

    Funded by: Medical Research Council: MR/K006584/1; Wellcome Trust: 093724, 095831

    Emerging infectious diseases 2013;19;5;736-42B

  • Population dynamics of HIV-2 in rural West Africa: comparison with HIV-1 and ongoing transmission at the heart of the epidemic.

    de Silva TI, van Tienen C, Onyango C, Jabang A, Vincent T, Loeff MF, Coutinho RA, Jaye A, Rowland-Jones S, Whittle H, Cotten M and Hué S

    Medical Research Council (UK) Laboratories, Fajara, The Gambia. thushandesilva@hotmail.com

    Objectives: To compare the population dynamics of HIV-2 and HIV-1, and to characterize ongoing HIV-2 transmission in rural Guinea-Bissau.

    Design: Phylogenetic and phylodynamic analyses using HIV-2 gag and env, and HIV-1 env sequences, combined with epidemiological data from a community cohort.

    Methods: Samples were obtained from surveys in 1989-1991, 1996-1997, 2003 and 2006-2007. Phylogenies were reconstructed using sequences from 103 HIV-2-infected and 56 HIV-1-infected patients using Bayesian Evolutionary Analysis by Sampling Trees (BEAST), a relaxed molecular clock and a Bayesian skyline coalescent model.

    Results: Bayesian skyline plots showed a strong increase in the 1990s of the HIV-1 effective population size (Ne) in the same period that the Ne of HIV-2 came into a plateau phase. The population dynamics of both viruses were remarkably similar following initial introduction. Incident infections were found more often in HIV-2 transmission clusters, with 55-58% of all individuals contributing to ongoing transmission. Some phylogenetically linked sexual partners had discordant viral loads (undetectable vs. detectable), suggesting host factors dictate the risk of disease progression in HIV-2. Multiple HIV-2 introductions into the cohort are evident, but ongoing transmission has occurred predominantly within the community.

    Conclusion: Comparison of HIV-1 and HIV-2 phylodynamics in the same community suggests both viruses followed similar growth patterns following introduction, and is consistent with the hypothesis that HIV-1 may have played a role in the decline of HIV-2 via competitive exclusion. The source of ongoing HIV-2 transmission in the cohort appears to be new HIV-2 cases, rather than the pool of older infections established during the early growth of HIV-2.

    Funded by: Medical Research Council: G0701313, G0801751, MC_U137884180, MC_UP_A900_1125

    AIDS (London, England) 2013;27;1;125-34

  • Potent autologous and heterologous neutralizing antibody responses occur in HIV-2 infection across a broad range of infection outcomes.

    de Silva TI, Aasa-Chapman M, Cotten M, Hué S, Robinson J, Bibollet-Ruche F, Sarge-Njie R, Berry N, Jaye A, Aaby P, Whittle H, Rowland-Jones S and Weiss R

    Medical Research Council (United Kingdom) Laboratories, Fajara, the Gambia. thushandesilva@hotmail.com

    Few studies have explored the role of neutralizing antibody (NAb) responses in controlling HIV-2 viremia and disease progression. Using a TZM-bl neutralization assay, we assessed heterologous and autologous NAb responses from a community cohort of HIV-2-infected individuals with a broad range of disease outcomes in rural Guinea-Bissau. All subjects (n = 40) displayed exceptionally high heterologous NAb titers (50% inhibitory plasma dilution or 50% inhibitory concentration [IC(50)], 1:7,000 to 1:1,000,000) against 5 novel primary HIV-2 envelopes and HIV-2 7312A, whereas ROD A and 3 primary envelopes were relatively resistant to neutralization. Most individuals also showed high autologous NAb against contemporaneous envelopes (78% of plasma-envelope combinations in 69 envelopes from 21 subjects), with IC(50)s above 1:10,000. No association between heterologous or autologous NAb titer and greater control of HIV-2 was found. A subset of envelopes was found to be more resistant to neutralization (by plasma and HIV-2 monoclonal antibodies). These envelopes were isolated from individuals with greater intrapatient sequence diversity and were associated with changes in potential N-linked glycosylation sites but not CD4 independence or CXCR4 use. Plasma collected from up to 15 years previously was able to potently neutralize recent autologous envelopes, suggesting a lack of escape from NAb and the persistence of neutralization-sensitive variants over time, despite significant NAb pressure. We conclude that despite the presence of broad and potent NAb responses in HIV-2-infected individuals, these are not the primary forces behind the dichotomous outcomes observed but reveal a limited capacity for adaptive selection and escape from host immunity in HIV-2 infection.

    Funded by: Medical Research Council: G0600007, G0801176, G0801751, MC_UP_A900_1121

    Journal of virology 2012;86;2;930-46

  • Molecular epidemiology of endemic human T-lymphotropic virus type 1 in a rural community in Guinea-Bissau.

    van Tienen C, de Silva TI, Alcantara LC, Onyango CO, Jarju S, Gonçalves N, Vincent T, Aaby P, Whittle H, Schim van der Loeff M and Cotten M

    Virology, Medical Research Council, Fajara, The Gambia. c.vantienen@erasmusmc.nl

    Background: Human T-Lymphotropic Virus Type 1 (HTLV-1) infection causes lethal adult T-cell leukemia (ATL) and severely debilitating HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in up to 5% of infected adults. HTLV-1 is endemic in parts of Africa and the highest prevalence in West Africa (5%) has been reported in Caio, a rural area in the North-West of Guinea-Bissau. It is not known which HTLV-1 variants are present in this community. Sequence data can provide insights in the molecular epidemiology and help to understand the origin and spread of HTLV-1.

    Objective: To gain insight into the molecular diversity of HTLV-1 in West Africa.

    Methods: HTLV-1 infected individuals were identified in community surveys between 1990-2007. The complete Long Terminal Repeat (LTR) and p24 coding region of HTLV-1 was sequenced from infected subjects. Socio-demographic data were obtained from community census and from interviews performed by fieldworkers. Phylogenetic analyses were performed to characterize the relationship between the Caio HTLV-1 and HTLV-1 from other parts of the world.

    Results: LTR and p24 sequences were obtained from 72 individuals (36 LTR, 24 p24 only and 12 both). Consistent with the low evolutionary change of HTLV-1, many of the sequences from unrelated individuals showed 100% nucleotide identity. Most (45 of 46) of the LTR sequences clustered with the Cosmopolitan HTLV-1 subtype 1a, subgroup D (1aD). LTR and p24 sequences from two subjects were divergent and formed a significant cluster with HTLV-1 subtype 1g, and with the most divergent African Simian T-cell Lymphotropic Virus, Tan90.

    Conclusions: The Cosmopolitan HTLV-1 1aD predominates in this rural West African community. However, HTLV-1 subtype 1g is also present. This subtype has not been described before in West Africa and may be more widespread than previously thought. These data are in line with the hypothesis that multiple monkey-to-man zoonotic events are contributing to HTLV-1 diversity.

    PLoS neglected tropical diseases 2012;6;6;e1690

  • HIV-2 capsids distinguish high and low virus load patients in a West African community cohort.

    Onyango CO, Leligdowicz A, Yokoyama M, Sato H, Song H, Nakayama EE, Shioda T, de Silva T, Townend J, Jaye A, Whittle H, Rowland-Jones S and Cotten M

    Medical Research Council Laboratories, Fajara, Atlantic Road, PO Box 273, The Gambia. conyango@mrc.gm

    HIV-2 causes AIDS similar to HIV-1, however a considerable proportion of HIV-2 infected patients show no disease and have low plasma virus load (VL). An analysis of HIV-2 capsid (p26) variation demonstrated that proline at p26 positions 119, 159 and 178 are more frequent in lower VL subjects while non-proline residues at all three sites are more frequent in subjects with high VL. In vitro replication levels of viruses bearing changes at the three sites suggested that these three residues influence virus replication by altering susceptibility to TRIM5alpha. These results provide new insights into HIV-2 pathogenesis.

    Funded by: Medical Research Council: G0801751, MC_U190085856

    Vaccine 2010;28 Suppl 2;B60-7

  • HIV-1 subtype distribution in the Gambia and the significant presence of CRF49_cpx, a novel circulating recombinant form.

    de Silva TI, Turner R, Hué S, Trikha R, van Tienen C, Onyango C, Jaye A, Foley B, Whittle H, Rowland-Jones SL and Cotten M

    Medical Research Council (UK) Laboratories, Atlantic Road, Fajara, The Gambia.

    Background: Detailed local HIV-1 sequence data are essential for monitoring the HIV epidemic, for maintaining sensitive sequence-based diagnostics, and to aid in designing vaccines.

    Results: Reported here are full envelope sequences derived from 38 randomly selected HIV-1 infections identified at a Gambian clinic between 1991 and 2009. Special care was taken to generate sequences from circulating viral RNA as uncloned products, either by limiting dilution or single genome amplification polymerase chain reaction (PCR). Within these 38 isolates, eight were subtyped as A and 18 as CRF02_AG. A small number of subtype B, C, D viruses were identified. Surprising, however, was the identification of six isolates with subtype J-like envelopes, a subtype found normally in Central Africa and the Democratic Republic of the Congo (DRC), with gag p24 regions that clustered with subtype A sequences. Near full-length sequence from three of these isolates confirmed that these represent a novel circulating recombinant form of HIV-1, now named CRF49_cpx.

    Conclusions: This study expands the HIV-1 sequence database from the Gambia and will provide important data for HIV diagnostics, patient care, and vaccine development.

    Funded by: Medical Research Council: G0801751, MC_U190085856

    Retrovirology 2010;7;82

  • An efficient proteomics method to identify the cellular targets of protein kinase inhibitors.

    Godl K, Wissing J, Kurtenbach A, Habenberger P, Blencke S, Gutbrod H, Salassidis K, Stein-Gerlach M, Missio A, Cotten M and Daub H

    Axxima Pharmaceuticals AG, Max-Lebsche-Platz 32, 81377 Munich, Germany.

    Small molecule inhibitors of protein kinases are widely used in signal transduction research and are emerging as a major class of drugs. Although interpretation of biological results obtained with these reagents critically depends on their selectivity, efficient methods for proteome-wide assessment of kinase inhibitor selectivity have not yet been reported. Here, we address this important issue and describe a method for identifying targets of the widely used p38 kinase inhibitor SB 203580. Immobilization of a suitable SB 203580 analogue and thoroughly optimized biochemical conditions for affinity chromatography permitted the dramatic enrichment and identification of several previously unknown protein kinase targets of SB 203580. In vitro kinase assays showed that cyclin G-associated kinase (GAK) and CK1 were almost as potently inhibited as p38alpha whereas RICK [Rip-like interacting caspase-like apoptosis-regulatory protein (CLARP) kinase/Rip2/CARDIAK] was even more sensitive to inhibition by SB 203580. The cellular kinase activity of RICK, a known signal transducer of inflammatory responses, was already inhibited by submicromolar concentrations of SB 203580 in intact cells. Therefore, our results warrant a reevaluation of the vast amount of data obtained with SB 203580 and might have significant implications on the development of p38 inhibitors as antiinflammatory drugs. Based on the procedures described here, efficient affinity purification techniques can be developed for other protein kinase inhibitors, providing crucial information about their cellular modes of action.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;26;15434-9

Carmen Diaz Soria

cds1@sanger.ac.uk PhD Student

I am on my second year of a 4-year Wellcome Trust PhD at the Sanger Institute. I started my PhD straight after finishing my undergraduate degree in Molecular Genetics at King’s College London. During my university studies, I had the opportunity to work at the pharmaceutical company Eli Lilly in Windlesham, Surrey. This experience enabled me to learn and develop an interest in laboratory research and most importantly, it inspired me to continue my studies in life sciences.

Research

At the present time, I have started my PhD project in the Virus Genomics group at the Sanger Institute. I focus on the study of two host factors: Interferon Inducible Transmembrane proteins 2 and 3 (IFITM2 and IFITM3). My aim is to understand the genetic diversity of IFITM proteins and test how polymorphisms in IFITM2 and IFITM3 alter host susceptibility to virus infection, specifically to dengue infection.

Astrid Gall

- Staff Scientist

I graduated with a veterinary degree and obtained a Dr. med. vet. degree (PhD equivalent) in Molecular Virology from the University of Veterinary Medicine, Hannover, Germany, in 2006. The work was carried out at the Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health. I stayed there as a postdoctoral scientist working on microarray diagnosis of influenza viruses. In 2009 I joined the Virus Genomics group at the Wellcome Trust Sanger Institute. I am also a Fellow and Tutor at Lucy Cavendish College, University of Cambridge.

Research

I lead the group’s research on Human Immunodeficiency Virus 1 (HIV-1) and manage large-scale sequencing efforts based on a universal method for deep sequencing of HIV-1 genomes across the global diversity I developed recently. Current research focuses on (i) evolutionary dynamics of HIV-1 within the host, (ii) interaction of the virus with the adaptive immune system and (iii) influence of viral and host factors on progression to AIDS. I have a wider interest in diversity, evolution and host-virus interaction of medically important, highly variable RNA viruses, concerning animal, human and zoonotic viruses.

References

  • Transmission and evolution of the Middle East respiratory syndrome coronavirus in Saudi Arabia: a descriptive genomic study.

    Cotten M, Watson SJ, Kellam P, Al-Rabeeah AA, Makhdoom HQ, Assiri A, Al-Tawfiq JA, Alhakeem RF, Madani H, AlRabiah FA, Al Hajjar S, Al-nassir WN, Albarrak A, Flemban H, Balkhy HH, Alsubaie S, Palser AL, Gall A, Bashford-Rogers R, Rambaut A, Zumla AI and Memish ZA

    Wellcome Trust Sanger Institute, Hinxton, UK.

    Background: Since June, 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) has, worldwide, caused 104 infections in people including 49 deaths, with 82 cases and 41 deaths reported from Saudi Arabia. In addition to confirming diagnosis, we generated the MERS-CoV genomic sequences obtained directly from patient samples to provide important information on MERS-CoV transmission, evolution, and origin.

    Methods: Full genome deep sequencing was done on nucleic acid extracted directly from PCR-confirmed clinical samples. Viral genomes were obtained from 21 MERS cases of which 13 had 100%, four 85-95%, and four 30-50% genome coverage. Phylogenetic analysis of the 21 sequences, combined with nine published MERS-CoV genomes, was done.

    Findings: Three distinct MERS-CoV genotypes were identified in Riyadh. Phylogeographic analyses suggest the MERS-CoV zoonotic reservoir is geographically disperse. Selection analysis of the MERS-CoV genomes reveals the expected accumulation of genetic diversity including changes in the S protein. The genetic diversity in the Al-Hasa cluster suggests that the hospital outbreak might have had more than one virus introduction.

    Interpretation: We present the largest number of MERS-CoV genomes (21) described so far. MERS-CoV full genome sequences provide greater detail in tracking transmission. Multiple introductions of MERS-CoV are identified and suggest lower R0 values. Transmission within Saudi Arabia is consistent with either movement of an animal reservoir, animal products, or movement of infected people. Further definition of the exposures responsible for the sporadic introductions of MERS-CoV into human populations is urgently needed.

    Funding: Saudi Arabian Ministry of Health, Wellcome Trust, European Community, and National Institute of Health Research University College London Hospitals Biomedical Research Centre.

    Funded by: Wellcome Trust: 093724

    Lancet 2013;382;9909;1993-2002

  • An elephantine viral problem.

    Gall A and Palser A

    This month's Genome Watch highlights how deep sequencing was used to generate the first full genomes of herpesviruses associated with a fatal disease in elephants.

    Nature reviews. Microbiology 2013;11;8;512

  • Restriction of V3 region sequence divergence in the HIV-1 envelope gene during antiretroviral treatment in a cohort of recent seroconverters.

    Gall A, Kaye S, Hué S, Bonsall D, Rance R, Baillie GJ, Fidler SJ, Weber JN, McClure MO, Kellam P and SPARTAC Trial Investigators

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

    Background: Dynamic changes in Human Immunodeficiency Virus 1 (HIV-1) sequence diversity and divergence are associated with immune control during primary infection and progression to AIDS. Consensus sequencing or single genome amplification sequencing of the HIV-1 envelope (env) gene, in particular the variable (V) regions, is used as a marker for HIV-1 genome diversity, but population diversity is only minimally, or semi-quantitatively sampled using these methods.

