Robert G Webster

St. Jude Children's Research Hospital, Memphis, Tennessee, United States

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Publications (809)4352.99 Total impact

  • Graham A D Blyth · Wing-Fuk Chan · Robert G Webster · Katharine E Magor ·
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    ABSTRACT: Importance: Immune IFITM genes are poorly conserved across species suggesting that selective pressure from host-specific viruses has driven this divergence. We wondered whether co-evolution between viruses and their natural host would result in evasion of IFITM restriction. Ducks are the natural host of avian influenza A viruses (IAV) and display little to no disease symptoms upon infection with most strains, including highly pathogenic avian influenza. We have characterized the duck IFITM locus, and identified IFITM3 as an important restrictor of several influenza A viruses, including avian strains. With only 38% amino acid identity to human IFITM3, duck IFITM3 possesses antiviral function against influenza. Thus, despite long co-evolution of virus and host effectors in the natural host, influenza evasion of IFITM3 restriction in ducks is not apparent.
    Journal of Virology 10/2015; DOI:10.1128/JVI.01593-15 · 4.44 Impact Factor
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    ABSTRACT: Importance: The number of humans infected with avian influenza viruses is increasing, raising concerns of the emergence of avian influenza viruses resistant to neuraminidase (NA) inhibitors (NAIs). As most studies have focused on NAI-resistance in human influenza viruses, here we investigated the molecular changes in NA that could confer NAI-resistance in avian viruses grown in immortalized monolayer cells, especially those of the N3, N7 and N9 subtypes, which have caused human infections. We identified not only numerous NAI-resistant substitutions previously reported in other NA subtypes but also several novel changes conferring reduced susceptibility to NAIs, which are subtype-specific. The findings indicate that some resistance markers are common across NA subtypes but other markers needs to be determined empirically for each subtype. The study also implies that antiviral surveillance monitoring could play a critical role in the clinical management of influenza infection and an essential component of pandemic preparedness.
    Journal of Virology 08/2015; 89(21). DOI:10.1128/JVI.01514-15 · 4.44 Impact Factor
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    ABSTRACT: The virologic factors that limit the transmission of swine influenza viruses between humans are unresolved. While it has been shown that acquisition of the neuraminidase (NA) and matrix (M) gene segments from a Eurasian-lineage swine virus was required for airborne transmission of the 2009 pandemic H1N1 virus (H1N1pdm09), we show here that an arginine to lysine change in the hemagglutinin (HA) was also necessary. This change at position 149 was distal to the receptor binding site but affected virus-receptor affinity and HA dynamics, allowing the virus to replicate more efficiently in nasal turbinate epithelium and subsequently transmit between ferrets. Receptor affinity should be considered as a factor limiting swine virus spread in humans.
    Scientific Reports 08/2015; 5:12828. DOI:10.1038/srep12828 · 5.58 Impact Factor
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    ABSTRACT: Wild waterfowl, including mallard ducks, are the natural reservoir of avian influenza A virus and they are resistant to strains that would cause fatal infection in chickens. Here we investigate potential involvement of TRIM proteins in the differential response of ducks and chickens to influenza. We examine a cluster of TRIM genes located on a single scaffold in the duck genome, which is a conserved synteny group with a TRIM cluster located in the extended MHC region in chickens and turkeys. We note a TRIM27-like gene is present in ducks, and absent in chickens and turkeys. Orthologous genes are predicted in many birds and reptiles, suggesting the gene has been lost in chickens and turkeys. Using quantitative real-time PCR (qPCR) we show that TRIM27-L, and the related TRIM27.1, are upregulated 5- and 9-fold at 1 day post-infection with highly pathogenic A/Vietnam/1203/2004. To assess whether TRIM27.1 or TRIM27-L are involved in modulation of antiviral gene expression, we overexpressed them in DF1 chicken cells, and neither show any direct effect on innate immune gene expression. However, when co-transfected with duck RIG-I-N (d2CARD) to constitutively activate the MAVS pathway, TRIM27.1 weakly decreases, while TRIM27-L strongly activates innate immune signaling leading to increased transcription of antiviral genes MX1 and IFN-β. Furthermore, when both are co-expressed, the activation of the MAVS signaling pathway by TRIM27-L over-rides the inhibition by TRIM27.1. Thus, ducks have an activating TRIM27-L to augment MAVS signaling following RIG-I detection, while chickens lack both TRIM27-L and RIG-I itself. