Professor Paul Kellam

Paul is the Virus Genomics team leader and is a Professor of Viral Pathogenesis at University College London. Paul's laboratory investigates genetic variation of viruses and the host organisms they infect to understand host-virus interactions and the functional consequences on virus pathogenesis.

Paul's research career has spanned pharmaceutical industry Research and Development and academic research, focusing throughout on the complex relationships between the human host and medically important viruses. Paul has a degree in Microbiology from Reading University and a PhD investigating HIV drug resistance from the Wellcome Foundation laboratories and Imperial College, London. In 1996 he joined Professor Weiss' laboratory at the Institute of Cancer Research (ICR) as a CRC research fellow. He moved to UCL in 1999, establishing his own laboratory focusing on how host and pathogen genetic variation and gene expression programs are integrated during disease and host responses to infection. At UCL, the Kellam laboratory focused on how the B-cell transcriptional environment influences the normal and tumour biology of the oncogenic herpesvirus KSHV and this work continues at the Sanger Institute.

In 2009 Paul established the Virus Genomics laboratory at the Wellcome Trust Sanger Institute to investigate genetic variation of hosts and viruses and to determine the molecular and pathogenic consequences on virus pathogenesis. Paul's laboratory combines molecular biology, virology and genetics with computational research to address basic biological questions in infection and immunity. In particular, the laboratory uses next-generation sequencing of virus genomes such as HIV, influenza virus, coronaviruses and human herpesviruses and next-generation sequencing of the human genomes from people infected with these viruses.

Selected Publications

2015 Publications

  • IVA: accurate de novo assembly of RNA virus genomes.

    Hunt M, Gall A, Ong SH, Brener J, Ferns B, Goulder P, Nastouli E, Keane JA, Kellam P and Otto TD

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, UK.

    Motivation: An accurate genome assembly from short read sequencing data is critical for downstream analysis, for example allowing investigation of variants within a sequenced population. However, assembling sequencing data from virus samples, especially RNA viruses, into a genome sequence is challenging due to the combination of viral population diversity and extremely uneven read depth caused by amplification bias in the inevitable reverse transcription and polymerase chain reaction amplification process of current methods.

    Results: We developed a new de novo assembler called IVA (Iterative Virus Assembler) designed specifically for read pairs sequenced at highly variable depth from RNA virus samples. We tested IVA on datasets from 140 sequenced samples from human immunodeficiency virus-1 or influenza-virus-infected people and demonstrated that IVA outperforms all other virus de novo assemblers.

    Availability and implementation: The software runs under Linux, has the GPLv3 licence and is freely available from


    Supplementary information: Supplementary data are available at Bioinformatics online.

    Bioinformatics (Oxford, England) 2015;31;14;2374-6

  • Immediate T-Helper 17 Polarization Upon Triggering CD11b/c on HIV-Exposed Dendritic Cells.

    Wilflingseder D, Schroll A, Hackl H, Gallasch R, Frampton D, Lass-Flörl C, Pancino G, Saez-Cirion A, Lambotte O, Weiss L, Kellam P, Trajanoski Z, Geijtenbeek T, Weiss G and Posch W

    Division of Hygiene and Medical Microbiology.

    Early on in human immunodeficiency virus (HIV) type 1 infection, gut T-helper (Th) 17 cells are massively depleted leading eventually to compromised intestinal barrier function and excessive immune activation. In contrast, the functional Th17 cell compartment of the gut is well-maintained in nonpathogenic simian immunodeficiency virus infection as well as HIV-1 long-term nonprogressors. Here, we show that dendritic cells (DCs) loaded with HIV-1 bearing high surface complement levels after incubation in plasma from HIV-infected individuals secreted significantly higher concentrations of Th17-polarizing cytokines than DCs exposed to nonopsonized HIV-1. The enhanced Th17-polarizing capacity of in vitro-generated and BDCA-1(+) DCs directly isolated from blood was linked to activation of ERK. In addition, C3a produced from DCs exposed to complement-opsonized HIV was associated with the higher Th17 polarization. Our in vitro and ex vivo data, therefore, indicate that complement opsonization of HIV-1 strengthens DC-mediated antiviral immune functions by simultaneously triggering Th17 expansion and intrinsic C3 formation via DC activation.

    The Journal of infectious diseases 2015;212;1;44-56

  • Discovery of a polyomavirus in European badgers (Meles meles) and the evolution of host range in the family Polyomaviridae.

    Hill SC, Murphy AA, Cotten M, Palser AL, Benson P, Lesellier S, Gormley E, Richomme C, Grierson S, Bhuachalla DN, Chambers M, Kellam P, Boschiroli ML, Ehlers B, Jarvis MA and Pybus OG

    Department of Zoology, University of Oxford, UK.

    Polyomaviruses infect a diverse range of mammalian and avian hosts, and are associated with a variety of symptoms. However, it is unknown whether the viruses are found in all mammalian families and the evolutionary history of the polyomaviruses is still unclear. Here, we report the discovery of a novel polyomavirus in the European badger (Meles meles), which to our knowledge represents the first polyomavirus to be characterized in the family Mustelidae, and within a European carnivoran. Although the virus was discovered serendipitously in the supernatant of a cell culture inoculated with badger material, we subsequently confirmed its presence in wild badgers. The European badger polyomavirus was tentatively named Meles meles polyomavirus 1 (MmelPyV1). The genome is 5187 bp long and encodes proteins typical of polyomaviruses. Phylogenetic analyses including all known polyomavirus genomes consistently group MmelPyV1 with California sea lion polyomavirus 1 across all regions of the genome. Further evolutionary analyses revealed phylogenetic discordance amongst polyomavirus genome regions, possibly arising from evolutionary rate heterogeneity, and a complex association between polyomavirus phylogeny and host taxonomic groups.

    The Journal of general virology 2015;96;Pt 6;1411-22

  • Bat and pig IFN-induced transmembrane protein 3 restrict cell entry by influenza virus and lyssaviruses.