    Results: Here we use second generation deep sequencing to determine inter-and intra-patient sequence heterogeneity and to quantify minor variants in a cohort of individuals either receiving or not receiving antiretroviral treatment following seroconversion; the SPARTAC trial. We show, through a cross-sectional study of sequence diversity of the env V3 in 30 antiretroviral-naive patients during primary infection that considerable population structure diversity exists, with some individuals exhibiting highly constrained plasma virus diversity. Diversity was independent of clinical markers (viral load, time from seroconversion, CD4 cell count) of infection. Serial sampling over 60 weeks of non-treated individuals that define three initially different diversity profiles showed that complex patterns of continuing HIV-1 sequence diversification and divergence could be readily detected. Evidence for minor sequence turnover, emergence of new variants and re-emergence of archived variants could be inferred from this analysis. Analysis of viral divergence over the same time period in patients who received short (12 weeks, ART12) or long course antiretroviral therapy (48 weeks, ART48) and a non-treated control group revealed that ART48 successfully suppressed viral divergence while ART12 did not have a significant effect.

    Conclusions: Deep sequencing is a sensitive and reliable method for investigating the diversity of the env V3 as an important component of HIV-1 genome diversity. Detailed insights into the complex early intra-patient dynamics of env V3 diversity and divergence were explored in antiretroviral-naïve recent seroconverters. Long course antiretroviral therapy, initiated soon after seroconversion and administered for 48 weeks, restricts HIV-1 divergence significantly. The effect of ART12 and ART48 on clinical markers of HIV infection and progression is currently investigated in the SPARTAC trial.

    Funded by: NIAID NIH HHS: R01 AI046995; Wellcome Trust

    Retrovirology 2013;10;8

  • Universal amplification, next-generation sequencing, and assembly of HIV-1 genomes.

    Gall A, Ferns B, Morris C, Watson S, Cotten M, Robinson M, Berry N, Pillay D and Kellam P

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom.

    Whole HIV-1 genome sequences are pivotal for large-scale studies of inter- and intrahost evolution, including the acquisition of drug resistance mutations. The ability to rapidly and cost-effectively generate large numbers of HIV-1 genome sequences from different populations and geographical locations and determine the effect of minority genetic variants is, however, a limiting factor. Next-generation sequencing promises to bridge this gap but is hindered by the lack of methods for the enrichment of virus genomes across the phylogenetic breadth of HIV-1 and methods for the robust assembly of the virus genomes from short-read data. Here we report a method for the amplification, next-generation sequencing, and unbiased de novo assembly of HIV-1 genomes of groups M, N, and O, as well as recombinants, that does not require prior knowledge of the sequence or subtype. A sensitivity of at least 3,000 copies/ml was determined by using plasma virus samples of known copy numbers. We applied our novel method to compare the genome diversities of HIV-1 groups, subtypes, and genes. The highest level of diversity was found in the env, nef, vpr, tat, and rev genes and parts of the gag gene. Furthermore, we used our method to investigate mutations associated with HIV-1 drug resistance in clinical samples at the level of the complete genome. Drug resistance mutations were detected as both major variant and minor species. In conclusion, we demonstrate the feasibility of our method for large-scale HIV-1 genome sequencing. This will enable the phylogenetic and phylodynamic resolution of the ongoing pandemic and efficient monitoring of complex HIV-1 drug resistance genotypes.

    Funded by: Wellcome Trust: S0753

    Journal of clinical microbiology 2012;50;12;3838-44

  • Evolutionary dynamics of local pandemic H1N1/2009 influenza virus lineages revealed by whole-genome analysis.

    Baillie GJ, Galiano M, Agapow PM, Myers R, Chiam R, Gall A, Palser AL, Watson SJ, Hedge J, Underwood A, Platt S, McLean E, Pebody RG, Rambaut A, Green J, Daniels R, Pybus OG, Kellam P and Zambon M

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom.

    Virus gene sequencing and phylogenetics can be used to study the epidemiological dynamics of rapidly evolving viruses. With complete genome data, it becomes possible to identify and trace individual transmission chains of viruses such as influenza virus during the course of an epidemic. Here we sequenced 153 pandemic influenza H1N1/09 virus genomes from United Kingdom isolates from the first (127 isolates) and second (26 isolates) waves of the 2009 pandemic and used their sequences, dates of isolation, and geographical locations to infer the genetic epidemiology of the epidemic in the United Kingdom. We demonstrate that the epidemic in the United Kingdom was composed of many cocirculating lineages, among which at least 13 were exclusively or predominantly United Kingdom clusters. The estimated divergence times of two of the clusters predate the detection of pandemic H1N1/09 virus in the United Kingdom, suggesting that the pandemic H1N1/09 virus was already circulating in the United Kingdom before the first clinical case. Crucially, three clusters contain isolates from the second wave of infections in the United Kingdom, two of which represent chains of transmission that appear to have persisted within the United Kingdom between the first and second waves. This demonstrates that whole-genome analysis can track in fine detail the behavior of individual influenza virus lineages during the course of a single epidemic or pandemic.

    Funded by: Medical Research Council: MC_U117512723; Wellcome Trust: 095831

    Journal of virology 2012;86;1;11-8

  • Disease-associated XMRV sequences are consistent with laboratory contamination.

    Hué S, Gray ER, Gall A, Katzourakis A, Tan CP, Houldcroft CJ, McLaren S, Pillay D, Futreal A, Garson JA, Pybus OG, Kellam P and Towers GJ

    MRC Centre for Medical Molecular Virology, Division of Infection and Immunity, University College London, 46 Cleveland St, London W1T 4JF, UK.

    Background: Xenotropic murine leukaemia viruses (MLV-X) are endogenous gammaretroviruses that infect cells from many species, including humans. Xenotropic murine leukaemia virus-related virus (XMRV) is a retrovirus that has been the subject of intense debate since its detection in samples from humans with prostate cancer (PC) and chronic fatigue syndrome (CFS). Controversy has arisen from the failure of some studies to detect XMRV in PC or CFS patients and from inconsistent detection of XMRV in healthy controls.

    Results: Here we demonstrate that Taqman PCR primers previously described as XMRV-specific can amplify common murine endogenous viral sequences from mouse suggesting that mouse DNA can contaminate patient samples and confound specific XMRV detection. To consider the provenance of XMRV we sequenced XMRV from the cell line 22Rv1, which is infected with an MLV-X that is indistinguishable from patient derived XMRV. Bayesian phylogenies clearly show that XMRV sequences reportedly derived from unlinked patients form a monophyletic clade with interspersed 22Rv1 clones (posterior probability >0.99). The cell line-derived sequences are ancestral to the patient-derived sequences (posterior probability >0.99). Furthermore, pol sequences apparently amplified from PC patient material (VP29 and VP184) are recombinants of XMRV and Moloney MLV (MoMLV) a virus with an envelope that lacks tropism for human cells. Considering the diversity of XMRV we show that the mean pairwise genetic distance among env and pol 22Rv1-derived sequences exceeds that of patient-associated sequences (Wilcoxon rank sum test: p = 0.005 and p < 0.001 for pol and env, respectively). Thus XMRV sequences acquire diversity in a cell line but not in patient samples. These observations are difficult to reconcile with the hypothesis that published XMRV sequences are related by a process of infectious transmission.

    Conclusions: We provide several independent lines of evidence that XMRV detected by sensitive PCR methods in patient samples is the likely result of PCR contamination with mouse DNA and that the described clones of XMRV arose from the tumour cell line 22Rv1, which was probably infected with XMRV during xenografting in mice. We propose that XMRV might not be a genuine human pathogen.

    Funded by: Medical Research Council: G0801172, G0801172(87743), G9721629; Wellcome Trust: 090940, WT076608, WT090940

    Retrovirology 2010;7;1;111

  • Rapid and highly sensitive neuraminidase subtyping of avian influenza viruses by use of a diagnostic DNA microarray.

    Gall A, Hoffmann B, Harder T, Grund C, Ehricht R and Beer M

    Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany.

    Rapid neuraminidase subtyping of avian influenza viruses from diagnostic samples is crucial considering the existence of permanently emerging and evolving strains. Here we report an easy-to-use, low-cost microarray for neuraminidase subtyping following fragment amplification by a generic, neuraminidase-specific reverse transcription-PCR (RT-PCR). This method enables highly specific characterization with a sensitivity equal to that of matrix gene-specific real-time RT-PCR.

    Journal of clinical microbiology 2009;47;9;2985-8

  • Design and validation of a microarray for detection, hemagglutinin subtyping, and pathotyping of avian influenza viruses.

    Gall A, Hoffmann B, Harder T, Grund C, Höper D and Beer M

    Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.

    Continuing threats of devastating outbreaks in poultry and of human infections caused by highly pathogenic avian influenza virus (HPAIV) H5N1 emphasize the need for the further development of rapid and reliable methods of virus detection and characterization. Here we report on the design and comprehensive validation of a low-density microarray as a diagnostic tool for the detection and typing of avian influenza virus (AIV). The array consists of one probe for the conserved matrix gene and 97 probes targeting the HA(0) cleavage-site region. Following fragment amplification by a generic PCR approach, the array enables AIV detection, hemagglutinin (HA) subtyping, and pathotyping within a single assay. For validation, a panel of 92 influenza A viruses which included 43 reference strains representing all 16 HA subtypes was used. All reference strains were correctly typed with respect to their HA subtypes and pathotypes, including HPAIV H5N1/Asia, which caused outbreaks in Germany in 2006 and 2007. In addition, differentiation of strains of the Eurasian and North American lineages of the H5 and H7 subtypes was possible. The sensitivity of the microarray for the matrix gene is comparable to that of real-time reverse transcription-PCR (RT-PCR). It is, however, 10- to 100-fold lower than that of real-time RT-PCR with respect to HA subtyping and pathotyping. The specificity of the array was excellent, as no pathogens relevant for differential diagnosis yielded a positive reaction. Validation with field samples included 19 cloacal swab specimens from wild and domestic birds. Influenza A virus was verified in all samples, whereas the HA subtypes could be determined for 14 samples. The results demonstrate that the microarray assay described complements current methods and can accelerate the diagnosis and characterization of AIV.

    Journal of clinical microbiology 2009;47;2;327-34

  • Universal primer set for amplification and sequencing of HA0 cleavage sites of all influenza A viruses.

    Gall A, Hoffmann B, Harder T, Grund C and Beer M

    Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany.

    Sequence analysis of the endoproteolytic cleavage site within the hemagglutinin (HA) precursor protein HA(0) is fundamental for studies of the molecular biology of influenza A viruses, in particular, for molecular pathotyping of subtype H5 and H7 isolates. A current problem for routine diagnostics is the emergence of new strains of the H5 or H7 subtype or even other subtypes which escape detection by commonly used reverse transcription-PCR (RT-PCR) protocols. Here, the first pan-HA (PanHA) RT-PCR assay targeting the HA(0) cleavage site of influenza A viruses of all 16 HA subtypes is reported. The assay was assessed in comparison to H5 and H7 subtype-specific RT-PCRs for the HA(0) cleavage site and a real-time RT-PCR detecting the M gene. A panel of 92 influenza A viruses was used for validation. Sequence data for influenza A viruses from 32 allantoic fluid samples and 11 diagnostic swab samples of all 16 HA subtypes were generated by direct sequencing of the PanHA RT-PCR products. The results demonstrate that the new PanHA RT-PCR assay--followed by cycle sequencing--can complement existing methods and strengthen the reliability of influenza A virus diagnostics, allowing both molecular pathotyping (H5 and H7) and subtyping (non-H5 or -H7) within a single approach.

    Journal of clinical microbiology 2008;46;8;2561-7

  • Persistence of viral RNA in rabbits which overcome an experimental RHDV infection detected by a highly sensitive multiplex real-time RT-PCR.

    Gall A, Hoffmann B, Teifke JP, Lange B and Schirrmeier H

    Friedrich-Loeffler-Institut, Institute of Diagnostic Virology, Boddenblick 5a, 17493 Greifswald-Insel Riems, Germany.

    An internally controlled multiplex real-time RT-PCR using TaqMan probes and external standards for absolute RNA quantification was developed as a new diagnostic tool for the detection of rabbit haemorrhagic disease virus (RHDV). The test revealed a specificity of 100%, an analytical sensitivity of 10 copies/well and a linearity over a range from 10(1) to 10(10) copies. The viral loads in organs, leukocytes, sera and excretions of seropositive, convalescent rabbits which were overcoming an experimental infection with RHDV were determined using the validated assay. As a result, viral RNA was demonstrated and quantified for at least 15 weeks. Thus, a persistence of viral RNA after experimental infection of rabbits could be shown for the first time. In contrast, neither antigen nor infectious virus could be detected by antigen-ELISA, immunohistochemistry or experimental transmission. Therefore, further experiments are necessary to prove that the persistence of RNA is linked with the persistence of infectious virus particles.

    Veterinary microbiology 2007;120;1-2;17-32

Saad Idris

- Wellcome Trust Clinical PhD Fellow

I graduated with a degree in Medicine and Surgery (MBBChir) with distinction from Cambridge University in 2006. I subsequently undertook specialist training as a clinical haematologist at Addenbrookes Hospital, Cambridge while gaining membership of the Royal College of Physicians (MRCP) and Fellowship of the Royal College of Pathologists (FRCPath). My specialist clinical interest lie in the treatment of lymphoproliferative disorders and haematopoeitic stem cell transplantation.

Research

I am currently undertaking my PhD jointly under the supervision of Prof Kellam at the Sanger Institute and Dr Okkenhaug at the Babraham institute. My research at the Sanger Institute is focusing on examining the immune repertoires in patients with lymphoproliferative disorders with the specific aim of using B cell receptor sequencing as a method of minimal residual disease monitoring in patients with aggressive B cell lymphoma. My work at the Babraham Institute is examining the role of the Phosphoinositide-3-kinase pathway in the development of normal and malignant B cells.

References

  • Micafungin for the treatment of invasive aspergillosis.

    Enoch DA, Idris SF, Aliyu SH, Micallef C, Sule O and Karas JA

    Clinical Microbiology & Public Health Laboratory, Public Health England, Addenbrookes Hospital, Cambridge CB2 2QW, UK. Electronic address: david.enoch@addenbrookes.nhs.uk.

    Invasive aspergillosis is a major cause of morbidity and mortality in immunocompromised patients, particularly those with neutropenia and those undergoing bone marrow or stem cell transplants. Micafungin is an echinocandin antifungal drug with activity against all major Candida spp. Currently, micafungin is indicated for treatment of invasive candidiasis, oesophageal candidiasis and prophylaxis of Candida infection in patients undergoing allogeneic haematopoietic stem cell transplantation or patients who are expected to have neutropenia. Micafungin demonstrates in vitro and in vivo activity against Aspergillus spp. It is currently not licensed to treat Aspergillus infections in the UK or USA. This review summarises the current evidence base surrounding the clinical use of micafungin in the treatment of invasive aspergillosis to consider the potential role of micafungin in these patients. There are currently no randomised studies comparing micafungin with standard antifungal therapy. Prospective non-randomised clinical studies, predominantly performed in Japan, involving 492 patients with aspergillosis and 455 febrile patients with chemotherapy-induced neutropenia suggest that micafungin may be as effective as comparator antifungal agents. Other clinical evidence is limited to case reports. Further experience in the form of randomised controlled trials is required to establish the exact role of micafungin in the context of currently available broad-spectrum antifungal agents.

    The Journal of infection 2014;68;6;507-26

  • The role of high-throughput technologies in clinical cancer genomics.

    Idris SF, Ahmad SS, Scott MA, Vassiliou GS and Hadfield J

    Department of Hematology/Oncology, Cambridge University NHS Hospitals Foundation Trust, Cambridge, CB2 0QQ, UK.

    Cancer is a genetic disease driven by both heritable and somatic alterations in DNA, which underpin not only oncogenesis but also progression and eventual metastasis. The major impetus for elucidating the nature and function of somatic mutations in cancer genomes is the potential for the development of effective targeted anticancer therapies. Over the last decade, high-throughput technologies have allowed us unprecedented access to a host of cancer genomes, leading to an influx of new information about their pathobiology. The challenge now is to integrate such emerging information into clinical practice to achieve tangible benefits for cancer patients. This review examines the roles array-based comparative genomic hybridization and next-generation sequencing are playing in furthering our understanding of both hematological and solid-organ tumors. Furthermore, the authors discuss the current challenges in translating the role of these technologies from bench to bedside.