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Molecular Immunology 08/2015; 67(2 Pt B). DOI:10.1016/j.molimm.2015.07.011 · 2.97 Impact Factor
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    ABSTRACT: Chickens are susceptible to infection with a limited number of Influenza A viruses and are a potential source of a human influenza pandemic. In particular, H5 and H7 haemagglutinin subtypes can evolve from low to highly pathogenic strains in gallinaceous poultry. Ducks on the other hand are a natural reservoir for these viruses and are able to withstand most avian influenza strains. Transcriptomic sequencing of lung and ileum tissue samples from birds infected with high (H5N1) and low (H5N2) pathogenic influenza viruses has allowed us to compare the early host response to these infections in both these species. Chickens (but not ducks) lack the intracellular receptor for viral ssRNA, RIG-I and the gene for an important RIG-I binding protein, RNF135. These differences in gene content partly explain the differences in host responses to low pathogenic and highly pathogenic avian influenza virus in chicken and ducks. We reveal very different patterns of expression of members of the interferon-induced transmembrane protein (IFITM) gene family in ducks and chickens. In ducks, IFITM1, 2 and 3 are strongly up regulated in response to highly pathogenic avian influenza, where little response is seen in chickens. Clustering of gene expression profiles suggests IFITM1 and 2 have an anti-viral response and IFITM3 may restrict avian influenza virus through cell membrane fusion. We also show, through molecular phylogenetic analyses, that avian IFITM1 and IFITM3 genes have been subject to both episodic and pervasive positive selection at specific codons. In particular, avian IFITM1 showed evidence of positive selection in the duck lineage at sites known to restrict influenza virus infection. Taken together these results support a model where the IFITM123 protein family and RIG-I all play a crucial role in the tolerance of ducks to highly pathogenic and low pathogenic strains of avian influenza viruses when compared to the chicken.
    BMC Genomics 08/2015; 16(1):574. DOI:10.1186/s12864-015-1778-8 · 3.99 Impact Factor
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    Zeynep A Koçer 1☯¤ · Robert Carter · Gang Wu · Jinghui Zhang · Robert G Webster ·
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    ABSTRACT: Among the influenza A viruses (IAVs) in wild aquatic birds, only H1, H2, and H3 subtypes have caused epidemics in humans. H1N1 viruses of avian origin have also caused 3 of 5 pandemics. To understand the reappearance of H1N1 in the context of pandemic emergence , we investigated whether avian H1N1 IAVs have contributed to the evolution of human, swine, and 2009 pandemic H1N1 IAVs. On the basis of phylogenetic analysis, we concluded that the polymerase gene segments (especially PB2 and PA) circulating in North American avian H1N1 IAVs have been reintroduced to swine multiple times, resulting in different lineages that led to the emergence of the 2009 pandemic H1N1 IAVs. Moreover, the similar topologies of hemagglutinin and nucleoprotein and neuraminidase and matrix gene segments suggest that each surface glycoprotein coevolved with an internal gene segment within the H1N1 subtype. The genotype of avian H1N1 IAVs of Charadriiformes origin isolated in 2009 differs from that of avian H1N1 IAVs of Anseriformes origin. When the anti-genic sites in the hemagglutinin of all 31 North American avian H1N1 IAVs were considered, 60%-80% of the amino acids at the antigenic sites were identical to those in 1918 and/or 2009 pandemic H1N1 viruses. Thus, although the pathogenicity of avian H1N1 IAVs could not be inferred from the phylogeny due to the small dataset, the evolutionary process within the H1N1 IAV subtype suggests that the circulation of H1N1 IAVs in wild birds poses a continuous threat for future influenza pandemics in humans.