    Benfield CT, Smith SE, Wright E, Wash RS, Ferrara F, Temperton NJ and Kellam P

    Department of Pathology and Pathogen Biology, The Royal Veterinary College, Hatfield, UK

    IFN-induced transmembrane protein 3 (IFITM3) is a restriction factor that blocks cytosolic entry of numerous viruses that utilize acidic endosomal entry pathways. In humans and mice, IFITM3 limits influenza-induced morbidity and mortality. Although many IFITM3-sensitive viruses are zoonotic, whether IFITMs function as antiviral restriction factors in mammalian species other than humans and mice is unknown. Here, IFITM3 orthologues in the microbat (Myotis myotis) and pig (Sus scrofa domesticus) were identified using rapid amplification of cDNA ends. Amino acid residues known to be important for IFITM3 function were conserved in the pig and microbat orthologues. Ectopically expressed pig and microbat IFITM3 co-localized with transferrin (early endosomes) and CD63 (late endosomes/multivesicular bodies). Pig and microbat IFITM3 restricted cell entry mediated by multiple influenza haemagglutinin subtypes and lyssavirus glycoproteins. Expression of pig or microbat IFITM3 in A549 cells reduced influenza virus yields and nucleoprotein expression. Conversely, small interfering RNA knockdown of IFITM3 in pig NPTr cells and primary microbat cells enhanced virus replication, demonstrating that these genes are functional in their species of origin at endogenous levels. In summary, we showed that IFITMs function as potent broad-spectrum antiviral effectors in two mammals - pigs and bats - identified as major reservoirs for emerging viruses.

    Funded by: Medical Research Council: G1000413; Wellcome Trust: 098051

    The Journal of general virology 2015;96;Pt 5;991-1005

  • Genome diversity of Epstein-Barr virus from multiple tumor types and normal infection.

    Palser AL, Grayson NE, White RE, Corton C, Correia S, Ba Abdullah MM, Watson SJ, Cotten M, Arrand JR, Murray PG, Allday MJ, Rickinson AB, Young LS, Farrell PJ and Kellam P

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

    Unlabelled: Epstein-Barr virus (EBV) infects most of the world's population and is causally associated with several human cancers, but little is known about how EBV genetic variation might influence infection or EBV-associated disease. There are currently no published wild-type EBV genome sequences from a healthy individual and very few genomes from EBV-associated diseases. We have sequenced 71 geographically distinct EBV strains from cell lines, multiple types of primary tumor, and blood samples and the first EBV genome from the saliva of a healthy carrier. We show that the established genome map of EBV accurately represents all strains sequenced, but novel deletions are present in a few isolates. We have increased the number of type 2 EBV genomes sequenced from one to 12 and establish that the type 1/type 2 classification is a major feature of EBV genome variation, defined almost exclusively by variation of EBNA2 and EBNA3 genes, but geographic variation is also present. Single nucleotide polymorphism (SNP) density varies substantially across all known open reading frames and is highest in latency-associated genes. Some T-cell epitope sequences in EBNA3 genes show extensive variation across strains, and we identify codons under positive selection, both important considerations for the development of vaccines and T-cell therapy. We also provide new evidence for recombination between strains, which provides a further mechanism for the generation of diversity. Our results provide the first global view of EBV sequence variation and demonstrate an effective method for sequencing large numbers of genomes to further understand the genetics of EBV infection.

    Importance: Most people in the world are infected by Epstein-Barr virus (EBV), and it causes several human diseases, which occur at very different rates in different parts of the world and are linked to host immune system variation. Natural variation in EBV DNA sequence may be important for normal infection and for causing disease. Here we used rapid, cost-effective sequencing to determine 71 new EBV sequences from different sample types and locations worldwide. We showed geographic variation in EBV genomes and identified the most variable parts of the genome. We identified protein sequences that seem to have been selected by the host immune system and detected variability in known immune epitopes. This gives the first overview of EBV genome variation, important for designing vaccines and immune therapy for EBV, and provides techniques to investigate relationships between viral sequence variation and EBV-associated diseases.

    Funded by: Wellcome Trust: 098051

    Journal of virology 2015;89;10;5222-37

  • Local evolutionary patterns of human respiratory syncytial virus derived from whole-genome sequencing.

    Agoti CN, Otieno JR, Munywoki PK, Mwihuri AG, Cane PA, Nokes DJ, Kellam P and Cotten M

    KEMRI-Wellcome Trust Research Programme, Kilifi, Kenya.

    Unlabelled: Human respiratory syncytial virus (RSV) is associated with severe childhood respiratory infections. A clear description of local RSV molecular epidemiology, evolution, and transmission requires detailed sequence data and can inform new strategies for virus control and vaccine development. We have generated 27 complete or nearly complete genomes of RSV from hospitalized children attending a rural coastal district hospital in Kilifi, Kenya, over a 10-year period using a novel full-genome deep-sequencing process. Phylogenetic analysis of the new genomes demonstrated the existence and cocirculation of multiple genotypes in both RSV A and B groups in Kilifi. Comparison of local versus global strains demonstrated that most RSV A variants observed locally in Kilifi were also seen in other parts of the world, while the Kilifi RSV B genomes encoded a high degree of variation that was not observed in other parts of the world. The nucleotide substitution rates for the individual open reading frames (ORFs) were highest in the regions encoding the attachment (G) glycoprotein and the NS2 protein. The analysis of RSV full genomes, compared to subgenomic regions, provided more precise estimates of the RSV sequence changes and revealed important patterns of RSV genomic variation and global movement. The novel sequencing method and the new RSV genomic sequences reported here expand our knowledge base for large-scale RSV epidemiological and transmission studies.

    Importance: The new RSV genomic sequences and the novel sequencing method reported here provide important data for understanding RSV transmission and vaccine development. Given the complex interplay between RSV A and RSV B infections, the existence of local RSV B evolution is an important factor in vaccine deployment.

    Funded by: Wellcome Trust: 077092, 084633, 100542, 102975

    Journal of virology 2015;89;7;3444-54

  • Host genetics of Epstein-Barr virus infection, latency and disease.

    Houldcroft CJ and Kellam P

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, UK; Division of Biological Anthropology, Department of Archaeology and Anthropology, University of Cambridge, Cambridge, UK.