    Funded by: Wellcome Trust: 095663

    Expert review of molecular diagnostics 2013;13;2;167-81

  • A simple screening tool allowing a zero tolerance approach to blood transfusion requests with incomplete special requirement information following a series of incidents.

    Billen A, Idris S, Pooley M, Lewin MJ and Besser MW

    Transfusion and apheresis science : official journal of the World Apheresis Association : official journal of the European Society for Haemapheresis 2012;46;3;347-8

  • Primary testicular lymphoma.

    Ahmad SS, Idris SF, Follows GA and Williams MV

    The Oncology Centre, Addenbrooke's Hospital, Cambridge, UK. Saif.ahmad@nhs.net

    Primary testicular non-Hodgkin lymphoma (PTL) comprises around 9% of testicular cancers and 1-2% of all non-Hodgkin lymphomas. Its incidence is increasing and it primarily affects older men, with a median age at presentation of around 67 years. By far the most common histological subtype is diffuse large B-cell lymphoma, accounting for 80-90% of PTLs. Most patients present with a unilateral testicular mass or swelling. Up to 90% of patients have stage I or II disease at diagnosis (60 and 30%, respectively) and bilateral testicular involvement is seen in around 35% of patients. PTL demonstrates a continuous pattern of relapse and propensity for extra-nodal sites such as the central nervous system and contralateral testis. Retrospective data have emphasised the importance of prophylactic radiotherapy in reducing recurrence rates within the contralateral testis. Recent outcome data from the prospective IELSG-10 trial have shown far better progression-free and overall survival than historical outcomes. This supports the use of orchidectomy followed by Rituximab- cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP), central nervous system prophylaxis and prophylactic radiotherapy to the contralateral testis with or without nodal radiotherapy in patients with limited disease. Central nervous system relapse remains a significant issue and future research should focus on identifying the best strategy to reduce its occurrence. Here we discuss the evidence supporting combination chemotherapy and radiotherapy in PTL.

    Clinical oncology (Royal College of Radiologists (Great Britain)) 2012;24;5;358-65

  • Cystic fibrosis neutrophils have normal intrinsic reactive oxygen species generation.

    McKeon DJ, Cadwallader KA, Idris S, Cowburn AS, Pasteur MC, Barker H, Haworth CS, Bilton D, Chilvers ER and Condliffe AM

    Respiratory Medicine Division, Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge, UK.

    Previous studies have identified abnormalities in the oxidative responses of the neutrophil in cystic fibrosis (CF), but it is unclear whether such changes relate to loss of membrane cystic fibrosis transmembrane conductance regulator (CFTR) or to the inflammatory environment present in this disease. The aim of the present study was to determine whether neutrophils from CF patients demonstrate an intrinsic abnormality of the respiratory burst. The respiratory burst activity of neutrophils isolated from stable DeltaF508 homozygote CF patients and matched healthy controls was quantified by both chemiluminscence and cytochrome C reduction. Expression of NADPH oxidase components and CFTR was determined by Western blotting and RT-PCR. The oxidative output from neutrophils from CF in response to receptor-linked and particulate stimuli did not differ from that of controls. Expression of NADPH oxidase components was identical in CF and non-CF neutrophils. While low levels of CFTR mRNA could be identified in the normal human neutrophil, we were unable to detect CFTR protein in human neutrophil lysates or immunoprecipitates. CFTR has no role in controlling neutrophil oxidative activity; previously reported differences in neutrophil function between CF and non-CF subjects most likely relate to the inflammatory milieu from which the cells were isolated.

    The European respiratory journal 2010;35;6;1264-72

  • Cholangiocarcinoma presenting as hemobilia and recurrent iron-deficiency anemia: a case report.

    Ahmad SS, Basheer FT, Idris SF, Hariraj R, Mathialagan R and Douds A

    Trent Cardiac Centre, Nottingham University Hospitals NHS Trust, NG5 1PB, UK. Saif83@hotmail.co.uk.

    Introduction: Iron-deficiency anemia is a relatively common presenting feature of several gastrointestinal malignancies. However, cholangiocarcinoma has rarely been reported as an underlying cause. The association of cholangiocarcinoma with the rare clinical finding of hemobilia is also highly unusual. To our knowledge, this is the first case report of cholangiocarcinoma presenting with acute hemobilia and chronic iron-deficiency anemia.

    We report the case of a Caucasian, 84-year-old woman presenting with recurrent, severe iron-deficiency anemia who was eventually diagnosed with intra-hepatic cholangiocarcinoma, following an acute episode of hemobilia. A right hepatectomy was subsequently performed with curative intent, and our patient has now fully recovered.

    Conclusion: This is a rare example of hemobilia and chronic iron-deficiency anemia in association with cholangiocarcinoma. We suggest that a diagnosis of cholangiocarcinoma should be considered in patients who present with iron-deficiency anemia of unknown cause, particularly in the presence of abnormal liver function.

    Journal of medical case reports 2010;4;133

  • Azithromycin therapy for neutrophilic airways disease: myth or magic?

    Idris SF, Chilvers ER, Haworth C, McKeon D and Condliffe AM

    Funded by: Medical Research Council; Wellcome Trust

    Thorax 2009;64;3;186-9

Pinky Langat

- PhD Student

I am currently in my second year of the Wellcome Trust Sanger Institute 4-year PhD programme. I graduated from McGill University in 2012 with a BSc Honours in Biochemistry and Minor in International Relations. During my undergraduate studies, I worked in a range of research areas focusing on the host genetics of cerebral malaria in the laboratory of Dr. Philippe Gros, the biophysical properties of photoswitchable fluorescent proteins in Dr. Amy S. Blum's group, and the ethics of sharing research data in public health emergencies with Dr. Ross Upshur at the University of Toronto.

Research

I work jointly between the Virus Genomics and Microbial Pathogenesis groups lead by Professors Paul Kellam and Gordon Dougan at the WTSI. Studying both influenza B virus and Salmonella Typhi bacterium, my PhD research aims to gain an understanding of i) the relationship between the molecular evolution and antigenic variation of pathogens and ii) the dynamics of host adaptive immune response to pathogen variants through antigen-specific immune repertoire analysis.

References

  • Susceptibility to lethal cerebral malaria is regulated by epistatic interaction between chromosome 4 (Berr6) and chromosome 1 (Berr7) loci in mice.

    Torre S, van Bruggen R, Kennedy JM, Berghout J, Bongfen SE, Langat P, Lathrop M, Vidal SM and Gros P

    Department of Human Genetics, McGill University, Montreal, Quebec, Canada.

    In humans, cerebral malaria is a rare but often lethal complication of infection with Plasmodium parasites, the occurrence of which is influenced by complex genetic factors of the host. We used a mouse model of experimental cerebral malaria (ECM) with Plasmodium berghei ANKA to study genetic factors regulating appearance of neurological symptoms and associated lethality. In a genome-wide screen of N-ethyl-N-nitrosourea-mutagenized mice derived from C57BL/6J (B6) and 129S1/SvImJ (129) mouse strains, we detected a strong interaction between the genetic backgrounds of these strains, which modulates ECM resistance. We have mapped a major gene locus to central chromosome 4 (log of the odds (LOD) 6.7; 79.6-97.3 Mb), which we designate Berr8. [corrected]. B6 alleles at Berr6 are associated with resistance, and are inherited in a co-dominant fashion. In mice heterozygous for Berr6 B6/129 alleles, resistance to ECM is strongly modulated by a second locus, Berr7, that maps to the proximal portion of chromosome 1 (LOD 4.03; 41.4 Mb). 129 alleles at Berr7 are associated with ECM resistance in a dosage-dependent fashion. Results are discussed in view of the possible role of this two-locus system in susceptibility to unrelated inflammatory conditions in mice and humans.

    Funded by: Canadian Institutes of Health Research

    Genes and immunity 2013;14;4;249-57

  • Conductance switching in the photoswitchable protein Dronpa.

    Korpany KV, Langat P, Kim DM, Edelman N, Cooper DR, Nadeau J and Blum AS

    Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, QC H3A 0B8, Canada.

    Dronpa, a photoswitchable GFP-like protein, was self-assembled onto gold substrates, and its conductance was measured using scanning tunneling microscopy (STM) and scanning tunneling spectroscopy (STS).

    Journal of the American Chemical Society 2012;134;39;16119-22

Swee Hoe Ong

- Senior Computational Biologist

I obtained my BSc in cell and molecular biology from the National University of Malaysia, followed by my MSc in bioinformatics from the National University of Singapore, and subsequently my PhD in molecular epidemiology from Universität Basel in Switzerland. I worked as a postdoctoral fellow at the Genome Institute of Singapore before joining the Sanger Institute in late 2013.

Research

My role in the Virus Genomics group at the Sanger Institute is to make sense out of virus sequence data, i.e. to understand the genomic diversity, molecular evolution, evolution dynamics and molecular epidemiology of important viral infectious agents such as HIV, HCV, HBV, MERS-CoV et cetera.

References

  • Spread, circulation, and evolution of the Middle East respiratory syndrome coronavirus.

    Cotten M, Watson SJ, Zumla AI, Makhdoom HQ, Palser AL, Ong SH, Al Rabeeah AA, Alhakeem RF, Assiri A, Al-Tawfiq JA, Albarrak A, Barry M, Shibl A, Alrabiah FA, Hajjar S, Balkhy HH, Flemban H, Rambaut A, Kellam P and Memish ZA

    Unlabelled: The Middle East respiratory syndrome coronavirus (MERS-CoV) was first documented in the Kingdom of Saudi Arabia (KSA) in 2012 and, to date, has been identified in 180 cases with 43% mortality. In this study, we have determined the MERS-CoV evolutionary rate, documented genetic variants of the virus and their distribution throughout the Arabian peninsula, and identified the genome positions under positive selection, important features for monitoring adaptation of MERS-CoV to human transmission and for identifying the source of infections. Respiratory samples from confirmed KSA MERS cases from May to September 2013 were subjected to whole-genome deep sequencing, and 32 complete or partial sequences (20 were ≥ 99% complete, 7 were 50 to 94% complete, and 5 were 27 to 50% complete) were obtained, bringing the total available MERS-CoV genomic sequences to 65. An evolutionary rate of 1.12 × 10(-3) substitutions per site per year (95% credible interval [95% CI], 8.76 × 10(-4); 1.37 × 10(-3)) was estimated, bringing the time to most recent common ancestor to March 2012 (95% CI, December 2011; June 2012). Only one MERS-CoV codon, spike 1020, located in a domain required for cell entry, is under strong positive selection. Four KSA MERS-CoV phylogenetic clades were found, with 3 clades apparently no longer contributing to current cases. The size of the population infected with MERS-CoV showed a gradual increase to June 2013, followed by a decline, possibly due to increased surveillance and infection control measures combined with a basic reproduction number (R0) for the virus that is less than 1.

    Importance: MERS-CoV adaptation toward higher rates of sustained human-to-human transmission appears not to have occurred yet. While MERS-CoV transmission currently appears weak, careful monitoring of changes in MERS-CoV genomes and of the MERS epidemic should be maintained. The observation of phylogenetically related MERS-CoV in geographically diverse locations must be taken into account in efforts to identify the animal source and transmission of the virus.

    Funded by: Wellcome Trust

    mBio 2014;5;1

  • Species identification and profiling of complex microbial communities using shotgun Illumina sequencing of 16S rRNA amplicon sequences.

    Ong SH, Kukkillaya VU, Wilm A, Lay C, Ho EX, Low L, Hibberd ML and Nagarajan N

    Genome Institute of Singapore, Genome #02-01, Singapore, Singapore.

    The high throughput and cost-effectiveness afforded by short-read sequencing technologies, in principle, enable researchers to perform 16S rRNA profiling of complex microbial communities at unprecedented depth and resolution. Existing Illumina sequencing protocols are, however, limited by the fraction of the 16S rRNA gene that is interrogated and therefore limit the resolution and quality of the profiling. To address this, we present the design of a novel protocol for shotgun Illumina sequencing of the bacterial 16S rRNA gene, optimized to amplify more than 90% of sequences in the Greengenes database and with the ability to distinguish nearly twice as many species-level OTUs compared to existing protocols. Using several in silico and experimental datasets, we demonstrate that despite the presence of multiple variable and conserved regions, the resulting shotgun sequences can be used to accurately quantify the constituents of complex microbial communities. The reconstruction of a significant fraction of the 16S rRNA gene also enabled high precision (>90%) in species-level identification thereby opening up potential application of this approach for clinical microbial characterization.

    PloS one 2013;8;4;e60811

  • Plasmablasts generated during repeated dengue infection are virus glycoprotein-specific and bind to multiple virus serotypes.

    Xu M, Hadinoto V, Appanna R, Joensson K, Toh YX, Balakrishnan T, Ong SH, Warter L, Leo YS, Wang CI and Fink K

    Singapore Immunology Network, Agency for Science, Technology and Research, 138648 Singapore.

    Dengue virus immune protection is specific to the serotype encountered and is thought to persist throughout one's lifetime. Many serotype cross-reactive memory B cells isolated from humans with previous dengue infection are specific for the nonstructural and the prM structural viral proteins, and they can enhance infection in vitro. However, plasmablasts circulating in enormous numbers during acute secondary infection have not been studied. In this study, we analyzed single plasmablasts from two patients by sorting the cells for Ig sequence analysis and for recombinant expression of Abs. In contrast to memory B cells, most plasmablast-derived Abs bound to the structural E protein of dengue, and protection experiments in mice revealed that virus serotypes encountered during past infections were neutralized more efficiently than were the serotypes of the current infection. Together with genetic analyses, we show evidence that plasmablasts in dengue patients are a polyclonal pool of activated E protein-specific memory B cells and that their specificity is not representative of the serum Abs secreted by long-lived plasma cells in the memory phase. These results contribute to the understanding of the phenomenon of original antigenic sin in dengue.

    Journal of immunology (Baltimore, Md. : 1950) 2012;189;12;5877-85

  • LoFreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from high-throughput sequencing datasets.

    Wilm A, Aw PP, Bertrand D, Yeo GH, Ong SH, Wong CH, Khor CC, Petric R, Hibberd ML and Nagarajan N

    Genome Institute of Singapore, 60 Biopolis Street, Genome, #02-01, Singapore 138672, Singapore.

    The study of cell-population heterogeneity in a range of biological systems, from viruses to bacterial isolates to tumor samples, has been transformed by recent advances in sequencing throughput. While the high-coverage afforded can be used, in principle, to identify very rare variants in a population, existing ad hoc approaches frequently fail to distinguish true variants from sequencing errors. We report a method (LoFreq) that models sequencing run-specific error rates to accurately call variants occurring in <0.05% of a population. Using simulated and real datasets (viral, bacterial and human), we show that LoFreq has near-perfect specificity, with significantly improved sensitivity compared with existing methods and can efficiently analyze deep Illumina sequencing datasets without resorting to approximations or heuristics. We also present experimental validation for LoFreq on two different platforms (Fluidigm and Sequenom) and its application to call rare somatic variants from exome sequencing datasets for gastric cancer. Source code and executables for LoFreq are freely available at http://sourceforge.net/projects/lofreq/.

    Nucleic acids research 2012;40;22;11189-201

  • A method for full genome sequencing of all four serotypes of the dengue virus.

    Christenbury JG, Aw PP, Ong SH, Schreiber MJ, Chow A, Gubler DJ, Vasudevan SG, Ooi EE and Hibberd ML

    Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, 8 College Rd., Singapore 169857, Singapore.

    The availability of whole genome sequencing has contributed to many aspects of dengue research, and its use in dengue virus (DENV) surveillance for early epidemic warning has been proposed. Methods to sequence the genomes of individual dengue serotypes have been described previously, but no single method is known to be applicable for all four serotypes. This report describes a method for sequencing the entire genome of all four DENV serotypes. Using tagged oligonucleotide primers designed for the 3' end, viral RNA was reverse transcribed into a cDNA spanning the entire genome of each of the four serotypes (DENV-1 to -4). This was followed by amplification of the entire cDNA in five overlapping amplicons. A sequence tag was added to the sense primer annealing to the 5' UTR sequence and the antisense primer annealing to the 3' UTR sequence to ensure no terminal nucleotides were omitted during PCR. Sixty-one virus isolates were sequenced: 58 DENV-2, one DENV-1, one DENV-4 and one DENV-3 published previously. The method described could be applied readily for viral biology studies and incorporated into proactive dengue virologic surveillance.