    PLoS ONE 07/2015; 10(7):e0133795. DOI:10.1371/journal.pone.0133795 · 3.23 Impact Factor
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    ABSTRACT: H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection , in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The mono-mer has some similarities to HA in the fusion pH conformation, and the monomer's formation, which results from the presence of isopro-panol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion.
    Proceedings of the National Academy of Sciences 07/2015; DOI:10.1073/pnas.1510816112 · 9.67 Impact Factor
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    ABSTRACT: Influenza A viruses of the H1N1 subtype have emerged from the avian influenza gene pool in aquatic birds and caused human pandemics at least twice during the past century. Despite this fact, surprisingly little is known about the H1N1 gene pool in the aquatic bird reservoir. A preliminary study showed that an H1N1 virus from a shorebird of the Charadriiformes order was transmitted between animals through the airborne route of infection, whereas an H1N1 virus from a bird of the Anseriformes order was not. Here we show that two of the three H1N1 viruses isolated from Charadriiformes species in 2009 were transmitted between animals through the airborne route of infection, and five H1N1 isolates from Anseriformes species were not. The one H1N1 virus from a Charadriiformes species that failed to transmit through the airborne route was a reassortant possessing multiple internal gene segments from Anseriformes species. The molecular differences between the airborne-transmissible and non-airborne-transmissible H1N1 viruses were multigenic, involving the selection of virus with human-like receptor-binding specificity (a2-6 sialic acid) and multiple differences in the polymerase complex, mainly in the PB2, PB1-F2, and nonstructural genes.
    Emerging Microbes and Infections 07/2015; 4(7). DOI:10.1038/emi.2015.40 · 2.26 Impact Factor
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    ABSTRACT: The emergence of influenza A virus (IAV) in domestic avian species and associated transmissions to mammals is unpredictable. In the Americas, the H7 IAVs are of particular concern, and there have been four separate outbreaks of highly pathogenic (HP) H7N3 in domestic poultry in North and South America between 2002 and 2012, with occasional spillover into humans. Here, we use long-term IAV surveillance in North American shorebirds at Delaware Bay, USA, from 1985 to 2012 and in ducks in Alberta, Canada, from 1976 to 2012 to determine which hemagglutinin (HA)–neuraminidase (NA) combinations predominated in Anseriformes (ducks) and Charadriiformes (shorebirds) and whether there is concordance between peaks of H7 prevalence and transmission in wild aquatic birds and the emergence of H7 IAVs in poultry and humans. Whole-genome sequencing supported phylogenetic and genomic constellation analyses to determine whether HP IAVs emerge in the context of specific internal gene segment sequences. Phylogenetic analysis of whole-genome sequences of the H7N3 influenza viruses from wild birds and HP H7N3 outbreaks in the Americas indicate that each HP outbreak was an independent emergence event and that the low pathogenic (LP) avian influenza precursors were most likely from dabbling ducks. The different polybasic cleavage sites in the four HP outbreaks support independent origins. At the 95% nucleotide percent identity-level phylogenetic analysis showed that the wild duck HA, PB1, and M sequences clustered with the poultry and human outbreak sequences. The genomic constellation analysis strongly suggests that gene segments/virus flow from wild birds to domestic poultry.