    Epstein-Barr virus (EBV) infects 95% of the adult population and is the cause of infectious mononucleosis. It is also associated with 1% of cancers worldwide, such as nasopharyngeal carcinoma, Hodgkin's lymphoma and Burkitt's lymphoma. Human and cancer genetic studies are now major forces determining gene variants associated with many cancers, including nasopharyngeal carcinoma and Hodgkin's lymphoma. Host genetics is also important in infectious disease; however, there have been no large-scale efforts towards understanding the contribution that human genetic variation plays in primary EBV infection and latency. This review covers 25 years of studies into host genetic susceptibility to EBV infection and disease, from candidate gene studies, to the first genome-wide association study of EBV antibody response, and an EBV-status stratified genome-wide association study of Hodgkin's lymphoma. Although many genes are implicated in EBV-related disease, studies are often small, not replicated or followed up in a different disease. Larger, appropriately powered genomic studies to understand the host response to EBV will be needed to move our understanding of the biology of EBV infection beyond the handful of genes currently identified. Fifty years since the discovery of EBV and its identification as a human oncogenic virus, a glimpse of the future is shown by the first whole-genome and whole-exome studies, revealing new human genes at the heart of the host-EBV interaction.

    Funded by: Medical Research Council: G0900209; Wellcome Trust: 098051

    Reviews in medical virology 2015;25;2;71-84

  • PANGEA-HIV: phylogenetics for generalised epidemics in Africa.

    Pillay D, Herbeck J, Cohen MS, de Oliveira T, Fraser C, Ratmann O, Brown AL, Kellam P and PANGEA-HIV Consortium

    Wellcome Trust-Africa Centre for Health and Population Studies, University of Kwazulu-Natal, Durban, South Africa. Electronic address:

    The Lancet. Infectious diseases 2015;15;3;259-61

  • Identification of a novel human rhinovirus C type by antibody capture VIDISCA-454.

    Jazaeri Farsani SM, Oude Munnink BB, Canuti M, Deijs M, Cotten M, Jebbink MF, Verhoeven J, Kellam P, Loens K, Goossens H, Ieven M and van der Hoek L

    Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Amsterdam 1105 AZ, the Netherlands.

    Causative agents for more than 30 percent of respiratory infections remain unidentified, suggesting that unknown respiratory pathogens might be involved. In this study, antibody capture VIDISCA-454 (virus discovery cDNA-AFLP combined with Roche 454 high-throughput sequencing) resulted in the discovery of a novel type of rhinovirus C (RV-C). The virus has an RNA genome of at least 7054 nt and carries the characteristics of rhinovirus C species. The gene encoding viral protein 1, which is used for typing, has only 81% nucleotide sequence identity with the closest known RV-C type, and, therefore, the virus represents the first member of a novel type, named RV-C54.

    Viruses 2015;7;1;239-51

2014 Publications

  • Molecular evolution of broadly neutralizing Llama antibodies to the CD4-binding site of HIV-1.

    McCoy LE, Rutten L, Frampton D, Anderson I, Granger L, Bashford-Rogers R, Dekkers G, Strokappe NM, Seaman MS, Koh W, Grippo V, Kliche A, Verrips T, Kellam P, Fassati A and Weiss RA

    Wohl Virion Centre and Medical Research Council (MRC) Centre for Medical Molecular Virology, Division of Infection and Immunity, University College London, London, United Kingdom.

    To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols.

    Funded by: Medical Research Council: G0900950, MC_EX_G0800785

    PLoS pathogens 2014;10;12;e1004552

  • Novel porcine-like human G26P[19] rotavirus identified in hospitalized paediatric diarrhoea patients in Ho Chi Minh City, Vietnam.

    My PV, Rabaa MA, Donato C, Cowley D, Phat VV, Dung TT, Anh PH, Vinh H, Bryant JE, Kellam P, Thwaites G, Woolhouse ME, Kirkwood CD and Baker S

    The Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

    During a hospital-based diarrhoeal disease study conducted in Ho Chi Minh City, Vietnam from 2009 to 2010, we identified four symptomatic children infected with G26P[19] rotavirus (RV)--an atypical variant that has not previously been reported in human gastroenteritis. To determine the genetic structure and investigate the origin of this G26P[19] strain, the whole genome of a representative example was characterized, revealing a novel genome constellation: G26-P[19]-I5-R1-C1-M1-A8-N1-T1-E1-H1. The genome segments were most closely related to porcine (VP7, VP4, VP6 and NSP1) and Wa-like porcine RVs (VP1-3 and NSP2-5). We proposed that this G26P[19] strain was the product of zoonotic transmission coupled with one or more reassortment events occurring in human and/or animal reservoirs. The identification of such strains has potential implications for vaccine efficacy in south-east Asia, and outlines the utility of whole-genome sequencing for studying RV diversity and zoonotic potential during disease surveillance.

    Funded by: Wellcome Trust: 089276/2/09/2, 095831, 100087, 100087/Z/12/Z, WT/093724/Z/10/Z

    The Journal of general virology 2014;95;Pt 12;2727-33

  • Respiratory tract samples, viral load, and genome fraction yield in patients with Middle East respiratory syndrome.

    Memish ZA, Al-Tawfiq JA, Makhdoom HQ, Assiri A, Alhakeem RF, Albarrak A, Alsubaie S, Al-Rabeeah AA, Hajomar WH, Hussain R, Kheyami AM, Almutairi A, Azhar EI, Drosten C, Watson SJ, Kellam P, Cotten M and Zumla A

    Global Centre for Mass Gatherings Medicine and Ministry of Health, Riyadh, Kingdom of Saudi Arabia and College of Medicine, Alfaisal University.

    Background: Analysis of clinical samples from patients with new viral infections is critical to confirm the diagnosis, to specify the viral load, and to sequence data necessary for characterizing the viral kinetics, transmission, and evolution. We analyzed samples from 112 patients infected with the recently discovered Middle East respiratory syndrome coronavirus (MERS-CoV).

    Methods: Respiratory tract samples from cases of MERS-CoV infection confirmed by polymerase chain reaction (PCR) were investigated to determine the MERS-CoV load and fraction of the MERS-CoV genome. These values were analyzed to determine associations with clinical sample type.

    Results: Samples from 112 individuals in which MERS-CoV was detected by PCR were analyzed, of which 13 were sputum samples, 64 were nasopharyngeal swab specimens, 30 were tracheal aspirates, and 3 were bronchoalveolar lavage specimens; 2 samples were of unknown origin. Tracheal aspirates yielded significantly higher MERS-CoV loads, compared with nasopharyngeal swab specimens (P = .005) and sputum specimens (P = .0001). Tracheal aspirates had viral loads similar to those in bronchoalveolar lavage samples (P = .3079). Bronchoalveolar lavage samples and tracheal aspirates had significantly higher genome fraction than nasopharyngeal swab specimens (P = .0095 and P = .0002, respectively) and sputum samples (P = .0009 and P = .0001, respectively). The genome yield from tracheal aspirates and bronchoalveolar lavage samples were similar (P = .1174).