    Journal of virological methods 2010;169;1;202-6

  • Genomic epidemiology of a dengue virus epidemic in urban Singapore.

    Schreiber MJ, Holmes EC, Ong SH, Soh HS, Liu W, Tanner L, Aw PP, Tan HC, Ng LC, Leo YS, Low JG, Ong A, Ooi EE, Vasudevan SG and Hibberd ML

    Novartis Institute for Tropical Diseases, Chromos, Singapore. mark.schreiber@novartis.com

    Dengue is one of the most important emerging diseases of humans, with no preventative vaccines or antiviral cures available at present. Although one-third of the world's population live at risk of infection, little is known about the pattern and dynamics of dengue virus (DENV) within outbreak situations. By exploiting genomic data from an intensively studied major outbreak, we are able to describe the molecular epidemiology of DENV at a uniquely fine-scaled temporal and spatial resolution. Two DENV serotypes (DENV-1 and DENV-3), and multiple component genotypes, spread concurrently and with similar epidemiological and evolutionary profiles during the initial outbreak phase of a major dengue epidemic that took place in Singapore during 2005. Although DENV-1 and DENV-3 differed in viremia and clinical outcome, there was no evidence for adaptive evolution before, during, or after the outbreak, indicating that ecological or immunological rather than virological factors were the key determinants of epidemic dynamics.

    Funded by: NIGMS NIH HHS: GM080533-01

    Journal of virology 2009;83;9;4163-73

  • Periodic re-emergence of endemic strains with strong epidemic potential-a proposed explanation for the 2004 Indonesian dengue epidemic.

    Ong SH, Yip JT, Chen YL, Liu W, Harun S, Lystiyaningsih E, Heriyanto B, Beckett CG, Mitchell WP, Hibberd ML, Suwandono A, Vasudevan SG and Schreiber MJ

    Novartis Institute for Tropical Diseases Pte. Ltd., Singapore, Singapore.

    Indonesia experienced a severe dengue epidemic in the first quarter of 2004 with 58,301 cases and 658 deaths reported to the WHO. All four dengue virus (DENV) serotypes were detected, with DENV-3 the predominant strain. To ascertain the molecular epidemiology of the DENV associated with the epidemic, complete genomes of 15 isolates were sequenced from patient serum collected in Jakarta during the epidemic, and two historical DENV-3 isolates from previous epidemics in 1988 and 1998 were selectively sequenced for comparative studies. Phylogenetic trees for all four serotypes indicate the viruses are endemic strains that have been circulating in Indonesia for a few decades. Whole-genome phylogeny showed the 2004 DENV-3 isolates share high similarity with those isolated in 1998 during a major epidemic in Sumatra. Together these subtype I DENV-3 strains form a Sumatran-Javan clade with demonstrated epidemic potential. No newly-acquired amino acid mutations were found while comparing genomes from the two epidemics. This suggests re-emergence of little-changed endemic strains as causative agents of the epidemic in 2004. Notably, the molecular evidence rules out change in the viral genomes as the trigger of the epidemic.

    Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 2008;8;2;191-204

  • IDBD: infectious disease biomarker database.

    Yang IS, Ryu C, Cho KJ, Kim JK, Ong SH, Mitchell WP, Kim BS, Oh HB and Kim KH

    Department of Life Sciences & Biotechnology, School of Life Sciences & Biotechnology, Korea University, Seoul, Korea.

    Biomarkers enable early diagnosis, guide molecularly targeted therapy and monitor the activity and therapeutic responses across a variety of diseases. Despite intensified interest and research, however, the overall rate of development of novel biomarkers has been falling. Moreover, no solution is yet available that efficiently retrieves and processes biomarker information pertaining to infectious diseases. Infectious Disease Biomarker Database (IDBD) is one of the first efforts to build an easily accessible and comprehensive literature-derived database covering known infectious disease biomarkers. IDBD is a community annotation database, utilizing collaborative Web 2.0 features, providing a convenient user interface to input and revise data online. It allows users to link infectious diseases or pathogens to protein, gene or carbohydrate biomarkers through the use of search tools. It supports various types of data searches and application tools to analyze sequence and structure features of potential and validated biomarkers. Currently, IDBD integrates 611 biomarkers for 66 infectious diseases and 70 pathogens. It is publicly accessible at http://biomarker.cdc.go.kr and http://biomarker.korea.ac.kr.

    Nucleic acids research 2008;36;Database issue;D455-60

  • DengueInfo: A web portal to dengue information resources.

    Schreiber MJ, Ong SH, Holland RC, Hibberd ML, Vasudevan SG, Mitchell WP and Holmes EC

    Novartis Institute for Tropical Diseases, Chromos #05-01, 10 Biopolis Road, Singapore 138670, Singapore. mark.schreiber@novartis.com

    DengueInfo (http://www.dengueinfo.org) is a web portal and database that brings clarity to dengue research by integrating the growing number of complete genome sequences of dengue virus with relevant literature and curated epidemiological information. Additionally, it represents a repository of on-going prospective and retrospective studies of dengue disease severity. We intend the database to be a flagship resource for the dengue community, providing standardized and high quality information and facilitating research into key aspects of dengue biology and assisting in its control. To aid this process we also introduce a standard nomenclature for dengue isolates inspired by globally accepted system used for influenza virus.

    Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 2007;7;4;540-1

  • A genomics approach to understanding host response during dengue infection.

    Hibberd ML, Ling L, Tolfvenstam T, Mitchell W, Wong C, Kuznetsov VA, George J, Ong SH, Ruan Y, Wei CL, Gu F, Fink J, Yip A, Liu W, Schreiber M and Vasudevan SG

    Genome Institute of Singapore, Biopolis, Singapore 138670.

    Dengue infection results in a wide clinical spectrum, ranging from asymptomatic, through fever (DF), to the life threatening complications haemorrhagic fever (DHF) and shock syndrome (DSS). Although we now understand that factors such as repeat infections and the type or magnitude of the host response are important in determining severity, the mechanisms of these actions remain largely unknown. Understanding this host-pathogen interaction may enable outcome prediction and new therapy options. Developments in biology now allow a 'systems approach' to be applied to this problem, utilizing whole genomes of both human and virus, in vitro and in vivo to enable a more complete picture of their interplay to be built up. We have developed a chip-based approach to viral sequencing, to increase efficiency and enable large numbers ofgenomes to be completed, together with a web-based interpretation tool. We have also applied human whole genome expression arrays (24000 genes) to characterize the types of host response made to infection and plan to investigate the role of host variation using human whole genome genetic association studies in the future. These technologies have identified novel host pathways involved in viral replication in vitro, and also host immune responses, such as the interferon signalling pathway, that are influenced by viral sequence. This data will be further refined through interlinking with similar data obtained from a large study of dengue patients, initiated in Singapore, that is able to look at the early host response to infection.

    Novartis Foundation symposium 2006;277;206-14; discussion 214-7, 251-3

Anne Palser

ap10@sanger.ac.uk Staff Scientist

I studied Biomedical Science at the University of Manchester and obtained a PhD in Neuroscience from Imperial College London studying the cell biology of multiple sclerosis. I worked at University College London as a postdoc and then moved to the Sanger Institute in 2009. I am currently a Staff Scientist in the Virus Genomics team.

Research

I lead the groups research on herpesviruses, focussing mainly on sequencing Epstein-Barr virus (EBV) and Kaposi's Sarcoma associated herpesvirus (KSHV), which are associated with several human tumours. We have developed large scale deep sequencing approaches to investigate worldwide genetic diversity in the virus and are also researching host factors that are involved in virus pathogenesis and tumour formation.

References

  • Spread, circulation, and evolution of the Middle East respiratory syndrome coronavirus.

    Cotten M, Watson SJ, Zumla AI, Makhdoom HQ, Palser AL, Ong SH, Al Rabeeah AA, Alhakeem RF, Assiri A, Al-Tawfiq JA, Albarrak A, Barry M, Shibl A, Alrabiah FA, Hajjar S, Balkhy HH, Flemban H, Rambaut A, Kellam P and Memish ZA

    Unlabelled: The Middle East respiratory syndrome coronavirus (MERS-CoV) was first documented in the Kingdom of Saudi Arabia (KSA) in 2012 and, to date, has been identified in 180 cases with 43% mortality. In this study, we have determined the MERS-CoV evolutionary rate, documented genetic variants of the virus and their distribution throughout the Arabian peninsula, and identified the genome positions under positive selection, important features for monitoring adaptation of MERS-CoV to human transmission and for identifying the source of infections. Respiratory samples from confirmed KSA MERS cases from May to September 2013 were subjected to whole-genome deep sequencing, and 32 complete or partial sequences (20 were ≥ 99% complete, 7 were 50 to 94% complete, and 5 were 27 to 50% complete) were obtained, bringing the total available MERS-CoV genomic sequences to 65. An evolutionary rate of 1.12 × 10(-3) substitutions per site per year (95% credible interval [95% CI], 8.76 × 10(-4); 1.37 × 10(-3)) was estimated, bringing the time to most recent common ancestor to March 2012 (95% CI, December 2011; June 2012). Only one MERS-CoV codon, spike 1020, located in a domain required for cell entry, is under strong positive selection. Four KSA MERS-CoV phylogenetic clades were found, with 3 clades apparently no longer contributing to current cases. The size of the population infected with MERS-CoV showed a gradual increase to June 2013, followed by a decline, possibly due to increased surveillance and infection control measures combined with a basic reproduction number (R0) for the virus that is less than 1.

    Importance: MERS-CoV adaptation toward higher rates of sustained human-to-human transmission appears not to have occurred yet. While MERS-CoV transmission currently appears weak, careful monitoring of changes in MERS-CoV genomes and of the MERS epidemic should be maintained. The observation of phylogenetically related MERS-CoV in geographically diverse locations must be taken into account in efforts to identify the animal source and transmission of the virus.

    Funded by: Wellcome Trust

    mBio 2014;5;1

  • Transmission and evolution of the Middle East respiratory syndrome coronavirus in Saudi Arabia: a descriptive genomic study.

    Cotten M, Watson SJ, Kellam P, Al-Rabeeah AA, Makhdoom HQ, Assiri A, Al-Tawfiq JA, Alhakeem RF, Madani H, AlRabiah FA, Al Hajjar S, Al-nassir WN, Albarrak A, Flemban H, Balkhy HH, Alsubaie S, Palser AL, Gall A, Bashford-Rogers R, Rambaut A, Zumla AI and Memish ZA

    Wellcome Trust Sanger Institute, Hinxton, UK.

    Background: Since June, 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) has, worldwide, caused 104 infections in people including 49 deaths, with 82 cases and 41 deaths reported from Saudi Arabia. In addition to confirming diagnosis, we generated the MERS-CoV genomic sequences obtained directly from patient samples to provide important information on MERS-CoV transmission, evolution, and origin.

    Methods: Full genome deep sequencing was done on nucleic acid extracted directly from PCR-confirmed clinical samples. Viral genomes were obtained from 21 MERS cases of which 13 had 100%, four 85-95%, and four 30-50% genome coverage. Phylogenetic analysis of the 21 sequences, combined with nine published MERS-CoV genomes, was done.

    Findings: Three distinct MERS-CoV genotypes were identified in Riyadh. Phylogeographic analyses suggest the MERS-CoV zoonotic reservoir is geographically disperse. Selection analysis of the MERS-CoV genomes reveals the expected accumulation of genetic diversity including changes in the S protein. The genetic diversity in the Al-Hasa cluster suggests that the hospital outbreak might have had more than one virus introduction.

    Interpretation: We present the largest number of MERS-CoV genomes (21) described so far. MERS-CoV full genome sequences provide greater detail in tracking transmission. Multiple introductions of MERS-CoV are identified and suggest lower R0 values. Transmission within Saudi Arabia is consistent with either movement of an animal reservoir, animal products, or movement of infected people. Further definition of the exposures responsible for the sporadic introductions of MERS-CoV into human populations is urgently needed.

    Funding: Saudi Arabian Ministry of Health, Wellcome Trust, European Community, and National Institute of Health Research University College London Hospitals Biomedical Research Centre.

    Funded by: Wellcome Trust: 093724

    Lancet 2013;382;9909;1993-2002

  • Network properties derived from deep sequencing of human B-cell receptor repertoires delineate B-cell populations.

    Bashford-Rogers RJ, Palser AL, Huntly BJ, Rance R, Vassiliou GS, Follows GA and Kellam P

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom;

    The adaptive immune response selectively expands B- and T-cell clones following antigen recognition by B- and T-cell receptors (BCR and TCR), respectively. Next-generation sequencing is a powerful tool for dissecting the BCR and TCR populations at high resolution, but robust computational analyses are required to interpret such sequencing. Here, we develop a novel computational approach for BCR repertoire analysis using established next-generation sequencing methods coupled with network construction and population analysis. BCR sequences organize into networks based on sequence diversity, with differences in network connectivity clearly distinguishing between diverse repertoires of healthy individuals and clonally expanded repertoires from individuals with chronic lymphocytic leukemia (CLL) and other clonal blood disorders. Network population measures defined by the Gini Index and cluster sizes quantify the BCR clonality status and are robust to sampling and sequencing depths. BCR network analysis therefore allows the direct and quantifiable comparison of BCR repertoires between samples and intra-individual population changes between temporal or spatially separated samples and over the course of therapy.

    Funded by: Wellcome Trust: 079249, 095663, 100140

    Genome research 2013;23;11;1874-84

  • An elephantine viral problem.

    Gall A and Palser A

    This month's Genome Watch highlights how deep sequencing was used to generate the first full genomes of herpesviruses associated with a fatal disease in elephants.

    Nature reviews. Microbiology 2013;11;8;512

  • Full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus.

    Cotten M, Lam TT, Watson SJ, Palser AL, Petrova V, Grant P, Pybus OG, Rambaut A, Guan Y, Pillay D, Kellam P and Nastouli E

    Wellcome Trust Sanger Institute, Hinxton, UK.

    A novel betacoronavirus associated with lethal respiratory and renal complications was recently identified in patients from several countries in the Middle East. We report the deep genome sequencing of the virus directly from a patient's sputum sample. Our high-throughput sequencing yielded a substantial depth of genome sequence assembly and showed the minority viral variants in the specimen. Detailed phylogenetic analysis of the virus genome (England/Qatar/2012) revealed its close relationship to European bat coronaviruses circulating among the bat species of the Vespertilionidae family. Molecular clock analysis showed that the 2 human infections of this betacoronavirus in June 2012 (EMC/2012) and September 2012 (England/Qatar/2012) share a common virus ancestor most likely considerably before early 2012, suggesting the human diversity is the result of multiple zoonotic events.

    Funded by: Medical Research Council: MR/K006584/1; Wellcome Trust: 093724, 095831

    Emerging infectious diseases 2013;19;5;736-42B

  • Evolutionary dynamics of local pandemic H1N1/2009 influenza virus lineages revealed by whole-genome analysis.

    Baillie GJ, Galiano M, Agapow PM, Myers R, Chiam R, Gall A, Palser AL, Watson SJ, Hedge J, Underwood A, Platt S, McLean E, Pebody RG, Rambaut A, Green J, Daniels R, Pybus OG, Kellam P and Zambon M

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom.

    Virus gene sequencing and phylogenetics can be used to study the epidemiological dynamics of rapidly evolving viruses. With complete genome data, it becomes possible to identify and trace individual transmission chains of viruses such as influenza virus during the course of an epidemic. Here we sequenced 153 pandemic influenza H1N1/09 virus genomes from United Kingdom isolates from the first (127 isolates) and second (26 isolates) waves of the 2009 pandemic and used their sequences, dates of isolation, and geographical locations to infer the genetic epidemiology of the epidemic in the United Kingdom. We demonstrate that the epidemic in the United Kingdom was composed of many cocirculating lineages, among which at least 13 were exclusively or predominantly United Kingdom clusters. The estimated divergence times of two of the clusters predate the detection of pandemic H1N1/09 virus in the United Kingdom, suggesting that the pandemic H1N1/09 virus was already circulating in the United Kingdom before the first clinical case. Crucially, three clusters contain isolates from the second wave of infections in the United Kingdom, two of which represent chains of transmission that appear to have persisted within the United Kingdom between the first and second waves. This demonstrates that whole-genome analysis can track in fine detail the behavior of individual influenza virus lineages during the course of a single epidemic or pandemic.