    Emerging Microbes and Infections 06/2015; 4(6):e35. DOI:10.1038/emi.2015.35 · 2.26 Impact Factor
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    ABSTRACT: The recently detected zoonotic H3N2 variant influenza A (H3N2v) viruses have caused 343 documented cases of human infection linked to contact with swine. An effective vaccine is needed for these viruses, which may acquire easy transmissibility among humans. However, viruses isolated from human cases do not replicate well in embryonated chicken eggs, posing an obstacle to egg-based vaccine production. To address this issue, we sought to identify egg-adaptive mutations in surface proteins that increase the yield of candidate vaccine viruses (CVVs) in eggs while preserving their immunizing effectiveness. After serial passage of a representative H3N2v isolate (A/Indiana/08/2011), we identified several egg-adaptive combinations of HA mutations and assessed the egg-based replication, antigenicity, and immunogenicity of A/Puerto Rico/8/34 (H1N1, PR8)-based 6+2 reverse genetics CVVs carrying these mutations. Here we demonstrate that the respective combined HA substitutions G1861V+N2461K, N1651K+G1861V, T1281N+N1651K+R762G, and T1281N+N1651K+I102M, all identified after egg passage, enhanced the replication of the CVVs in eggs without substantially affecting their antigenicity or immunogenicity. The mutations were stable, and the mutant viruses acquired no additional substitutions during six subsequent egg passages. We found two crucial mutations, G186V, which was previously defined, and N246K, which in combination improved virus yield in eggs without significantly impacting antigenicity or immunogenicity. This combination of egg-adaptive mutations appears to most effectively generate high egg-based yields of influenza A/Indiana/08/2011-like CVVs. Copyright © 2015. Published by Elsevier Ltd.
    Vaccine 05/2015; 33(28). DOI:10.1016/j.vaccine.2015.05.011 · 3.62 Impact Factor
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    ABSTRACT: Human infection with avian influenza A(H7N9) virus is associated mainly with the exposure to infected poultry. The factors that allow interspecies transmission but limit human-to-human transmission are unknown. Here we show that A/Anhui/1/2013(H7N9) influenza virus infection of chickens (natural hosts) is asymptomatic and that it generates a high genetic diversity. In contrast, diversity is tightly restricted in infected ferrets, limiting further adaptation to a fully transmissible form. Airborne transmission in ferrets is accompanied by the mutations in PB1, NP and NA genes that reduce viral polymerase and neuraminidase activity. Therefore, while A(H7N9) virus can infect mammals, further adaptation appears to incur a fitness cost. Our results reveal that a tight genetic bottleneck during avian-to-mammalian transmission is a limiting factor in A(H7N9) influenza virus adaptation to mammals. This previously unrecognized biological mechanism limiting species jumps provides a measure of adaptive potential and may serve as a risk assessment tool for pandemic preparedness.
    Nature Communications 04/2015; 6:6553. DOI:10.1038/ncomms7553 · 11.47 Impact Factor
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    Jeremy C Jones · Stephanie Sonnberg · Richard J Webby · Robert G Webster ·
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    ABSTRACT: Low pathogenicity avian influenza A(H7N9) virus has been detected in poultry since 2013, and the virus has caused >450 infections in humans. The mode of subtype H7N9 virus transmission between avian species remains largely unknown, but various wild birds have been implicated as a source of transmission. H7N9 virus was recently detected in a wild sparrow in Shanghai, China, and passerine birds, such as finches, which share space and resources with wild migratory birds, poultry, and humans, can be productively infected with the virus. We demonstrate that interspecies transmission of H7N9 virus occurs readily between society finches and bobwhite quail but only sporadically between finches and chickens. Inoculated finches are better able to infect naive poultry than the reverse. Transmission occurs through shared water but not through the airborne route. It is therefore conceivable that passerine birds may serve as vectors for dissemination of H7N9 virus to domestic poultry.