    Conclusions: Lower respiratory tract samples yield significantly higher MERS-CoV loads and genome fractions than upper respiratory tract samples.

    Funded by: Department of Health; Wellcome Trust

    The Journal of infectious diseases 2014;210;10;1590-4

  • Accumulation of human-adapting mutations during circulation of A(H1N1)pdm09 influenza virus in humans in the United Kingdom.

    Elderfield RA, Watson SJ, Godlee A, Adamson WE, Thompson CI, Dunning J, Fernandez-Alonso M, Blumenkrantz D, Hussell T, MOSAIC Investigators, Zambon M, Openshaw P, Kellam P and Barclay WS

    Section of Virology, Faculty of Medicine, Imperial College London, London, United Kingdom.

    Unlabelled: The influenza pandemic that emerged in 2009 provided an unprecedented opportunity to study adaptation of a virus recently acquired from an animal source during human transmission. In the United Kingdom, the novel virus spread in three temporally distinct waves between 2009 and 2011. Phylogenetic analysis of complete viral genomes showed that mutations accumulated over time. Second- and third-wave viruses replicated more rapidly in human airway epithelial (HAE) cells than did the first-wave virus. In infected mice, weight loss varied between viral isolates from the same wave but showed no distinct pattern with wave and did not correlate with viral load in the mouse lungs or severity of disease in the human donor. However, second- and third-wave viruses induced less alpha interferon in the infected mouse lungs. NS1 protein, an interferon antagonist, had accumulated several mutations in second- and third-wave viruses. Recombinant viruses with the third-wave NS gene induced less interferon in human cells, but this alone did not account for increased virus fitness in HAE cells. Mutations in HA and NA genes in third-wave viruses caused increased binding to α-2,6-sialic acid and enhanced infectivity in human mucus. A recombinant virus with these two segments replicated more efficiently in HAE cells. A mutation in PA (N321K) enhanced polymerase activity of third-wave viruses and also provided a replicative advantage in HAE cells. Therefore, multiple mutations allowed incremental changes in viral fitness, which together may have contributed to the apparent increase in severity of A(H1N1)pdm09 influenza virus during successive waves.

    Importance: Although most people infected with the 2009 pandemic influenza virus had mild or unapparent symptoms, some suffered severe and devastating disease. The reasons for this variability were unknown, but the numbers of severe cases increased during successive waves of human infection in the United Kingdom. To determine the causes of this variation, we studied genetic changes in virus isolates from individual hospitalized patients. There were no consistent differences between these viruses and those circulating in the community, but we found multiple evolutionary changes that in combination over time increased the virus's ability to infect human cells. These adaptations may explain the remarkable ability of A(H1N1)pdm09 virus to continue to circulate despite widespread immunity and the apparent increase in severity of influenza over successive waves of infection.

    Funded by: Medical Research Council: G0802752, MC_G1001212; Wellcome Trust: 090382/Z/09/Z, 097117

    Journal of virology 2014;88;22;13269-83

  • Evidence for camel-to-human transmission of MERS coronavirus.

    Madani TA, Azhar EI and Hashem AM

    The New England journal of medicine 2014;371;14;1360

  • Phylogenetic studies of transmission dynamics in generalized HIV epidemics: an essential tool where the burden is greatest?

    Dennis AM, Herbeck JT, Brown AL, Kellam P, de Oliveira T, Pillay D, Fraser C and Cohen MS

    *Division of Infectious Diseases, University of North Carolina at Chapel Hill, Chapel Hill, NC; †Department of Microbiology, University of Washington, Seattle, WA; ‡Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, United Kingdom; §Wellcome Trust Sanger Institute, Cambridge, United Kingdom; ‖Division of Infection and Immunity, University College London, London, United Kingdom; ¶Wellcome Trust-Africa Centre for Health and Population Studies, University of Kwazula-Natal, ZA; and #Department of Infectious Disease Epidemiology, Imperial College London, London, United Kingdom.

    Efficient and effective HIV prevention measures for generalized epidemics in sub-Saharan Africa have not yet been validated at the population level. Design and impact evaluation of such measures requires fine-scale understanding of local HIV transmission dynamics. The novel tools of HIV phylogenetics and molecular epidemiology may elucidate these transmission dynamics. Such methods have been incorporated into studies of concentrated HIV epidemics to identify proximate and determinant traits associated with ongoing transmission. However, applying similar phylogenetic analyses to generalized epidemics, including the design and evaluation of prevention trials, presents additional challenges. Here we review the scope of these methods and present examples of their use in concentrated epidemics in the context of prevention. Next, we describe the current uses for phylogenetics in generalized epidemics and discuss their promise for elucidating transmission patterns and informing prevention trials. Finally, we review logistic and technical challenges inherent to large-scale molecular epidemiological studies of generalized epidemics and suggest potential solutions.

    Funded by: NCATS NIH HHS: KL2TR000084; NIAID NIH HHS: P30 AI027757, P30 AI050410, P30AI027757; NIMHD NIH HHS: L60 MD005444; Wellcome Trust: 097410

    Journal of acquired immune deficiency syndromes (1999) 2014;67;2;181-95

  • Deep sequencing of norovirus genomes defines evolutionary patterns in an urban tropical setting.

    Cotten M, Petrova V, Phan MV, Rabaa MA, Watson SJ, Ong SH, Kellam P and Baker S

    The Wellcome Trust Sanger Institute, Hinxton, United Kingdom.

    Unlabelled: Norovirus is a highly transmissible infectious agent that causes epidemic gastroenteritis in susceptible children and adults. Norovirus infections can be severe and can be initiated from an exceptionally small number of viral particles. Detailed genome sequence data are useful for tracking norovirus transmission and evolution. To address this need, we have developed a whole-genome deep-sequencing method that generates entire genome sequences from small amounts of clinical specimens. This novel approach employs an algorithm for reverse transcription and PCR amplification primer design using all of the publically available norovirus sequence data. Deep sequencing and de novo assembly were used to generate norovirus genomes from a large set of diarrheal patients attending three hospitals in Ho Chi Minh City, Vietnam, over a 2.5-year period. Positive-selection analysis and direct examination of protein changes in the virus over time identified codons in the regions encoding proteins VP1, p48 (NS1-2), and p22 (NS4) under positive selection and expands the known targets of norovirus evolutionary pressure.