    Funded by: Medical Research Council: MC_U117512723; Wellcome Trust: 095831

    Journal of virology 2012;86;1;11-8

  • Marked endotheliotropism of highly pathogenic avian influenza virus H5N1 following intestinal inoculation in cats.

    Reperant LA, van de Bildt MW, van Amerongen G, Leijten LM, Watson S, Palser A, Kellam P, Eissens AC, Frijlink HW, Osterhaus AD and Kuiken T

    Department of Ecology and Evolutionary Biology, Princeton University, Princeton, New Jersey, USA.

    Highly pathogenic avian influenza virus (HPAIV) H5N1 can infect mammals via the intestine; this is unusual since influenza viruses typically infect mammals via the respiratory tract. The dissemination of HPAIV H5N1 following intestinal entry and associated pathogenesis are largely unknown. To assess the route of spread of HPAIV H5N1 to other organs and to determine its associated pathogenesis, we inoculated infected chicken liver homogenate directly into the intestine of cats by use of enteric-coated capsules. Intestinal inoculation of HPAIV H5N1 resulted in fatal systemic disease. The spread of HPAIV H5N1 from the lumen of the intestine to other organs took place via the blood and lymphatic vascular systems but not via neuronal transmission. Remarkably, the systemic spread of the virus via the vascular system was associated with massive infection of endothelial and lymphendothelial cells, resulting in widespread hemorrhages. This is unique for influenza in mammals and resembles the pathogenesis of HPAIV infection in terrestrial poultry. It contrasts with the pathogenesis of systemic disease from the same virus following entry via the respiratory tract, where lesions are characterized mainly by necrosis and inflammation and are associated with the presence of influenza virus antigen in parenchymal, not endothelial cells. The marked endotheliotropism of the virus following intestinal inoculation indicates that the pathogenesis of systemic influenza virus infection in mammals may differ according to the portal of entry.

    Journal of virology 2012;86;2;1158-65

  • A viral discovery methodology for clinical biopsy samples utilising massively parallel next generation sequencing.

    Daly GM, Bexfield N, Heaney J, Stubbs S, Mayer AP, Palser A, Kellam P, Drou N, Caccamo M, Tiley L, Alexander GJ, Bernal W and Heeney JL

    Department of Veterinary Medicine, The University of Cambridge, Cambridge, United Kingdom.

    Here we describe a virus discovery protocol for a range of different virus genera, that can be applied to biopsy-sized tissue samples. Our viral enrichment procedure, validated using canine and human liver samples, significantly improves viral read copy number and increases the length of viral contigs that can be generated by de novo assembly. This in turn enables the Illumina next generation sequencing (NGS) platform to be used as an effective tool for viral discovery from tissue samples.

    Funded by: Wellcome Trust

    PloS one 2011;6;12;e28879

  • Specific capture and whole-genome sequencing of viruses from clinical samples.

    Depledge DP, Palser AL, Watson SJ, Lai IY, Gray ER, Grant P, Kanda RK, Leproust E, Kellam P and Breuer J

    Division of Infection and Immunity, University College London, London, United Kingdom. d.depledge@ucl.ac.uk

    Whole genome sequencing of viruses directly from clinical samples is integral for understanding the genetics of host-virus interactions. Here, we report the use of sample sparing target enrichment (by hybridisation) for viral nucleic acid separation and deep-sequencing of herpesvirus genomes directly from a range of clinical samples including saliva, blood, virus vesicles, cerebrospinal fluid, and tumour cell lines. We demonstrate the effectiveness of the method by deep-sequencing 13 highly cell-associated human herpesvirus genomes and generating full length genome alignments at high read depth. Moreover, we show the specificity of the method enables the study of viral population structures and their diversity within a range of clinical samples types.

    Funded by: Department of Health; Medical Research Council: G07008, G0700814, G0900950; Wellcome Trust: 081703MA

    PloS one 2011;6;11;e27805

  • Neural cell adhesion molecule stimulates survival of premyelinating oligodendrocytes via the fibroblast growth factor receptor.

    Palser AL, Norman AL, Saffell JL and Reynolds R

    Department of Cellular and Molecular Neuroscience, Wolfson Neuroscience Laboratories, Imperial College London, Hammersmith Hospital Campus, London, United Kingdom.

    Axonal signals are critical in promoting the survival and maturation of oligodendrocytes during myelination, with contact-dependent signals thought to play a key role. However, the exact nature of these signals remains unclear. Neural cell adhesion molecule (NCAM) is expressed by both axons and oligodendrocytes and is ideally localized to transduce signals from the axon. This study sought to investigate the influence of NCAM on premyelinating oligodendrocytes in vitro. Both a soluble molecule comprising the extracellular domain of NCAM and a peptide derived from the fibroblast growth factor receptor (FGFR) binding motif within the first fibronectin domain stimulated a dose-dependent increase in survival of premyelinating oligodendrocytes in vitro. The survival effect was blocked by a mitogen-activated protein kinase (MAPK) inhibitor and an FGFR inhibitor, suggesting that activation of MAPK signalling pathways following interaction with the FGFR is involved in the survival effect of NCAM. Furthermore, NCAM presented in a cellular monolayer induced an increase in radial process outgrowth of oligodendrocyte progenitor cells. These data suggest that NCAM may play a role in axon-oligodendrocyte signalling during myelination, leading to an increase in oligodendrocyte survival and process outgrowth following axonal contact.

    Funded by: Medical Research Council

    Journal of neuroscience research 2009;87;15;3356-68

My Phan

- Postdoctoral Fellow

I graduated from the Flinders University of South Australia (Australia) with a Bachelor degree in Medical Science major in Biochemistry and Molecular Biology. Subsequently, I obtained a DPhil degree (University of Oxford, Oxford, United Kingdom), studying various aspects of acute childhood gastroenteritis in Ho Chi Minh City (Vietnam) with a focus on enteric viruses such as rotaviruses and noroviruses. In 2014, I joined the Virus Genomics group as a postdoctoral research fellow.

Research

I am currently involving in the VIZIONS (Vietnam Initiative on Zoonotic Infections) project, which aims at utilising the genome scale biology to understand the origin and dynamic evolution of infectious diseases in Vietnam. My research focuses on investigating enteric viruses, such as rotaviruses, to add insights into the viral genomic diversity in human and animals, and hence to understand the elements involved in the emergence of novel viral variants in Vietnam.

References

  • The prevalence and genetic diversity of group A rotaviruses on pig farms in the Mekong Delta region of Vietnam.

    Pham HA, Carrique-Mas JJ, Nguyen VC, Ngo TH, Nguyet LA, Do TD, Vo BH, Phan VT, Rabaa MA, Farrar J, Baker S and Bryant JE

    Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Viet Nam.

    Group A rotaviruses (ARoVs) are a common cause of severe diarrhea among children worldwide and the cause of approximately 45% of pediatric hospitalizations for acute diarrhea in Vietnam. ARoVs are known to cause significant economic losses to livestock producers by reducing growth performance and production efficiencies, however little is known about the implications of asymptomatic endemic circulation of ARoV. We aimed to determine the prevalence and predominant circulating genotypes of ARoVs on pig farms in a southern province of Vietnam. We found overall animal-level and farm-level prevalence of 32.7% (239/730) and 74% (77/104), respectively, and identified six different G types and 4 P types in various combinations (G2, G3, G4, G5, G9, G11 and P[6], P[13], P[23], and P[34]). There was no significant association between ARoV infection and clinical disease in pigs, suggesting that endemic asymptomatic circulation of ARoV may complicate rotavirus disease attribution during outbreaks of diarrhea in swine. Sequence analysis of the detected ARoVs suggested homology to recent human clinical cases and extensive genetic diversity. The epidemiological relevance of these findings for veterinary practitioners and to ongoing pediatric ARoV vaccine initiatives in Vietnam merits further study.

    Funded by: Wellcome Trust

    Veterinary microbiology 2014;170;3-4;258-65

  • Identification of possible virulence marker from Campylobacter jejuni isolates.

    Harrison JW, Dung TT, Siddiqui F, Korbrisate S, Bukhari H, Tra MP, Hoang NV, Carrique-Mas J, Bryant J, Campbell JI, Studholme DJ, Wren BW, Baker S, Titball RW and Champion OL

    A novel protein translocation system, the type-6 secretion system (T6SS), may play a role in virulence of Campylobacter jejuni. We investigated 181 C. jejuni isolates from humans, chickens, and environmental sources in Vietnam, Thailand, Pakistan, and the United Kingdom for T6SS. The marker was most prevalent in human and chicken isolates from Vietnam.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/1024631/1; Wellcome Trust: WT097835MF

    Emerging infectious diseases 2014;20;6;1026-9

  • The policy of free healthcare for children under the age of 6 years in Vietnam: assessment of the uptake for children hospitalised with acute diarrhoea in Ho Chi Minh City.

    Shieh M, Thompson C, Phan VT, Van TT, Tediosi F, Merson L, Farrar JJ, Ha MT, Ho LV, Pham TN and Baker S

    Novartis Vaccines Institute for Global Health, Siena, Italy.

    Objective: To assess the proportion of, and reasons for, households not utilising the policy of free healthcare for children under 6 years of age (FCCU6) for hospitalisation with diarrhoea, and assess the risk of catastrophic expenditure for households that forgo FCCU6 and pay out of pocket.

    Methods: Invoices detailing insurance information and charges incurred from 472 hospitalised diarrhoeal cases in one paediatric hospital in Ho Chi Minh City were retrieved. Hospital charges and the utilisation of elective services were analysed for patients utilising and not utilising FCCU6. Associations between socio-economic factors with non-utilisation of FCCU6 were evaluated.

    Results: Overall, 29% of patients were FCCU6 non-users. The FCCU6 non-users paid a median hospital charge of $29.13 (interquartile range, IQR: $18.57-46.24), consuming no more than 1.4% of a medium-income household's annual income. Seventy per cent of low-income FCCU6 non-users utilised less-expensive elective services, whereas only 43% of medium income patients and 21% of high-income patients did (P = 0.036). Patients from larger households and those with a parent working in government were more likely to use FCCU6.

    Conclusions: The rate of FCCU6 non-usage in this study population was 29%. A significant proportion of those that did not use FCCU6 was from lower income households and may perceive a justifiable cost-benefit ratio when forgoing FCCU6. Although a single diarrhoeal hospitalisation is unlikely to induce a catastrophic expenditure, FCCU6 non-usage may disproportionately increase the risk of catastrophic expenditure for lower income households over multiple illnesses.

    Funded by: Wellcome Trust: 093724, 100087

    Tropical medicine & international health : TM & IH 2013;18;12;1444-51

  • Tracking the establishment of local endemic populations of an emergent enteric pathogen.

    Holt KE, Thieu Nga TV, Thanh DP, Vinh H, Kim DW, Vu Tra MP, Campbell JI, Hoang NV, Vinh NT, Minh PV, Thuy CT, Nga TT, Thompson C, Dung TT, Nhu NT, Vinh PV, Tuyet PT, Phuc HL, Lien NT, Phu BD, Ai NT, Tien NM, Dong N, Parry CM, Hien TT, Farrar JJ, Parkhill J, Dougan G, Thomson NR and Baker S

    Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC 3010, Australia.

    Shigella sonnei is a human-adapted pathogen that is emerging globally as the dominant agent of bacterial dysentery. To investigate local establishment, we sequenced the genomes of 263 Vietnamese S. sonnei isolated over 15 y. Our data show that S. sonnei was introduced into Vietnam in the 1980s and has undergone localized clonal expansion, punctuated by genomic fixation events through periodic selective sweeps. We uncover geographical spread, spatially restricted frontier populations, and convergent evolution through local gene pool sampling. This work provides a unique, high-resolution insight into the microevolution of a pioneering human pathogen during its establishment in a new host population.

    Funded by: Wellcome Trust: 093724, 098051, 100087

    Proceedings of the National Academy of Sciences of the United States of America 2013;110;43;17522-7

  • The dynamics of GII.4 Norovirus in Ho Chi Minh City, Vietnam.

    Tra My PV, Lam HM, Thompson CN, Phuc HL, Tuyet PT, Vinh H, Hoang NV, Minh P, Vinh NT, Thuy CT, Nga TT, Hau NT, Chinh NT, Thuong TC, Tuan HM, Campbell JI, Clements AC, Farrar J, Boni MF and Baker S

    Wellcome Trust Major Overseas Programme, Oxford University Clinical Research Unit, 764 Vo Van Kiet, District 5, Ho Chi Minh City, Viet Nam.

    Norovirus (NoV) is a major cause of epidemic gastroenteritis in industrialized countries, yet the epidemiological significance of NoV in industrializing countries remains poorly understood. The spatiotemporal distribution of NoV genotypes identified in 2054 enrolled children was investigated between May 2009 and December 2010, in Ho Chi Minh City (HCMC), Vietnam. A total of 315 NoV extracted from stool samples were genotyped and GPS mapped to their source. Genogroup II NoV, particularly GII.4, were predominant, and the GII.4 strains could be subgrouped into GII.4-2006b (Minerva) and GII.4-2010 (New Orleans) variants. There was no spatiotemporal structure among the endemic GII strains; yet a significant spatiotemporal signal corresponding with the novel introduction of GII.4-2010 variant was detected. These data show that NoV GII.4 variants are highly endemic in HCMC and describe a scenario of rapid NoV strain replacement occurring in HCMC in early 2010.

    Funded by: Wellcome Trust: 089276, 093724, 100087

    Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 2013;18;335-43

  • Epidemiological features and risk factors of Salmonella gastroenteritis in children resident in Ho Chi Minh City, Vietnam.

    Thompson CN, Phan VT, Le TP, Pham TN, Hoang LP, Ha V, Nguyen VM, Pham VM, Nguyen TV, Cao TT, Tran TT, Nguyen TT, Dao MT, Campbell JI, Nguyen TC, Tang CT, Ha MT, Farrar J and Baker S

    Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, Oxford University, Oxford, UK.

    Non-typhoidal Salmonella are an important but poorly characterized cause of paediatric diarrhoea in developing countries. We conducted a hospital-based case-control study in children aged <5 years in Ho Chi Minh City to define the epidemiology and examine risk factors associated with Salmonella diarrhoeal infections. From 1419 diarrhoea cases and 571 controls enrolled between 2009 and 2010, 77 (5∙4%) diarrhoea cases were stool culture-positive for non-typhoidal Salmonella. Salmonella patients were more likely to be younger than controls (median age 10 and 12 months, respectively) [odds ratio (OR) 0∙97; 95% confidence interval (CI) 0∙94-0∙99], to report a recent diarrhoeal contact (8∙1% cases, 1∙8% controls; OR 5∙98, 95% CI 1∙8-20∙4) and to live in a household with >2 children (cases 20∙8%, controls 10∙2%; OR 2∙32, 95% CI 1∙2-4∙7). Our findings indicate that Salmonella are an important cause of paediatric gastroenteritis in this setting and we suggest that transmission may occur through direct human contact in the home.

    Funded by: Wellcome Trust: 089276, 093724

    Epidemiology and infection 2013;141;8;1604-13

  • The validation and utility of a quantitative one-step multiplex RT real-time PCR targeting rotavirus A and norovirus.

    Dung TT, Phat VV, Nga TV, My PV, Duy PT, Campbell JI, Thuy CT, Hoang NV, Van Minh P, Le Phuc H, Tuyet PT, Vinh H, Kien DT, Huy Hle A, Vinh NT, Nga TT, Hau NT, Chinh NT, Thuong TC, Tuan HM, Simmons C, Farrar JJ and Baker S

    The Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Oxford University Clinical Research Unit, Ho Chi Minh City, Viet Nam.

    Rotavirus (RoV) and Norovirus (NoV) are the main causes of viral gastroenteritis. Currently, there is no validated multiplex real-time PCR that can detect and quantify RoV and NoV simultaneously. The aim of the study was to develop, validate, and internally control a multiplex one-step RT real-time PCR to detect and quantify RoV and NoV in stool samples. PCR sensitivity was assessed by comparing amplification against the current gold standard, enzyme immunoassay (EIA), on stool samples from 94 individuals with diarrhea and 94 individuals without diarrhea. PCR detected 10% more RoV positive samples than EIA in stools samples from patients with diarrhea. PCR detected 23% more NoV genogroup II positive samples from individuals with diarrhea and 9% more from individuals without diarrhea than EIA, respectively. Genotyping of the PCR positive/EIA negative samples suggested the higher rate of PCR positivity, in comparison to EIA, was due to increased sensitivity, rather than nonspecific hybridization. Quantitation demonstrated that the viral loads of RoV and NoV in the stools of diarrheal patients were an order of magnitude greater than in individuals without diarrhea. This internally controlled real-time PCR method is robust, exhibits a high degree of reproducibility, and may have a greater utility and sensitivity than commercial EIA kits.