    Emerging Infectious Diseases 04/2015; 21(4):619-628. DOI:10.3201/eid2104.141703 · 6.75 Impact Factor
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    ABSTRACT: Background: An effective vaccine is urgently needed against the H7N9 avian influenza virus. We evaluated the immunogenicity and protective efficacy of a split-virion H7N9 vaccine with or without the oil-in-water adjuvants in ferrets. Methods: Ferrets were vaccinated with 2 doses of unadjuvanted, MF59 or AS03-adjuvanted A/Shanghai/2/2013 (H7N9) vaccine, and the induction of antibodies to hemagglutinin (HA) or neuraminidase proteins was evaluated. Ferrets were then challenged with wild-type H7N9 virus to assess the vaccine's protective efficacy. The vaccine composition and integrity was also evaluated in vitro. Results: Adjuvanted vaccines stimulated robust serum antibody titers against HA and neuraminidase compared with the unadjuvanted vaccines. Although there was a difference in adjuvanticity between AS03 and MF59 at a lower dose (3.75 µg of HA), both adjuvants induced comparable antibody responses after 2 doses of 15 µg. On challenge, ferrets that received adjuvanted vaccines showed lower viral burden than the control or unadjuvanted vaccine group. In vitro examinations revealed that the vaccine contained visible split-virus particles and retained the native conformation of HA recognizable by polyclonal and monoclonal antibodies. Conclusions: The adjuvanted H7N9 vaccines demonstrated superior immunogenicity and protective efficacy against H7N9 infection in ferrets and hold potential as a vaccination regimen.
    The Journal of Infectious Diseases 02/2015; 212(4). DOI:10.1093/infdis/jiv099 · 6.00 Impact Factor
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    ABSTRACT: Unlabelled: Influenza A and B viruses are human pathogens that are regarded to cause almost equally significant disease burdens. Neuraminidase (NA) inhibitors (NAIs) are the only class of drugs available to treat influenza A and B virus infections, so the development of NAI-resistant viruses with superior fitness is a public health concern. The fitness of NAI-resistant influenza B viruses has not been widely studied. Here we examined the replicative capacity and relative fitness in normal human bronchial epithelial (NHBE) cells of recombinant influenza B/Yamanashi/166/1998 viruses containing a single amino acid substitution in NA generated by reverse genetics (rg) that is associated with NAI resistance. The replication in NHBE cells of viruses with reduced inhibition by oseltamivir (recombinant virus with the E119A mutation generated by reverse genetics [rg-E119A], rg-D198E, rg-I222T, rg-H274Y, rg-N294S, and rg-R371K, N2 numbering) or zanamivir (rg-E119A and rg-R371K) failed to be inhibited by the presence of the respective NAI. In a fluorescence-based assay, detection of rg-E119A was easily masked by the presence of NAI-susceptible virus. We coinfected NHBE cells with NAI-susceptible and -resistant viruses and used next-generation deep sequencing to reveal the order of relative fitness compared to that of recombinant wild-type (WT) virus generated by reverse genetics (rg-WT): rg-H274Y > rg-WT > rg-I222T > rg-N294S > rg-D198E > rg-E119A ≫ rg-R371K. Based on the lack of attenuated replication of rg-E119A in NHBE cells in the presence of oseltamivir or zanamivir and the fitness advantage of rg-H274Y over rg-WT, we emphasize the importance of these substitutions in the NA glycoprotein. Human infections with influenza B viruses carrying the E119A or H274Y substitution could limit the therapeutic options for those infected; the emergence of such viruses should be closely monitored. Importance: Influenza B viruses are important human respiratory pathogens contributing to a significant portion of seasonal influenza virus infections worldwide. The development of resistance to a single class of available antivirals, the neuraminidase (NA) inhibitors (NAIs), is a public health concern. Amino acid substitutions in the NA glycoprotein of influenza B virus not only can confer antiviral resistance but also can alter viral fitness. Here we used normal human bronchial epithelial (NHBE) cells, a model of the human upper respiratory tract, to examine the replicative capacities and fitness of NAI-resistant influenza B viruses. We show that virus with an E119A NA substitution can replicate efficiently in NHBE cells in the presence of oseltamivir or zanamivir and that virus with the H274Y NA substitution has a relative fitness greater than that of the wild-type NAI-susceptible virus. This study is the first to use NHBE cells to determine the fitness of NAI-resistant influenza B viruses.