    Importance: The high transmissibility and rapid evolutionary rate of norovirus, combined with a short-lived host immune responses, are thought to be the reasons why the virus causes the majority of pediatric viral diarrhea cases. The evolutionary patterns of this RNA virus have been described in detail for only a portion of the virus genome and never for a virus from a detailed urban tropical setting. We provide a detailed sequence description of the noroviruses circulating in three Ho Chi Minh City hospitals over a 2.5-year period. This study identified patterns of virus change in known sites of host immune response and identified three additional regions of the virus genome under selection that were not previously recognized. In addition, the method described here provides a robust full-genome sequencing platform for community-based virus surveillance.

    Funded by: Wellcome Trust: 100087, WT/093724

    Journal of virology 2014;88;19;11056-69

  • Genomic diversity of Epstein-Barr virus genomes isolated from primary nasopharyngeal carcinoma biopsy samples.

    Kwok H, Wu CW, Palser AL, Kellam P, Sham PC, Kwong DL and Chiang AK

    Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

    Unlabelled: Undifferentiated nasopharyngeal carcinoma (NPC) has a 100% association with Epstein-Barr virus (EBV). However, only three EBV genomes isolated from NPC patients have been sequenced to date, and the role of EBV genomic variations in the pathogenesis of NPC is unclear. We sought to obtain the sequences of EBV genomes in multiple NPC biopsy specimens in the same geographic location in order to reveal their sequence diversity. Three published EBV (B95-8, C666-1, and HKNPC1) genomes were first resequenced using the sequencing workflow of target enrichment of EBV DNA by hybridization, followed by next-generation sequencing, de novo assembly, and joining of contigs by Sanger sequencing. The sequences of eight NPC biopsy specimen-derived EBV (NPC-EBV) genomes, designated HKNPC2 to HKNPC9, were then determined. They harbored 1,736 variations in total, including 1,601 substitutions, 64 insertions, and 71 deletions, compared to the reference EBV. Furthermore, genes encoding latent, early lytic, and tegument proteins and glycoproteins were found to contain nonsynonymous mutations of potential biological significance. Phylogenetic analysis showed that the HKNPC6 and -7 genomes, which were isolated from tumor biopsy specimens of advanced metastatic NPC cases, were distinct from the other six NPC-EBV genomes, suggesting the presence of at least two parental lineages of EBV among the NPC-EBV genomes. In conclusion, much greater sequence diversity among EBV isolates derived from NPC biopsy specimens is demonstrated on a whole-genome level through a complete sequencing workflow. Large-scale sequencing and comparison of EBV genomes isolated from NPC and normal subjects should be performed to assess whether EBV genomic variations contribute to NPC pathogenesis.

    Importance: This study established a sequencing workflow from EBV DNA capture and sequencing to de novo assembly and contig joining. We reported eight newly sequenced EBV genomes isolated from primary NPC biopsy specimens and revealed the sequence diversity on a whole-genome level among these EBV isolates. At least two lineages of EBV strains are observed, and recombination among these lineages is inferred. Our study has demonstrated the value of, and provided a platform for, genome sequencing of EBV.

    Journal of virology 2014;88;18;10662-72

  • Community case clusters of Middle East respiratory syndrome coronavirus in Hafr Al-Batin, Kingdom of Saudi Arabia: a descriptive genomic study.

    Memish ZA, Cotten M, Watson SJ, Kellam P, Zumla A, Alhakeem RF, Assiri A, Rabeeah AA and Al-Tawfiq JA

    Global Centre for Mass Gatherings Medicine (GCMGM), Ministry of Health, Riyadh, Kingdom of Saudi Arabia (KSA); College of Medicine, Alfaisal University, Riyadh, Kingdom of Saudi Arabia. Electronic address:

    The Middle East respiratory syndrome coronavirus (MERS-CoV) was first described in September 2012 and to date 86 deaths from a total of 206 cases of MERS-CoV infection have been reported to the WHO. Camels have been implicated as the reservoir of MERS-CoV, but the exact source and mode of transmission for most patients remain unknown. During a 3 month period, June to August 2013, there were 12 positive MERS-CoV cases reported from the Hafr Al-Batin region district in the north east region of the Kingdom of Saudi Arabia. In addition to the different regional camel festivals in neighboring countries, Hafr Al-Batin has the biggest camel market in the entire Kingdom and hosts an annual camel festival. Thus, we conducted a detailed epidemiological, clinical and genomic study to ascertain common exposure and transmission patterns of all cases of MERS-CoV reported from Hafr Al-Batin. Analysis of previously reported genetic data indicated that at least two of the infected contacts could not have been directly infected from the index patient and alternate source should be considered. While camels appear as the likely source, other sources have not been ruled out. More detailed case control studies with detailed case histories, epidemiological information and genomic analysis are being conducted to delineate the missing pieces in the transmission dynamics of MERS-CoV outbreak.

    International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases 2014;23;63-8

  • IFITM proteins-cellular inhibitors of viral entry.

    Smith S, Weston S, Kellam P and Marsh M

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

    Interferon inducible transmembrane (IFITM) proteins are a recently discovered family of cellular anti-viral proteins that restrict the replication of a number of enveloped and non-enveloped viruses. IFITM proteins are located in the plasma membrane and endosomal membranes, the main portals of entry for many viruses. Biochemical and membrane fusion studies suggest IFITM proteins have the ability to inhibit viral entry, possibly by modulating the fluidity of cellular membranes. Here we discuss the IFITM proteins, recent work on their mode of action, and future directions for research.

    Funded by: Medical Research Council: G1000413; Wellcome Trust: 098051

    Current opinion in virology 2014;4;71-7

  • 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

  • RNA-seq analysis of host and viral gene expression highlights interaction between varicella zoster virus and keratinocyte differentiation.