    Funded by: Wellcome Trust: 089276, 093724, 100087

    Journal of virological methods 2013;187;1;138-43

  • The emergence of rotavirus G12 and the prevalence of enteric viruses in hospitalized pediatric diarrheal patients in southern Vietnam.

    Tra My PV, Rabaa MA, Vinh H, Holmes EC, Hoang NV, Vinh NT, Phuong le T, Tham NT, Bay PV, Campbell JI, Farrar J and Baker S

    Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam. mypvt@oucru.org

    Diarrhea is a major cause of childhood morbidity and mortality in developing countries, and the majority of infections are of viral etiology. We aimed to compare the etiological prevalence of the major enteric viruses in an urban and a rural setting in southern Vietnam. We simultaneously screened fecal specimens from 362 children in Ho Chi Minh City and Dong Thap province that were hospitalized with acute diarrhea over a 1-month-long period for four viral gastrointestinal pathogens. Rotavirus was the most common pathogen identified, but there was a differential prevalence of rotavirus and norovirus between the urban and rural locations. Furthermore, rotavirus genotyping and phylogenetic analysis again differentiated the genotypes by the sampling location. Our data show a disproportional distribution of enteric viral pathogens in urban and rural locations, and we provide evidence of continual importation of new rotavirus strains into southern Vietnam and report the emergence of rotavirus genotype G12.

    Funded by: Wellcome Trust

    The American journal of tropical medicine and hygiene 2011;85;4;768-75

  • The burden and characteristics of enteric fever at a healthcare facility in a densely populated area of Kathmandu.

    Karkey A, Arjyal A, Anders KL, Boni MF, Dongol S, Koirala S, My PV, Nga TV, Clements AC, Holt KE, Duy PT, Day JN, Campbell JI, Dougan G, Dolecek C, Farrar J, Basnyat B and Baker S

    Oxford University Clinical Research Unit, Patan Academy of Health Sciences, Lagankhel, Kathmandu, Nepal.

    Enteric fever, caused by Salmonella enterica serovars Typhi and Paratyphi A (S. Typhi and S. Paratyphi A) remains a major public health problem in many settings. The disease is limited to locations with poor sanitation which facilitates the transmission of the infecting organisms. Efficacious and inexpensive vaccines are available for S. Typhi, yet are not commonly deployed to control the disease. Lack of vaccination is due partly to uncertainty of the disease burden arising from a paucity of epidemiological information in key locations. We have collected and analyzed data from 3,898 cases of blood culture-confirmed enteric fever from Patan Hospital in Lalitpur Sub-Metropolitan City (LSMC), between June 2005 and May 2009. Demographic data was available for a subset of these patients (n = 527) that were resident in LSMC and who were enrolled in trials. We show a considerable burden of enteric fever caused by S. Typhi (2,672; 68.5%) and S. Paratyphi A (1,226; 31.5%) at this Hospital over a four year period, which correlate with seasonal fluctuations in rainfall. We found that local population density was not related to incidence and we identified a focus of infections in the east of LSMC. With data from patients resident in LSMC we found that the median age of those with S. Typhi (16 years) was significantly less than S. Paratyphi A (20 years) and that males aged 15 to 25 were disproportionately infected. Our findings provide a snapshot into the epidemiological patterns of enteric fever in Kathmandu. The uneven distribution of enteric fever patients within the population suggests local variation in risk factors, such as contaminated drinking water. These findings are important for initiating a vaccination scheme and improvements in sanitation. We suggest any such intervention should be implemented throughout the LSMC area.

    Funded by: Medical Research Council: G0600718; Wellcome Trust

    PloS one 2010;5;11;e13988

  • A changing picture of shigellosis in southern Vietnam: shifting species dominance, antimicrobial susceptibility and clinical presentation.

    Vinh H, Nhu NT, Nga TV, Duy PT, Campbell JI, Hoang NV, Boni MF, My PV, Parry C, Nga TT, Van Minh P, Thuy CT, Diep TS, Phuong le T, Chinh MT, Loan HT, Tham NT, Lanh MN, Mong BL, Anh VT, Bay PV, Chau NV, Farrar J and Baker S

    The Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam. vinhh@oucru.org

    Background: Shigellosis remains considerable public health problem in some developing countries. The nature of Shigellae suggests that they are highly adaptable when placed under selective pressure in a human population. This is demonstrated by variation and fluctuations in serotypes and antimicrobial resistance profile of organisms circulating in differing setting in endemic locations. Antimicrobial resistance in the genus Shigella is a constant threat, with reports of organisms in Asia being resistant to multiple antimicrobials and new generation therapies.

    Methods: Here we compare microbiological, clinical and epidemiological data from patients with shigellosis over three different periods in southern Vietnam spanning 14 years.

    Results: Our data demonstrates a shift in dominant infecting species (S. flexneri to S. sonnei) and resistance profile of the organisms circulating in southern Vietnam. We find that there was no significant variation in the syndromes associated with either S. sonnei or S. flexneri, yet the clinical features of the disease are more severe in later observations.

    Conclusions: Our findings show a change in clinical presentation of shigellosis in this setting, as the disease may be now more pronounced, this is concurrent with a change in antimicrobial resistance profile. These data highlight the socio-economic development of southern Vietnam and should guide future vaccine development and deployment strategies.

    Current Controlled Trials ISRCTN55945881.

    Funded by: Medical Research Council: G0600718; Wellcome Trust

    BMC infectious diseases 2009;9;204

Neneh Sallah

- PhD Student

I am currently a 2nd Year PhD student funded by the Wellcome Trust Sanger Institute. I graduated from the University of Manchester in 2011 with a BSc (Hons) in Microbiology during which I spent summers at the Medical Research Council (MRC), The Gambia unit investigating Tuberculosis genotype diversity and transmission led by Dr. Bouke de Jong. From 2011-2012 I returned to the MRC, mainly investigating potential vectors of diarrheal diseases and the genotypic diversity of Rotavirus in The Gambia supervised by Dr. Martin Antonio.

Research

I am jointly supervised by Dr. Ines Barroso (Metabolic disease group) and Prof. Paul Kellam (Virus Genomics). My research is mainly focused on exploring the genetics of host-virus interactions in Kaposi’s Sarcoma-associated Herpesvirus (KSHV) infection. I am currently investigating how variation in host and virus genes affect virus biological function particularly genes that play a role in viral reactivation and tumour development.

References

  • High genotypic diversity among rotavirus strains infecting Gambian children.

    Kwambana BA, Ikumapayi UN, Sallah N, Dione M, Jarju S, Panchalingham S, Jafali J, Lamin M, Betts M, Adeyemi M, Akinsola A, Bittaye O, Jasseh M, Kotloff KL, Levine MM, Nataro JP, Corrah T, Hossain MJ, Saha D and Antonio M

    From the *Medical Research Council Unit, Fajara, The Gambia; †Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD; and ‡Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, VA.

    Background: Rotavirus is the leading cause of diarrhea in children <5 years of age. In light of the implementation of rotavirus vaccines of limited valency, it is important to characterize the genotypic diversity of circulating rotavirus in sub-Saharan Africa.

    Methods: We collected stool samples from children 0-59 months of age who presented at the health centres as cases with moderate-to-severe diarrhea in the Upper River Region of The Gambia. Stool samples were also collected from age, sex and area-matched healthy controls. All stool samples were assayed for rotavirus antigens by enzyme-linked immunosorbent assay and genotyping was done using reverse transcriptase polymerase chain reaction.

    Results: We enrolled 1029 cases and 1569 controls during the 3-year study period (2008-2010). The detection rate of rotavirus among the cases was 20% (204/1029) and 3% (42/1569) among controls. At least 18 genotypes were found and the predominant genotypes were G2P[6] (28%), G1P[8] (26%) and G1P[10] (10%). The rare identified genotypes (<1%) were G2P[14], G8P[6], G9P[6] and G4P[10]. There was also a strong positive association between rotavirus infection and the dry season (odds ratio: 9.83, 95% confidence interval: 6.18-15.63, P < 0.001). A significant increase in the odds of rotavirus and G1P[8] detection with the use of untreated water and the presence of cats, rodents and cows in the child's residence was also found.

    Conclusion: This study provides important baseline data for the genotypes circulating before vaccine implementation. The wide diversity of genotypes circulating in The Gambia implies the need for vigilant effectiveness surveillance following the implementation of RotaTeq in August 2013.

    The Pediatric infectious disease journal 2014;33 Suppl 1;S69-75

  • Immunogenic Mycobacterium africanum strains associated with ongoing transmission in The Gambia.

    Gehre F, Antonio M, Otu JK, Sallah N, Secka O, Faal T, Owiafe P, Sutherland JS, Adetifa IM, Ota MO, Kampmann B, Corrah T and de Jong BC

    In West Africa, Mycobacterium tuberculosis strains co-circulate with M. africanum, and both pathogens cause pulmonary tuberculosis in humans. Given recent findings that M. tuberculosis T-cell epitopes are hyperconserved, we hypothesized that more immunogenic strains have increased capacity to spread within the human host population. We investigated the relationship between the composition of the mycobacterial population in The Gambia, as measured by spoligotype analysis, and the immunogenicity of these strains as measured by purified protein derivative-induced interferon-γ release in ELISPOT assays of peripheral blood mononuclear cells. We found a positive correlation between strains with superior spreading capacity and their relative immunogenicity. Although our observation is true for M. tuberculosis and M. africanum strains, the association was especially pronounced in 1 M. africanum sublineage, characterized by spoligotype shared international type 181, which is responsible for 20% of all tuberculosis cases in the region and therefore poses a major public health threat in The Gambia.

    Funded by: Medical Research Council: MC_UP_A900_1122

    Emerging infectious diseases 2013;19;10;1598-1604

  • Chrysomya putoria, a putative vector of diarrheal diseases.

    Lindsay SW, Lindsay TC, Duprez J, Hall MJ, Kwambana BA, Jawara M, Nurudeen IU, Sallah N, Wyatt N, D'Alessandro U, Pinder M and Antonio M

    School of Biological and Biomedical Sciences, Durham University, Durham City, United Kingdom. S.W.Lindsay@durham.ac.uk

    Background: Chrysomya spp are common blowflies in Africa, Asia and parts of South America and some species can reproduce in prodigious numbers in pit latrines. Because of their strong association with human feces and their synanthropic nature, we examined whether these flies are likely to be vectors of diarrheal pathogens.

    Flies were sampled using exit traps placed over the drop holes of latrines in Gambian villages. Odor-baited fly traps were used to determine the relative attractiveness of different breeding and feeding media. The presence of bacteria on flies was confirmed by culture and bacterial DNA identified using PCR. A median of 7.00 flies/latrine/day (IQR = 0.0-25.25) was collected, of which 95% were Chrysomya spp, and of these nearly all were Chrysomya putoria (99%). More flies were collected from traps with feces from young children (median = 3.0, IQR = 1.75-10.75) and dogs (median = 1.50, IQR = 0.0-13.25) than from herbivores (median = 0.0, IQR = 0.0-0.0; goat, horse, cow and calf; p<0.001). Flies were strongly attracted to raw meat (median = 44.5, IQR = 26.25-143.00) compared with fish (median = 0.0, IQR = 0.0-19.75, ns), cooked and uncooked rice, and mangoes (median = 0.0, IQR = 0.0-0.0; p<0.001). Escherichia coli were cultured from the surface of 21% (15/72 agar plates) of Chrysomya spp and 10% of these were enterotoxigenic. Enteroaggregative E. coli were identified by PCR in 2% of homogenized Chrysomya spp, Shigella spp in 1.4% and Salmonella spp in 0.6% of samples.

    The large numbers of C. putoria that can emerge from pit latrines, the presence of enteric pathogens on flies, and their strong attraction to raw meat and fish suggests these flies may be common vectors of diarrheal diseases in Africa.

    Funded by: Medical Research Council: MC_U190074190, MC_U190081991, MC_UP_A900_1119

    PLoS neglected tropical diseases 2012;6;11;e1895

Sarah Smith

- PhD Student

I graduated from the University of York with a BSc in molecular biology. During the third year of this four year programme, I completed a year of research in the Gene Medicine group at the Nuffield Department of Clinical Laboratory Sciences (University of Oxford). I am currently in my 4th year of a Wellcome Trust funded PhD at the Wellcome Trust Sanger Institute.

Research

I work in the viral genomics group at the Sanger Institute where we investigate host and virus genetic variation in order to understand the biology of virus spread, host species adaption, and zoonosis. Specifically I am investigating one family of virus restriction factors (VRFs), the Interferon Inducible Transmembrane (IFITM) proteins, with the aim of understanding the genetic variation that results in virus restriction both within humans and between different animal species.

References

  • Chicken interferon-inducible transmembrane protein 3 restricts influenza viruses and lyssaviruses in vitro.

    Smith SE, Gibson MS, Wash RS, Ferrara F, Wright E, Temperton N, Kellam P and Fife M

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom.

    Interferon-inducible transmembrane protein 3 (IFITM3) is an effector protein of the innate immune system. It confers potent, cell-intrinsic resistance to infection by diverse enveloped viruses both in vitro and in vivo, including influenza viruses, West Nile virus, and dengue virus. IFITM3 prevents cytosolic entry of these viruses by blocking complete virus envelope fusion with cell endosome membranes. Although the IFITM locus, which includes IFITM1, -2, -3, and -5, is present in mammalian species, this locus has not been unambiguously identified or functionally characterized in avian species. Here, we show that the IFITM locus exists in chickens and is syntenic with the IFITM locus in mammals. The chicken IFITM3 protein restricts cell infection by influenza A viruses and lyssaviruses to a similar level as its human orthologue. Furthermore, we show that chicken IFITM3 is functional in chicken cells and that knockdown of constitutive expression in chicken fibroblasts results in enhanced infection by influenza A virus. Chicken IFITM2 and -3 are constitutively expressed in all tissues examined, whereas IFITM1 is only expressed in the bursa of Fabricius, gastrointestinal tract, cecal tonsil, and trachea. Despite being highly divergent at the amino acid level, IFITM3 proteins of birds and mammals can restrict replication of viruses that are able to infect different host species, suggesting IFITM proteins may provide a crucial barrier for zoonotic infections.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/J004448/1; Medical Research Council: G0600369, G1000413; PHS HHS: 098051

    Journal of virology 2013;87;23;12957-66

  • The CD225 domain of IFITM3 is required for both IFITM protein association and inhibition of influenza A virus and dengue virus replication.

    John SP, Chin CR, Perreira JM, Feeley EM, Aker AM, Savidis G, Smith SE, Elia AE, Everitt AR, Vora M, Pertel T, Elledge SJ, Kellam P and Brass AL

    Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    The interferon-induced transmembrane protein 3 (IFITM3) gene is an interferon-stimulated gene that inhibits the replication of multiple pathogenic viruses in vitro and in vivo. IFITM3 is a member of a large protein superfamily, whose members share a functionally undefined area of high amino acid conservation, the CD225 domain. We performed mutational analyses of IFITM3 and identified multiple residues within the CD225 domain, consisting of the first intramembrane domain (intramembrane domain 1 [IM1]) and a conserved intracellular loop (CIL), that are required for restriction of both influenza A virus (IAV) and dengue virus (DENV) infection in vitro. Two phenylalanines within IM1 (F75 and F78) also mediate a physical association between IFITM proteins, and the loss of this interaction decreases IFITM3-mediated restriction. By extension, similar IM1-mediated associations may contribute to the functions of additional members of the CD225 domain family. IFITM3's distal N-terminal domain is also needed for full antiviral activity, including a tyrosine (Y20), whose alteration results in mislocalization of a portion of IFITM3 to the cell periphery and surface. Comparative analyses demonstrate that similar molecular determinants are needed for IFITM3's restriction of both IAV and DENV. However, a portion of the CIL including Y99 and R87 is preferentially needed for inhibition of the orthomyxovirus. Several IFITM3 proteins engineered with rare single-nucleotide polymorphisms demonstrated reduced expression or mislocalization, and these events were associated with enhanced viral replication in vitro, suggesting that possessing such alleles may impact an individual's risk for viral infection. On the basis of this and other data, we propose a model for IFITM3-mediated restriction.