    Journal of Virology 02/2015; 89(8). DOI:10.1128/JVI.02473-14 · 4.44 Impact Factor

Publication Stats

58k Citations
4,352.99 Total Impact Points


  • 1971-2015
    • St. Jude Children's Research Hospital
      • Department of Infectious Diseases
      Memphis, Tennessee, United States
  • 2012
    • The Scripps Research Institute
      La Jolla, California, United States
  • 2005-2012
    • Shantou University
      • International Institute of Infection and Immunity
      Swatow, Guangdong, China
    • Centers for Disease Control and Prevention
      Atlanta, Michigan, United States
    • National Institute of Veterinary Research, Vietnam
      Hà Nội, Ha Nội, Vietnam
  • 2010-2011
    • University of Alberta
      • Department of Biological Sciences
      Edmonton, Alberta, Canada
    • Korea Research Institute of Bioscience and Biotechnology KRIBB
      • Viral Infectious Disease Research Center
      Anzan, Gyeonggi Province, South Korea
  • 1977-2011
    • The University of Hong Kong
      • Department of Microbiology
      Hong Kong, Hong Kong
  • 2005-2010
    • Ivanovsky Institute of Virology
      Moskva, Moscow, Russia
  • 2002-2010
    • University of Tennessee
      • Department of Pathology
      Knoxville, Tennessee, United States
    • Philipps-Universität Marburg
      • Institut für Virologie
      Marburg, Hesse, Germany
    • Istituto Superiore di Sanità
      • Department of Infectious, Parasitic and Immune-mediated Diseases
      Roma, Latium, Italy
  • 2009
    • Chungbuk National University
      • College of Medicine and Medical Research Institute
      Tyundyu, North Chungcheong, South Korea
  • 2006
    • The University of Tennessee Medical Center at Knoxville
      Knoxville, Tennessee, United States
    • University of Michigan
      • School of Public Health
      Ann Arbor, Michigan, United States
  • 2004
    • University of Maryland, College Park
      • Virginia-Maryland Regional College of Veterinary Medicine
      CGS, Maryland, United States
  • 1977-2004
    • Queen Mary Hospital
      Hong Kong, Hong Kong
  • 2001
    • The University of Memphis
      Memphis, Tennessee, United States
  • 1999
    • Emory University
      Atlanta, Georgia, United States
    • University of Wisconsin, Madison
      • Department of Pathobiological Sciences
      Mississippi, United States
  • 1992-1997
    • University of Massachusetts Medical School
      • Department of Pathology
      Worcester, MA, United States
  • 1996
    • Kimron Veterinary Institute
      Beit Dajan, Central District, Israel
  • 1994
    • Sapporo University
      Sapporo, Hokkaidō, Japan
    • Erasmus Universiteit Rotterdam
      • Department of Virology
      Rotterdam, South Holland, Netherlands
  • 1987-1990
    • University of Alabama at Birmingham
      • Department of Microbiology
      Birmingham, AL, United States
    • Georgia Poultry Laboratory Network
      Georgia, United States
  • 1986-1990
    • University of Otago
      • Virus Research Unit
      Taieri, Otago, New Zealand
    • Hokkaido University
      • Laboratory of Radiation Biology
      Sapporo, Hokkaidō, Japan
  • 1988
    • Johns Hopkins University
      Baltimore, Maryland, United States
    • University of Louisville
      • Department of Microbiology and Immunology
      Louisville, Kentucky, United States
  • 1983-1987
    • National Institute for Biological Standards and Control
      • Division of Virology
      Potters Bar, England, United Kingdom
  • 1985
    • The University of Tennessee Health Science Center
      • Department of Microbiology, Immunology and Biochemistry
      Memphis, Tennessee, United States
  • 1981
    • Oxford University Hospitals NHS Trust
      • Nuffield Department of Medicine
      Oxford, England, United Kingdom
  • 1978-1981
    • Wistar Institute
      Filadelfia, Pennsylvania, United States
  • 1980
    • MRC National Institute for Medical Research
      Londinium, England, United Kingdom
  • 1963-1979
    • Australian National University
      • John Curtin School of Medical Research
      Canberra, Australian Capital Territory, Australia