    Jones M, Dry IR, Frampton D, Singh M, Kanda RK, Yee MB, Kellam P, Hollinshead M, Kinchington PR, O'Toole EA and Breuer J

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

    Varicella zoster virus (VZV) is the etiological agent of chickenpox and shingles, diseases characterized by epidermal skin blistering. Using a calcium-induced keratinocyte differentiation model we investigated the interaction between epidermal differentiation and VZV infection. RNA-seq analysis showed that VZV infection has a profound effect on differentiating keratinocytes, altering the normal process of epidermal gene expression to generate a signature that resembles patterns of gene expression seen in both heritable and acquired skin-blistering disorders. Further investigation by real-time PCR, protein analysis and electron microscopy revealed that VZV specifically reduced expression of specific suprabasal cytokeratins and desmosomal proteins, leading to disruption of epidermal structure and function. These changes were accompanied by an upregulation of kallikreins and serine proteases. Taken together VZV infection promotes blistering and desquamation of the epidermis, both of which are necessary to the viral spread and pathogenesis. At the same time, analysis of the viral transcriptome provided evidence that VZV gene expression was significantly increased following calcium treatment of keratinocytes. Using reporter viruses and immunohistochemistry we confirmed that VZV gene and protein expression in skin is linked with cellular differentiation. These studies highlight the intimate host-pathogen interaction following VZV infection of skin and provide insight into the mechanisms by which VZV remodels the epidermal environment to promote its own replication and spread.

    Funded by: Medical Research Council: G0501446, G0700814, G0900950, G9721629; NEI NIH HHS: EY08098; NINDS NIH HHS: NS064022; Wellcome Trust: 081703/B/06/Z

    PLoS pathogens 2014;10;1;e1003896

  • A membrane topology model for human interferon inducible transmembrane protein 1.

    Weston S, Czieso S, White IJ, Smith SE, Kellam P and Marsh M

    MRC Laboratory for Molecular Cell Biology, University College London, London, United Kingdom.

    InterFeron Inducible TransMembrane proteins 1-3 (IFITM1, IFITM2 and IFITM3) are a family of proteins capable of inhibiting the cellular entry of numerous human and animal viruses. IFITM1-3 are unique amongst the currently described viral restriction factors in their apparent ability to block viral entry. This restrictive property is dependant on the localisation of the proteins to plasma and endosomal membranes, which constitute the main portals of viral entry into cells. The topology of the IFITM proteins within cell membranes is an unresolved aspect of their biology. Here we present data from immunofluorescence microscopy, protease cleavage, biotin-labelling and immuno-electron microscopy assays, showing that human IFITM1 has a membrane topology in which the N-terminal domain resides in the cytoplasm, and the C-terminal domain is extracellular. Furthermore, we provide evidence that this topology is conserved for all of the human interferon-induced IFITM proteins. This model is consistent with that recently proposed for murine IFITM3, but differs from that proposed for murine IFITM1.

    Funded by: Medical Research Council: G1000413; Wellcome Trust: 098051

    PloS one 2014;9;8;e104341

  • Capturing needles in haystacks: a comparison of B-cell receptor sequencing methods.

    Bashford-Rogers RJ, Palser AL, Idris SF, Carter L, Epstein M, Callard RE, Douek DC, Vassiliou GS, Follows GA, Hubank M and Kellam P

    Background: Deep-sequencing methods are rapidly developing in the field of B-cell receptor (BCR) and T-cell receptor (TCR) diversity. These promise to revolutionise our understanding of adaptive immune dynamics, identify novel antibodies, and allow monitoring of minimal residual disease. However, different methods for BCR and TCR enrichment and amplification have been proposed. Here we perform the first systematic comparison between different methods of enrichment, amplification and sequencing for generating BCR and TCR repertoires using large sample numbers.

    Results: Resampling from the same RNA or cDNA pool results in highly correlated and reproducible repertoires, but resampling low frequency clones leads to stochastic variance. Repertoires generated by different sequencing methods (454 Roche and Illumina MiSeq) and amplification methods (multiplex PCR, 5' Rapid amplification of cDNA ends (5'RACE), and RNA-capture) are highly correlated, and resulting IgHV gene frequencies between the different methods were not significantly different. Read length has an impact on captured repertoire structure, and ultimately full-length BCR sequences are most informative for repertoire analysis as diversity outside of the CDR is very useful for phylogenetic analysis. Additionally, we show RNA-based BCR repertoires are more informative than using DNA.

    Conclusions: Repertoires generated by different sequencing and amplification methods are consistent, but we show that read lengths, depths and error profiles should be considered in experimental design, and multiple sampling approaches could be employed to minimise stochastic sampling variation. This detailed investigation of immune repertoire sequencing methods is essential for informing basic and clinical research.

    Funded by: Wellcome Trust: 095663

    BMC immunology 2014;15;29

  • Complete Genome Sequence of the WHO International Standard for HIV-1 RNA Determined by Deep Sequencing.

    Gall A, Morris C, Kellam P and Berry N

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

    The World Health Organization (WHO) International Standard for HIV-1 RNA nucleic acid assays was characterized by complete genome deep sequencing analysis. The entire coding sequence and flanking long terminal repeats (LTRs), including minority species, were assigned subtype B. This information will aid the design, development, and evaluation of HIV-1 RNA amplification assays.

    Funded by: Medical Research Council: G0600007

    Genome announcements 2014;2;1

  • Full genome virus detection in fecal samples using sensitive nucleic acid preparation, deep sequencing, and a novel iterative sequence classification algorithm.

    Cotten M, Oude Munnink B, Canuti M, Deijs M, Watson SJ, Kellam P and van der Hoek L

    Wellcome Trust Sanger Institute, Hinxton, United Kingdom.

    We have developed a full genome virus detection process that combines sensitive nucleic acid preparation optimised for virus identification in fecal material with Illumina MiSeq sequencing and a novel post-sequencing virus identification algorithm. Enriched viral nucleic acid was converted to double-stranded DNA and subjected to Illumina MiSeq sequencing. The resulting short reads were processed with a novel iterative Python algorithm SLIM for the identification of sequences with homology to known viruses. De novo assembly was then used to generate full viral genomes. The sensitivity of this process was demonstrated with a set of fecal samples from HIV-1 infected patients. A quantitative assessment of the mammalian, plant, and bacterial virus content of this compartment was generated and the deep sequencing data were sufficient to assembly 12 complete viral genomes from 6 virus families. The method detected high levels of enteropathic viruses that are normally controlled in healthy adults, but may be involved in the pathogenesis of HIV-1 infection and will provide a powerful tool for virus detection and for analyzing changes in the fecal virome associated with HIV-1 progression and pathogenesis.