    Funded by: Howard Hughes Medical Institute; NIAID NIH HHS: 1R01AI091786, R01 AI091786; Wellcome Trust

    Journal of virology 2013;87;14;7837-52

  • Sherlock Genomes - viral investigator.

    Smith SE and Wash RS

    Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK. microbes@sanger.ac.uk.

    This month's Genome Watch highlights how deep sequencing technologies have vastly reduced the time and prior knowledge needed to generate viral genomes.

    Nature reviews. Microbiology 2013;11;3;150

  • IFITM3 restricts the morbidity and mortality associated with influenza.

    Everitt AR, Clare S, Pertel T, John SP, Wash RS, Smith SE, Chin CR, Feeley EM, Sims JS, Adams DJ, Wise HM, Kane L, Goulding D, Digard P, Anttila V, Baillie JK, Walsh TS, Hume DA, Palotie A, Xue Y, Colonna V, Tyler-Smith C, Dunning J, Gordon SB, GenISIS Investigators, MOSAIC Investigators, Smyth RL, Openshaw PJ, Dougan G, Brass AL and Kellam P

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton CB10 1SA, UK.

    The 2009 H1N1 influenza pandemic showed the speed with which a novel respiratory virus can spread and the ability of a generally mild infection to induce severe morbidity and mortality in a subset of the population. Recent in vitro studies show that the interferon-inducible transmembrane (IFITM) protein family members potently restrict the replication of multiple pathogenic viruses. Both the magnitude and breadth of the IFITM proteins' in vitro effects suggest that they are critical for intrinsic resistance to such viruses, including influenza viruses. Using a knockout mouse model, we now test this hypothesis directly and find that IFITM3 is essential for defending the host against influenza A virus in vivo. Mice lacking Ifitm3 display fulminant viral pneumonia when challenged with a normally low-pathogenicity influenza virus, mirroring the destruction inflicted by the highly pathogenic 1918 'Spanish' influenza. Similar increased viral replication is seen in vitro, with protection rescued by the re-introduction of Ifitm3. To test the role of IFITM3 in human influenza virus infection, we assessed the IFITM3 alleles of individuals hospitalized with seasonal or pandemic influenza H1N1/09 viruses. We find that a statistically significant number of hospitalized subjects show enrichment for a minor IFITM3 allele (SNP rs12252-C) that alters a splice acceptor site, and functional assays show the minor CC genotype IFITM3 has reduced influenza virus restriction in vitro. Together these data reveal that the action of a single intrinsic immune effector, IFITM3, profoundly alters the course of influenza virus infection in mouse and humans.

    Funded by: Cancer Research UK: 13031; Chief Scientist Office; Medical Research Council: G0600511, G0800767, G0800777, G0802752, G0901697, MC_G1001212, MC_U122785833; NIAID NIH HHS: R01 AI091786, R01AI091786; Wellcome Trust: 090382, 090382/Z/09/Z, 090385/Z/09/Z, 098051

    Nature 2012;484;7395;519-23

  • Shuttle vector system for Methanococcus maripaludis with improved transformation efficiency.

    Walters AD, Smith SE and Chong JP

    Department of Biology (Area 5), University of York, Wentworth Way, York YO10 5DD, United Kingdom.

    We have identified an open reading frame and DNA element that are sufficient to maintain shuttle vectors in Methanococcus maripaludis. Strain S0001, containing ORF1 from pURB500 integrated into the M. maripaludis genome, supports a significantly smaller shuttle vector, pAW42, and a 7,000-fold increase in transformation efficiency for pURB500-based vectors.

    Funded by: Cancer Research UK: C23949/A10945

    Applied and environmental microbiology 2011;77;7;2549-51

Rachael Wash

- Staff Scientist

I have a BA in Natural Sciences and a PhD from the University of Cambridge, and an MSc in Equine Science from the University of Wales, Aberystwyth. After my MSc I joined the Animal Health Trust, Newmarket to carry out research in equine herpes virus-1 pathogenesis. I also completed my PhD research there, developing recombinant vaccines for African horse sickness virus. I joined the Wellcome Trust Sanger Institute as a postdoc in 2009 to focus on host-virus interactions and have since become a staff scientist in the Virus Genomics team.

Research

I am interested in respiratory viruses and identifying host factors, which affect the outcome of infection. My research projects include the use of siRNA screening, exome analysis of extreme phenotypes and work with macrophages derived from embryonic stem cells to determine host factors that play a role in virus replication or restriction. My research also focuses on a family of host proteins, Interferon Inducible Transmembrane (IFITM) proteins that are known to restrict virus infections, developing further understanding of how human variation can affect their functionality.

References

  • Chicken interferon-inducible transmembrane protein 3 restricts influenza viruses and lyssaviruses in vitro.

    Smith SE, Gibson MS, Wash RS, Ferrara F, Wright E, Temperton N, Kellam P and Fife M

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom.

    Interferon-inducible transmembrane protein 3 (IFITM3) is an effector protein of the innate immune system. It confers potent, cell-intrinsic resistance to infection by diverse enveloped viruses both in vitro and in vivo, including influenza viruses, West Nile virus, and dengue virus. IFITM3 prevents cytosolic entry of these viruses by blocking complete virus envelope fusion with cell endosome membranes. Although the IFITM locus, which includes IFITM1, -2, -3, and -5, is present in mammalian species, this locus has not been unambiguously identified or functionally characterized in avian species. Here, we show that the IFITM locus exists in chickens and is syntenic with the IFITM locus in mammals. The chicken IFITM3 protein restricts cell infection by influenza A viruses and lyssaviruses to a similar level as its human orthologue. Furthermore, we show that chicken IFITM3 is functional in chicken cells and that knockdown of constitutive expression in chicken fibroblasts results in enhanced infection by influenza A virus. Chicken IFITM2 and -3 are constitutively expressed in all tissues examined, whereas IFITM1 is only expressed in the bursa of Fabricius, gastrointestinal tract, cecal tonsil, and trachea. Despite being highly divergent at the amino acid level, IFITM3 proteins of birds and mammals can restrict replication of viruses that are able to infect different host species, suggesting IFITM proteins may provide a crucial barrier for zoonotic infections.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/J004448/1; Medical Research Council: G0600369, G1000413; PHS HHS: 098051

    Journal of virology 2013;87;23;12957-66

  • Sherlock Genomes - viral investigator.

    Smith SE and Wash RS

    Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK. microbes@sanger.ac.uk.

    This month's Genome Watch highlights how deep sequencing technologies have vastly reduced the time and prior knowledge needed to generate viral genomes.

    Nature reviews. Microbiology 2013;11;3;150

  • IFITM3 restricts the morbidity and mortality associated with influenza.

    Everitt AR, Clare S, Pertel T, John SP, Wash RS, Smith SE, Chin CR, Feeley EM, Sims JS, Adams DJ, Wise HM, Kane L, Goulding D, Digard P, Anttila V, Baillie JK, Walsh TS, Hume DA, Palotie A, Xue Y, Colonna V, Tyler-Smith C, Dunning J, Gordon SB, GenISIS Investigators, MOSAIC Investigators, Smyth RL, Openshaw PJ, Dougan G, Brass AL and Kellam P

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton CB10 1SA, UK.

    The 2009 H1N1 influenza pandemic showed the speed with which a novel respiratory virus can spread and the ability of a generally mild infection to induce severe morbidity and mortality in a subset of the population. Recent in vitro studies show that the interferon-inducible transmembrane (IFITM) protein family members potently restrict the replication of multiple pathogenic viruses. Both the magnitude and breadth of the IFITM proteins' in vitro effects suggest that they are critical for intrinsic resistance to such viruses, including influenza viruses. Using a knockout mouse model, we now test this hypothesis directly and find that IFITM3 is essential for defending the host against influenza A virus in vivo. Mice lacking Ifitm3 display fulminant viral pneumonia when challenged with a normally low-pathogenicity influenza virus, mirroring the destruction inflicted by the highly pathogenic 1918 'Spanish' influenza. Similar increased viral replication is seen in vitro, with protection rescued by the re-introduction of Ifitm3. To test the role of IFITM3 in human influenza virus infection, we assessed the IFITM3 alleles of individuals hospitalized with seasonal or pandemic influenza H1N1/09 viruses. We find that a statistically significant number of hospitalized subjects show enrichment for a minor IFITM3 allele (SNP rs12252-C) that alters a splice acceptor site, and functional assays show the minor CC genotype IFITM3 has reduced influenza virus restriction in vitro. Together these data reveal that the action of a single intrinsic immune effector, IFITM3, profoundly alters the course of influenza virus infection in mouse and humans.

    Funded by: Cancer Research UK: 13031; Chief Scientist Office; Medical Research Council: G0600511, G0800767, G0800777, G0802752, G0901697, MC_G1001212, MC_U122785833; NIAID NIH HHS: R01 AI091786, R01AI091786; Wellcome Trust: 090382, 090382/Z/09/Z, 090385/Z/09/Z, 098051

    Nature 2012;484;7395;519-23

  • Evolutionary dynamics of local pandemic H1N1/2009 influenza virus lineages revealed by whole-genome analysis.

    Baillie GJ, Galiano M, Agapow PM, Myers R, Chiam R, Gall A, Palser AL, Watson SJ, Hedge J, Underwood A, Platt S, McLean E, Pebody RG, Rambaut A, Green J, Daniels R, Pybus OG, Kellam P and Zambon M

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom.

    Virus gene sequencing and phylogenetics can be used to study the epidemiological dynamics of rapidly evolving viruses. With complete genome data, it becomes possible to identify and trace individual transmission chains of viruses such as influenza virus during the course of an epidemic. Here we sequenced 153 pandemic influenza H1N1/09 virus genomes from United Kingdom isolates from the first (127 isolates) and second (26 isolates) waves of the 2009 pandemic and used their sequences, dates of isolation, and geographical locations to infer the genetic epidemiology of the epidemic in the United Kingdom. We demonstrate that the epidemic in the United Kingdom was composed of many cocirculating lineages, among which at least 13 were exclusively or predominantly United Kingdom clusters. The estimated divergence times of two of the clusters predate the detection of pandemic H1N1/09 virus in the United Kingdom, suggesting that the pandemic H1N1/09 virus was already circulating in the United Kingdom before the first clinical case. Crucially, three clusters contain isolates from the second wave of infections in the United Kingdom, two of which represent chains of transmission that appear to have persisted within the United Kingdom between the first and second waves. This demonstrates that whole-genome analysis can track in fine detail the behavior of individual influenza virus lineages during the course of a single epidemic or pandemic.

    Funded by: Medical Research Council: MC_U117512723; Wellcome Trust: 095831

    Journal of virology 2012;86;1;11-8

  • A modified vaccinia Ankara virus (MVA) vaccine expressing African horse sickness virus (AHSV) VP2 protects against AHSV challenge in an IFNAR -/- mouse model.

    Castillo-Olivares J, Calvo-Pinilla E, Casanova I, Bachanek-Bankowska K, Chiam R, Maan S, Nieto JM, Ortego J and Mertens PP

    Institute for Animal Health, Pirbright, Woking, Surrey, United Kingdom. javier.castillo-olivares@bbsrc.ac.uk

    African horse sickness (AHS) is a lethal viral disease of equids, which is transmitted by Culicoides midges that become infected after biting a viraemic host. The use of live attenuated vaccines has been vital for the control of this disease in endemic regions. However, there are safety concerns over their use in non-endemic countries. Research efforts over the last two decades have therefore focused on developing alternative vaccines based on recombinant baculovirus or live viral vectors expressing structural components of the AHS virion. However, ethical and financial considerations, relating to the use of infected horses in high biosecurity installations, have made progress very slow. We have therefore assessed the potential of an experimental mouse-model for AHSV infection for vaccine and immunology research. We initially characterised AHSV infection in this model, then tested the protective efficacy of a recombinant vaccine based on modified vaccinia Ankara expressing AHS-4 VP2 (MVA-VP2).

    Funded by: Biotechnology and Biological Sciences Research Council

    PloS one 2011;6;1;e16503

  • Induction of antibody responses to African horse sickness virus (AHSV) in ponies after vaccination with recombinant modified vaccinia Ankara (MVA).

    Chiam R, Sharp E, Maan S, Rao S, Mertens P, Blacklaws B, Davis-Poynter N, Wood J and Castillo-Olivares J

    Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk, United Kingdom.

    Background: African horse sickness virus (AHSV) causes a non-contagious, infectious disease in equids, with mortality rates that can exceed 90% in susceptible horse populations. AHSV vaccines play a crucial role in the control of the disease; however, there are concerns over the use of polyvalent live attenuated vaccines particularly in areas where AHSV is not endemic. Therefore, it is important to consider alternative approaches for AHSV vaccine development. We have carried out a pilot study to investigate the ability of recombinant modified vaccinia Ankara (MVA) vaccines expressing VP2, VP7 or NS3 genes of AHSV to stimulate immune responses against AHSV antigens in the horse.

    VP2, VP7 and NS3 genes from AHSV-4/Madrid87 were cloned into the vaccinia transfer vector pSC11 and recombinant MVA viruses generated. Antigen expression or transcription of the AHSV genes from cells infected with the recombinant viruses was confirmed. Pairs of ponies were vaccinated with MVAVP2, MVAVP7 or MVANS3 and both MVA vector and AHSV antigen-specific antibody responses were analysed. Vaccination with MVAVP2 induced a strong AHSV neutralising antibody response (VN titre up to a value of 2). MVAVP7 also induced AHSV antigen-specific responses, detected by western blotting. NS3 specific antibody responses were not detected.

    Conclusions: This pilot study demonstrates the immunogenicity of recombinant MVA vectored AHSV vaccines, in particular MVAVP2, and indicates that further work to investigate whether these vaccines would confer protection from lethal AHSV challenge in the horse is justifiable.

    Funded by: Biotechnology and Biological Sciences Research Council

    PloS one 2009;4;6;e5997

  • Use of polarised equine endothelial cell cultures and an in vitro thrombosis model for potential characterisation of EHV-1 strain variation.

    Chiam R, Smid L, Kydd JH, Smith KC, Platt A and Davis-Poynter NJ

    Animal Health Trust, Centre for Preventive Medicine, Lanwades Park, Kentford, Newmarket, Suffolk CB8 7UU, UK.

    Equine herpesvirus-1 (EHV-1) is responsible for respiratory disease and abortion in pregnant mares. Some high virulence isolates of EHV-1 also cause neurological disease. The pathogenesis of both abortion and neurological disease relates in part, to thrombus formation occurring in the pregnant uterus and central nervous system. The differences in disease outcome may relate to differing abilities of high and low virulence EHV-1 isolates to cause cell-associated viraemia, infect endothelial cells and cause thrombosis at sites distant from the respiratory tract. This study attempted to identify in vitro assays, which could be used to characterise the interaction between these isolates, equine endothelial cells and clotting factors. No significant difference was found between the growth kinetics of high and low virulence isolates of EHV-1 in polarised endothelial cells. For both isolates, virus was released preferentially from the apical surface of the polarised cells. The functional effects of viral infection on endothelial cells, with reference to virally-induced thrombosis were then investigated. Endothelial cells were grown on microcarrier beads, infected with EHV-1 and assayed for procoagulant activity. No significant difference in clotting time was observed between mock and EHV-1 infected endothelial cells in microcarrier cultures. Thus the degree of thrombosis may reflect a more complex interaction between endothelial cells, circulating leucocytes and other factors in the microenvironment.

    Veterinary microbiology 2006;113;3-4;243-9

Simon Watson

- Senior Computational Biologist

I obtained my bachelor’s degree in Human Genetics at the University of Leeds in 2004, and subsequently undertook a Master’s degree in Bioinformatics and Computational Biology. Following this, I attained my PhD at University College London in computational virology, where I investigated the application of molecular dynamics to simulate the biochemical effect of saquinavir-resistance mutations in HIV-1 protease. Since then I have been employed as a bioinformatician in the Virus Genomics group at the Wellcome Trust Sanger Institute, leading the group’s influenza research.