    Funded by: Wellcome Trust

    PloS one 2014;9;4;e93269

  • Generation and characterization of influenza A viruses with altered polymerase fidelity.

    Cheung PP, Watson SJ, Choy KT, Fun Sia S, Wong DD, Poon LL, Kellam P, Guan Y, Malik Peiris JS and Yen HL

    Li Ka Shing Faculty of Medicine, Centre of Influenza Research, School of Public Health, The University of Hong Kong, No. 21 Sassoon Road, Pokfulam, Hong Kong SAR, China.

    Genetic diversity of influenza A viruses (IAV) acquired through the error-prone RNA-dependent RNA polymerase (RdRP) or through genetic reassortment enables perpetuation of IAV in humans through epidemics or pandemics. Here, to assess the biological significance of genetic diversity acquired through RdRP, we characterize an IAV fidelity variant derived from passaging a seasonal H3N2 virus in the presence of ribavirin, a purine analogue that increases guanosine-to-adenosine mutations. We demonstrate that a single PB1-V43I mutation increases selectivity to guanosine in A/Wuhan/359/95 (H3N2) and A/Vietnam/1203/04 (H5N1) viruses. The H5N1 PB1-V43I-recombinant virus replicates to comparable titres as the wild-type virus in vitro or in the mouse lungs. However, a decrease in viral population diversity at day 3 post inoculation is associated with a tenfold reduced lethality and neurotropism in mice. Applying a fidelity variant with reduced mutational frequency, we provide direct experimental evidence for the role of genetic diversity in IAV pathogenesis.

    Funded by: NIAID NIH HHS: HHSN272201400006C, N01AI70005; PHS HHS: HHSN27220140006C; Wellcome Trust

    Nature communications 2014;5;4794

  • Host genetic variants and gene expression patterns associated with Epstein-Barr virus copy number in lymphoblastoid cell lines.

    Houldcroft CJ, Petrova V, Liu JZ, Frampton D, Anderson CA, Gall A and Kellam P

    Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom; Division of Biological Anthropology, Department of Archaeology and Anthropology, University of Cambridge, Cambridge, United Kingdom.

    Lymphoblastoid cell lines (LCLs) are commonly used in molecular genetics, supplying DNA for the HapMap and 1000 Genomes Projects, used to test chemotherapeutic agents, and informing the basis of a number of population genetics studies of gene expression. The process of transforming human B cells into LCLs requires the presence of Epstein-Barr virus (EBV), a double-stranded DNA virus which through B-cell immortalisation maintains an episomal virus genome in every cell of an LCL at variable copy numbers. Previous studies have reported that EBV alters host-gene expression and EBV copy number may be under host genetic control. We performed a genome-wide association study of EBV genome copy number in LCLs and found the phenotype to be highly heritable, although no individual SNPs achieved a significant association with EBV copy number. The expression of two host genes (CXCL16 and AGL) was positively correlated and expression of ADARB2 was negatively correlated with EBV copy number in a genotype-independent manner. This study shows an association between EBV copy number and the gene expression profile of LCLs, and suggests that EBV copy number should be considered as a covariate in future studies of host gene expression in LCLs.

    Funded by: Medical Research Council: G0900209; Wellcome Trust: 098051

    PloS one 2014;9;10;e108384

  • Human immunodeficiency virus Tat associates with a specific set of cellular RNAs.

    Bouwman RD, Palser A, Parry CM, Coulter E, Rasaiyaah J, Kellam P and Jenner RG

    MRC Centre for Medical Molecular Virology, Division of Infection and Immunity, University College London, London WC1E 6BT, UK.

    Background: Human Immunodeficiency Virus 1 (HIV-1) exhibits a wide range of interactions with the host cell but whether viral proteins interact with cellular RNA is not clear. A candidate interacting factor is the trans-activator of transcription (Tat) protein. Tat is required for expression of virus genes but activates transcription through an unusual mechanism; binding to an RNA stem-loop, the transactivation response element (TAR), with the host elongation factor P-TEFb. HIV-1 Tat has also been shown to alter the expression of host genes during infection, contributing to viral pathogenesis but, whether Tat also interacts with cellular RNAs is unknown.

    Results: Using RNA immunoprecipitation coupled with microarray analysis, we have discovered that HIV-1 Tat is associated with a specific set of human mRNAs in T cells. mRNAs bound by Tat share a stem-loop structural element and encode proteins with common biological roles. In contrast, we do not find evidence that Tat associates with microRNAs or the RNA-induced silencing complex (RISC). The interaction of Tat with cellular RNA requires an intact RNA binding domain and Tat RNA binding is linked to an increase in RNA abundance in cell lines and during infection of primary CD4+ T cells by HIV.

    Conclusions: We conclude that Tat interacts with a specific set of human mRNAs in T cells, many of which show changes in abundance in response to Tat and HIV infection. This work uncovers a previously unrecognised interaction between HIV and its host that may contribute to viral alteration of the host cellular environment.

    Funded by: Medical Research Council: G0600081, G0802068; Wellcome Trust

    Retrovirology 2014;11;53

  • 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: 095831

    mBio 2014;5;1

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 (London, England) 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

  • Amphotericin B increases influenza A virus infection by preventing IFITM3-mediated restriction.

    Lin TY, Chin CR, Everitt AR, Clare S, Perreira JM, Savidis G, Aker AM, John SP, Sarlah D, Carreira EM, Elledge SJ, Kellam P and Brass AL

    Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA 01655, USA.

    The IFITMs inhibit influenza A virus (IAV) replication in vitro and in vivo. Here, we establish that the antimycotic heptaen, amphotericin B (AmphoB), prevents IFITM3-mediated restriction of IAV, thereby increasing viral replication. Consistent with its neutralization of IFITM3, a clinical preparation of AmphoB, AmBisome, reduces the majority of interferon's protective effect against IAV in vitro. Mechanistic studies reveal that IFITM1 decreases host-membrane fluidity, suggesting both a possible mechanism for IFITM-mediated restriction and its negation by AmphoB. Notably, we reveal that mice treated with AmBisome succumbed to a normally mild IAV infection, similar to animals deficient in Ifitm3. Therefore, patients receiving antifungal therapy with clinical preparations of AmphoB may be functionally immunocompromised and thus more vulnerable to influenza, as well as other IFITM3-restricted viral infections.

    Funded by: NIAID NIH HHS: 1R01AI091786, R01 AI091786; NIDDK NIH HHS: T32 DK007191; Wellcome Trust

    Cell reports 2013;5;4;895-908

  • 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

  • Modeling the association of space, time, and host species with variation of the HA, NA, and NS genes of H5N1 highly pathogenic avian influenza viruses isolated from birds in Romania in 2005-2007.

    Alkhamis M, Perez A, Batey N, Howard W, Baillie G, Watson S, Franz S, Focosi-Snyman R, Onita I, Cioranu R, Turcitu M, Kellam P, Brown IH and Breed AC

    Center for Animal Disease Modeling and Surveillance (CADMS), School of Veterinary Medicine, One Shields Avenue, University of California, Davis, CA 95616, USA.

    Molecular characterization studies of a diverse collection of avian influenza viruses (AIVs) have demonstrated that AIVs' greatest genetic variability lies in the HA, NA, and NS genes. The objective here was to quantify the association between geographical locations, periods of time, and host species and pairwise nucleotide variation in the HA, NA, and NS genes of 70 isolates of H5N1 highly pathogenic avian influenza virus (HPAIV) collected from October 2005 to December 2007 from birds in Romania. A mixed-binomial Bayesian regression model was used to quantify the probability of nucleotide variation between isolates and its association with space, time, and host species. As expected for the three target genes, a higher probability of nucleotide differences (odds ratios [ORs] > 1) was found between viruses sampled from places at greater geographical distances from each other, viruses sampled over greater periods of time, and viruses derived from different species. The modeling approach in the present study maybe useful in further understanding the molecular epidemiology of H5N1 HPAI virus in bird populations. The methodology presented here will be useful in predicting the most likely genetic distance for any of the three gene segments of viruses that have not yet been isolated or sequenced based on space, time, and host species during the course of an epidemic.

    Funded by: Wellcome Trust: 079643, 093724

    Avian diseases 2013;57;3;612-21

  • 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.

    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; NIDDK NIH HHS: T32 DK007191; 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.

    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.

    Clinical relevance: 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: BB/H014306/1; 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 ( 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

  • Autologous antibody capture to enrich immunogenic viruses for viral discovery.

    Oude Munnink BB, Jazaeri Farsani SM, Deijs M, Jonkers J, Verhoeven JT, Ieven M, Goossens H, de Jong MD, Berkhout B, Loens K, Kellam P, Bakker M, Canuti M, Cotten M and van der Hoek L

    Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands.

    Discovery of new viruses has been boosted by novel deep sequencing technologies. Currently, many viruses can be identified by sequencing without knowledge of the pathogenicity of the virus. However, attributing the presence of a virus in patient material to a disease in the patient can be a challenge. One approach to meet this challenge is identification of viral sequences based on enrichment by autologous patient antibody capture. This method facilitates identification of viruses that have provoked an immune response within the patient and may increase the sensitivity of the current virus discovery techniques. To demonstrate the utility of this method, virus discovery deep sequencing (VIDISCA-454) was performed on clinical samples from 19 patients: 13 with a known respiratory viral infection and 6 with a known gastrointestinal viral infection. Patient sera was collected from one to several months after the acute infection phase. Input and antibody capture material was sequenced and enrichment was assessed. In 18 of the 19 patients, viral reads from immunogenic viruses were enriched by antibody capture (ranging between 1.5x to 343x in respiratory material, and 1.4x to 53x in stool). Enriched reads were also determined in an identity independent manner by using a novel algorithm Xcompare. In 16 of the 19 patients, 21% to 100% of the enriched reads were derived from infecting viruses. In conclusion, the technique provides a novel approach to specifically identify immunogenic viral sequences among the bulk of sequences which are usually encountered during virus discovery metagenomics.

    Funded by: Wellcome Trust: 093724

    PloS one 2013;8;11;e78454

  • Defining the range of pathogens susceptible to Ifitm3 restriction using a knockout mouse model.

    Everitt AR, Clare S, McDonald JU, Kane L, Harcourt K, Ahras M, Lall A, Hale C, Rodgers A, Young DB, Haque A, Billker O, Tregoning JS, Dougan G and Kellam P

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

    The interferon-inducible transmembrane (IFITM) family of proteins has been shown to restrict a broad range of viruses in vitro and in vivo by halting progress through the late endosomal pathway. Further, single nucleotide polymorphisms (SNPs) in its sequence have been linked with risk of developing severe influenza virus infections in humans. The number of viruses restricted by this host protein has continued to grow since it was first demonstrated as playing an antiviral role; all of which enter cells via the endosomal pathway. We therefore sought to test the limits of antimicrobial restriction by Ifitm3 using a knockout mouse model. We showed that Ifitm3 does not impact on the restriction or pathogenesis of bacterial (Salmonella typhimurium, Citrobacter rodentium, Mycobacterium tuberculosis) or protozoan (Plasmodium berghei) pathogens, despite in vitro evidence. However, Ifitm3 is capable of restricting respiratory syncytial virus (RSV) in vivo either through directly restricting RSV cell infection, or by exerting a previously uncharacterised function controlling disease pathogenesis. This represents the first demonstration of a virus that enters directly through the plasma membrane, without the need for the endosomal pathway, being restricted by the IFITM family; therefore further defining the role of these antiviral proteins.

    Funded by: Medical Research Council: G0501670, MC_U117581288, U117581288

    PloS one 2013;8;11;e80723

  • 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: Department of Health: DHCS/04/G121/68; Medical Research Council: G0600371, 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.

    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; Department of Health: DHCS/04/G121/68; Medical Research Council: G0600371, G0600511, G0800767, G0800777, G0802752, G0901697, G1000758, MC_G1001212, MC_U122785833; NIAID NIH HHS: R01 AI091786, R01AI091786; NIDDK NIH HHS: P30 DK043351, T32 DK007191; 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.

    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

    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: Medical Research Council: G0801822; 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.

    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.

    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.

    Methodology/principal findings: 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.

    Conclusions/significance: 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.

    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.

    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.

    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.

    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.

    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:

    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.

    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.

    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.

    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

    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.

    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.

    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.

    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


    Supplementary information:

    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.

    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


    Funded by: Biotechnology and Biological Sciences Research Council: G18886

    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.

    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.

    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 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.

    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.

    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.

    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.

    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

    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.

    Study design/methods: 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] 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 (London, England) 1990;336;8715;585-90

Reviews, invited commentaries and book chapters

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