Research

I am in charge of the group's influenza research: investigating the diversity, ecology, and molecular evolution of different subtypes of influenza virus in human, swine and avian hosts, with particular interest in intra-host viral population dynamics. I also provide informatics assistance to the molecular biologists in the Virus Genomics group, helping to assemble and analyse NGS whole-genome sequence data for a range of viruses. More recently, I was involved in the MERS coronavirus outbreak in the Middle East, using deep-sequencing technologies to reconstruct MERS-CoV genomes from clinical patients, and characterise the molecular epidemiology of the emerging outbreak in real-time.

References

  • Transmission and evolution of the Middle East respiratory syndrome coronavirus in Saudi Arabia: a descriptive genomic study.

    Cotten M, Watson SJ, Kellam P, Al-Rabeeah AA, Makhdoom HQ, Assiri A, Al-Tawfiq JA, Alhakeem RF, Madani H, AlRabiah FA, Al Hajjar S, Al-nassir WN, Albarrak A, Flemban H, Balkhy HH, Alsubaie S, Palser AL, Gall A, Bashford-Rogers R, Rambaut A, Zumla AI and Memish ZA

    Wellcome Trust Sanger Institute, Hinxton, UK.

    Background: Since June, 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) has, worldwide, caused 104 infections in people including 49 deaths, with 82 cases and 41 deaths reported from Saudi Arabia. In addition to confirming diagnosis, we generated the MERS-CoV genomic sequences obtained directly from patient samples to provide important information on MERS-CoV transmission, evolution, and origin.

    Methods: Full genome deep sequencing was done on nucleic acid extracted directly from PCR-confirmed clinical samples. Viral genomes were obtained from 21 MERS cases of which 13 had 100%, four 85-95%, and four 30-50% genome coverage. Phylogenetic analysis of the 21 sequences, combined with nine published MERS-CoV genomes, was done.

    Findings: Three distinct MERS-CoV genotypes were identified in Riyadh. Phylogeographic analyses suggest the MERS-CoV zoonotic reservoir is geographically disperse. Selection analysis of the MERS-CoV genomes reveals the expected accumulation of genetic diversity including changes in the S protein. The genetic diversity in the Al-Hasa cluster suggests that the hospital outbreak might have had more than one virus introduction.

    Interpretation: We present the largest number of MERS-CoV genomes (21) described so far. MERS-CoV full genome sequences provide greater detail in tracking transmission. Multiple introductions of MERS-CoV are identified and suggest lower R0 values. Transmission within Saudi Arabia is consistent with either movement of an animal reservoir, animal products, or movement of infected people. Further definition of the exposures responsible for the sporadic introductions of MERS-CoV into human populations is urgently needed.

    Funding: Saudi Arabian Ministry of Health, Wellcome Trust, European Community, and National Institute of Health Research University College London Hospitals Biomedical Research Centre.

    Funded by: Wellcome Trust: 093724

    Lancet 2013;382;9909;1993-2002

  • Hospital outbreak of Middle East respiratory syndrome coronavirus.

    Assiri A, McGeer A, Perl TM, Price CS, Al Rabeeah AA, Cummings DA, Alabdullatif ZN, Assad M, Almulhim A, Makhdoom H, Madani H, Alhakeem R, Al-Tawfiq JA, Cotten M, Watson SJ, Kellam P, Zumla AI, Memish ZA and KSA MERS-CoV Investigation Team

    Global Center for Mass Gatherings Medicine, Ministry of Health, Riyadh, Saudi Arabia.

    Background: In September 2012, the World Health Organization reported the first cases of pneumonia caused by the novel Middle East respiratory syndrome coronavirus (MERS-CoV). We describe a cluster of health care-acquired MERS-CoV infections.

    Methods: Medical records were reviewed for clinical and demographic information and determination of potential contacts and exposures. Case patients and contacts were interviewed. The incubation period and serial interval (the time between the successive onset of symptoms in a chain of transmission) were estimated. Viral RNA was sequenced.

    Results: Between April 1 and May 23, 2013, a total of 23 cases of MERS-CoV infection were reported in the eastern province of Saudi Arabia. Symptoms included fever in 20 patients (87%), cough in 20 (87%), shortness of breath in 11 (48%), and gastrointestinal symptoms in 8 (35%); 20 patients (87%) presented with abnormal chest radiographs. As of June 12, a total of 15 patients (65%) had died, 6 (26%) had recovered, and 2 (9%) remained hospitalized. The median incubation period was 5.2 days (95% confidence interval [CI], 1.9 to 14.7), and the serial interval was 7.6 days (95% CI, 2.5 to 23.1). A total of 21 of the 23 cases were acquired by person-to-person transmission in hemodialysis units, intensive care units, or in-patient units in three different health care facilities. Sequencing data from four isolates revealed a single monophyletic clade. Among 217 household contacts and more than 200 health care worker contacts whom we identified, MERS-CoV infection developed in 5 family members (3 with laboratory-confirmed cases) and in 2 health care workers (both with laboratory-confirmed cases).

    Conclusions: Person-to-person transmission of MERS-CoV can occur in health care settings and may be associated with considerable morbidity. Surveillance and infection-control measures are critical to a global public health response.

    Funded by: NIGMS NIH HHS: U01 GM070708, U54 GM088491; Wellcome Trust: 093724

    The New England journal of medicine 2013;369;5;407-16

  • Full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus.

    Cotten M, Lam TT, Watson SJ, Palser AL, Petrova V, Grant P, Pybus OG, Rambaut A, Guan Y, Pillay D, Kellam P and Nastouli E

    Wellcome Trust Sanger Institute, Hinxton, UK.

    A novel betacoronavirus associated with lethal respiratory and renal complications was recently identified in patients from several countries in the Middle East. We report the deep genome sequencing of the virus directly from a patient's sputum sample. Our high-throughput sequencing yielded a substantial depth of genome sequence assembly and showed the minority viral variants in the specimen. Detailed phylogenetic analysis of the virus genome (England/Qatar/2012) revealed its close relationship to European bat coronaviruses circulating among the bat species of the Vespertilionidae family. Molecular clock analysis showed that the 2 human infections of this betacoronavirus in June 2012 (EMC/2012) and September 2012 (England/Qatar/2012) share a common virus ancestor most likely considerably before early 2012, suggesting the human diversity is the result of multiple zoonotic events.

    Funded by: Medical Research Council: MR/K006584/1; Wellcome Trust: 093724, 095831

    Emerging infectious diseases 2013;19;5;736-42B

  • Viral population analysis and minority-variant detection using short read next-generation sequencing.

    Watson SJ, Welkers MR, Depledge DP, Coulter E, Breuer JM, de Jong MD and Kellam P

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

    RNA viruses within infected individuals exist as a population of evolutionary-related variants. Owing to evolutionary change affecting the constitution of this population, the frequency and/or occurrence of individual viral variants can show marked or subtle fluctuations. Since the development of massively parallel sequencing platforms, such viral populations can now be investigated to unprecedented resolution. A critical problem with such analyses is the presence of sequencing-related errors that obscure the identification of true biological variants present at low frequency. Here, we report the development and assessment of the Quality Assessment of Short Read (QUASR) Pipeline (http://sourceforge.net/projects/quasr) specific for virus genome short read analysis that minimizes sequencing errors from multiple deep-sequencing platforms, and enables post-mapping analysis of the minority variants within the viral population. QUASR significantly reduces the error-related noise in deep-sequencing datasets, resulting in increased mapping accuracy and reduction of erroneous mutations. Using QUASR, we have determined influenza virus genome dynamics in sequential samples from an in vitro evolution of 2009 pandemic H1N1 (A/H1N1/09) influenza from samples sequenced on both the Roche 454 GSFLX and Illumina GAIIx platforms. Importantly, concordance between the 454 and Illumina sequencing allowed unambiguous minority-variant detection and accurate determination of virus population turnover in vitro.

    Funded by: Medical Research Council: G0700814; Wellcome Trust

    Philosophical transactions of the Royal Society of London. Series B, Biological sciences 2013;368;1614;20120205

  • Universal amplification, next-generation sequencing, and assembly of HIV-1 genomes.

    Gall A, Ferns B, Morris C, Watson S, Cotten M, Robinson M, Berry N, Pillay D and Kellam P

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom.

    Whole HIV-1 genome sequences are pivotal for large-scale studies of inter- and intrahost evolution, including the acquisition of drug resistance mutations. The ability to rapidly and cost-effectively generate large numbers of HIV-1 genome sequences from different populations and geographical locations and determine the effect of minority genetic variants is, however, a limiting factor. Next-generation sequencing promises to bridge this gap but is hindered by the lack of methods for the enrichment of virus genomes across the phylogenetic breadth of HIV-1 and methods for the robust assembly of the virus genomes from short-read data. Here we report a method for the amplification, next-generation sequencing, and unbiased de novo assembly of HIV-1 genomes of groups M, N, and O, as well as recombinants, that does not require prior knowledge of the sequence or subtype. A sensitivity of at least 3,000 copies/ml was determined by using plasma virus samples of known copy numbers. We applied our novel method to compare the genome diversities of HIV-1 groups, subtypes, and genes. The highest level of diversity was found in the env, nef, vpr, tat, and rev genes and parts of the gag gene. Furthermore, we used our method to investigate mutations associated with HIV-1 drug resistance in clinical samples at the level of the complete genome. Drug resistance mutations were detected as both major variant and minor species. In conclusion, we demonstrate the feasibility of our method for large-scale HIV-1 genome sequencing. This will enable the phylogenetic and phylodynamic resolution of the ongoing pandemic and efficient monitoring of complex HIV-1 drug resistance genotypes.

    Funded by: Wellcome Trust: S0753

    Journal of clinical microbiology 2012;50;12;3838-44

  • Evolutionary dynamics of local pandemic H1N1/2009 influenza virus lineages revealed by whole-genome analysis.

    Baillie GJ, Galiano M, Agapow PM, Myers R, Chiam R, Gall A, Palser AL, Watson SJ, Hedge J, Underwood A, Platt S, McLean E, Pebody RG, Rambaut A, Green J, Daniels R, Pybus OG, Kellam P and Zambon M

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom.

    Virus gene sequencing and phylogenetics can be used to study the epidemiological dynamics of rapidly evolving viruses. With complete genome data, it becomes possible to identify and trace individual transmission chains of viruses such as influenza virus during the course of an epidemic. Here we sequenced 153 pandemic influenza H1N1/09 virus genomes from United Kingdom isolates from the first (127 isolates) and second (26 isolates) waves of the 2009 pandemic and used their sequences, dates of isolation, and geographical locations to infer the genetic epidemiology of the epidemic in the United Kingdom. We demonstrate that the epidemic in the United Kingdom was composed of many cocirculating lineages, among which at least 13 were exclusively or predominantly United Kingdom clusters. The estimated divergence times of two of the clusters predate the detection of pandemic H1N1/09 virus in the United Kingdom, suggesting that the pandemic H1N1/09 virus was already circulating in the United Kingdom before the first clinical case. Crucially, three clusters contain isolates from the second wave of infections in the United Kingdom, two of which represent chains of transmission that appear to have persisted within the United Kingdom between the first and second waves. This demonstrates that whole-genome analysis can track in fine detail the behavior of individual influenza virus lineages during the course of a single epidemic or pandemic.

    Funded by: Medical Research Council: MC_U117512723; Wellcome Trust: 095831

    Journal of virology 2012;86;1;11-8

  • Marked endotheliotropism of highly pathogenic avian influenza virus H5N1 following intestinal inoculation in cats.

    Reperant LA, van de Bildt MW, van Amerongen G, Leijten LM, Watson S, Palser A, Kellam P, Eissens AC, Frijlink HW, Osterhaus AD and Kuiken T

    Department of Ecology and Evolutionary Biology, Princeton University, Princeton, New Jersey, USA.

    Highly pathogenic avian influenza virus (HPAIV) H5N1 can infect mammals via the intestine; this is unusual since influenza viruses typically infect mammals via the respiratory tract. The dissemination of HPAIV H5N1 following intestinal entry and associated pathogenesis are largely unknown. To assess the route of spread of HPAIV H5N1 to other organs and to determine its associated pathogenesis, we inoculated infected chicken liver homogenate directly into the intestine of cats by use of enteric-coated capsules. Intestinal inoculation of HPAIV H5N1 resulted in fatal systemic disease. The spread of HPAIV H5N1 from the lumen of the intestine to other organs took place via the blood and lymphatic vascular systems but not via neuronal transmission. Remarkably, the systemic spread of the virus via the vascular system was associated with massive infection of endothelial and lymphendothelial cells, resulting in widespread hemorrhages. This is unique for influenza in mammals and resembles the pathogenesis of HPAIV infection in terrestrial poultry. It contrasts with the pathogenesis of systemic disease from the same virus following entry via the respiratory tract, where lesions are characterized mainly by necrosis and inflammation and are associated with the presence of influenza virus antigen in parenchymal, not endothelial cells. The marked endotheliotropism of the virus following intestinal inoculation indicates that the pathogenesis of systemic influenza virus infection in mammals may differ according to the portal of entry.

    Journal of virology 2012;86;2;1158-65

  • Analysis of high-depth sequence data for studying viral diversity: a comparison of next generation sequencing platforms using Segminator II.

    Archer J, Baillie G, Watson SJ, Kellam P, Rambaut A and Robertson DL

    Computational and Evolutionary Biology, Faculty of Life Sciences, University of Manchester, Manchester, UK. john.archer@manchester.ac.uk

    Background: Next generation sequencing provides detailed insight into the variation present within viral populations, introducing the possibility of treatment strategies that are both reactive and predictive. Current software tools, however, need to be scaled up to accommodate for high-depth viral data sets, which are often temporally or spatially linked. In addition, due to the development of novel sequencing platforms and chemistries, each with implicit strengths and weaknesses, it will be helpful for researchers to be able to routinely compare and combine data sets from different platforms/chemistries. In particular, error associated with a specific sequencing process must be quantified so that true biological variation may be identified.

    Results: Segminator II was developed to allow for the efficient comparison of data sets derived from different sources. We demonstrate its usage by comparing large data sets from 12 influenza H1N1 samples sequenced on both the 454 Life Sciences and Illumina platforms, permitting quantification of platform error. For mismatches median error rates at 0.10 and 0.12%, respectively, suggested that both platforms performed similarly. For insertions and deletions median error rates within the 454 data (at 0.3 and 0.2%, respectively) were significantly higher than those within the Illumina data (0.004 and 0.006%, respectively). In agreement with previous observations these higher rates were strongly associated with homopolymeric stretches on the 454 platform. Outside of such regions both platforms had similar indel error profiles. Additionally, we apply our software to the identification of low frequency variants.

    Conclusion: We have demonstrated, using Segminator II, that it is possible to distinguish platform specific error from biological variation using data derived from two different platforms. We have used this approach to quantify the amount of error present within the 454 and Illumina platforms in relation to genomic location as well as location on the read. Given that next generation data is increasingly important in the analysis of drug-resistance and vaccine trials, this software will be useful to the pathogen research community. A zip file containing the source code and jar file is freely available for download from http://www.bioinf.manchester.ac.uk/segminator/.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/H012419/1; Wellcome Trust: 095831

    BMC bioinformatics 2012;13;47

  • Specific capture and whole-genome sequencing of viruses from clinical samples.

    Depledge DP, Palser AL, Watson SJ, Lai IY, Gray ER, Grant P, Kanda RK, Leproust E, Kellam P and Breuer J

    Division of Infection and Immunity, University College London, London, United Kingdom. d.depledge@ucl.ac.uk

    Whole genome sequencing of viruses directly from clinical samples is integral for understanding the genetics of host-virus interactions. Here, we report the use of sample sparing target enrichment (by hybridisation) for viral nucleic acid separation and deep-sequencing of herpesvirus genomes directly from a range of clinical samples including saliva, blood, virus vesicles, cerebrospinal fluid, and tumour cell lines. We demonstrate the effectiveness of the method by deep-sequencing 13 highly cell-associated human herpesvirus genomes and generating full length genome alignments at high read depth. Moreover, we show the specificity of the method enables the study of viral population structures and their diversity within a range of clinical samples types.

    Funded by: Department of Health; Medical Research Council: G07008, G0700814, G0900950; Wellcome Trust: 081703MA

    PloS one 2011;6;11;e27805

* quick link - http://q.sanger.ac.uk/virusgen