Adam Rubrum

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

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Publications (19)113.6 Total impact

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    ABSTRACT: The cleavage motif in the hemagglutinin (HA) protein of highly pathogenic H5 and H7 subtypes of avian influenza viruses is characterized by a peptide insertion or a multibasic cleavage site (MBCS). Here, we isolated an H4N2 virus from quails (Quail/CA12) with two additional arginines in the HA cleavage site, PEKRRTR/G, forming an MBCS-like motif. Quail/CA12 is a reassortant virus with the HA and neuraminidase (NA) gene most similar to a duck-isolated H4N2 virus, PD/CA06 with a monobasic HA cleavage site. Quail/CA12 required exogenous trypsin for efficient growth in culture and caused no clinical illness in infected chickens. Quail/CA12 had high binding preference for α2,6-linked sialic acids and showed higher replication and transmission ability in chickens and quails than PD/CA06. Although the H4N2 virus remained low pathogenic, these data suggests that the acquisition of MBCS in the field is not restricted to H5 or H7 subtypes.
    Virology. 08/2014; 468-470C:72-80.
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    ABSTRACT: Avian influenza virus subtype H9N2 has been circulating in the Middle East since the 1990s. For uncertain reasons, H9N2 was not detected in Egyptian farms until the end of 2010. Circulation of H9N2 viruses in Egyptian poultry in the presence of the enzootic highly pathogenic H5N1 subtype adds a huge risk factor to the Egyptian poultry industry. In this study, 22 H9N2 viruses collected from 2011 to 2013 in Egypt were isolated and sequenced. The genomic signatures and protein sequences of these isolates were analyzed. Multiple mammalian-host-associated mutations were detected that favor transmission from avian to mammalian hosts. Other mutations related to virulence were also identified. Phylogenetic data showed that Egyptian H9N2 viruses were closely related to viruses isolated from neighboring Middle Eastern countries, and their HA gene resembled those of viruses of the G1-like lineage. No reassortment was detected with H5N1 subtypes. Serological analysis of H9N2 virus revealed antigenic conservation among Egyptian isolates. Accordingly, continuous surveillance that results in genetic and antigenic characterization of H9N2 in Egypt is warranted.
    Archives of virology. 07/2014;
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    ABSTRACT: The H7N9 influenza virus caused significant mortality and morbidity in infected humans during an outbreak in China in 2013 stimulating vaccine development efforts. As previous H7-based vaccines have been poorly immunogenic in humans we sought to determine the immunogenic and protective properties of an inactivated whole virus vaccine derived from a 2013 H7N9 virus in ferrets. As whole virus vaccine preparations have been shown to be more immunogenic in humans, but less likely to be used, than split or surface antigen formulations, we vaccinated ferrets with a single dose of 15, 30, or 50μg of the vaccine and subsequently challenged with wild-type A/Anhui/1/2013 (H7N9) either by direct instillation or by contact with infected animals. Although ferrets vaccinated with higher doses of vaccine had higher serum hemagglutinin inhibition (HI) titers, the titers were still low. During subsequent instillation challenge, however, ferrets vaccinated with 50μg of vaccine showed no illness and shed significantly less virus than mock vaccinated controls. All vaccinated ferrets had lower virus loads in their lungs as compared to controls. In a separate study where unvaccinated-infected ferrets were placed in the same cage with vaccinated-uninfected ferrets, vaccination did not prevent infection in the contact ferrets, although they showed a trend of lower viral load. Overall, we conclude that inactivated whole-virus H7N9 vaccine was able to reduce the severity of infection and viral load, despite the lack of hemagglutinin-inhibiting antibodies.
    Vaccine. 06/2014;
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    ABSTRACT: CClade 2.2 highly pathogenic H5N1 viruses have been in continuous circulation in Egyptian poultry since 2006. Their persistence caused significant genetic drift that led to the re-classification of these viruses into sub-clades 2.2.1 and 2.2.1.1. Here, we conducted full genome sequence and phylogenetic analyses of 45 H5N1 isolated during 2006-2013 through systematic surveillance in Egypt, and 53 viruses that were previously sequenced and available in the public domain. Results indicate that H5N1 viruses in Egypt continue to evolve and a new distinct cluster has emerged. Mutations affecting viral virulence, pathogenicity, transmission, receptor-binding preference, and drug resistance were studied. In light of our findings that H5N1 in Egypt continues to evolve, surveillance and molecular studies need to be sustained.
    Journal of General Virology 04/2014; · 3.13 Impact Factor
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    ABSTRACT: Continuous circulation of influenza A(H5N1) virus among poultry in Egypt has created an epicenter in which the viruses evolve into newer subclades and continue to cause disease in humans. To detect influenza viruses in Egypt, since 2009 we have actively surveyed various regions and poultry production sectors. From August 2010 through January 2013, >11,000 swab samples were collected; 10% were positive by matrix gene reverse transcription PCR. During this period, subtype H9N2 viruses emerged, cocirculated with subtype H5N1 viruses, and frequently co-infected the same avian host. Genetic and antigenic analyses of viruses revealed that influenza A(H5N1) clade 2.2.1 viruses are dominant and that all subtype H9N2 viruses are G1-like. Cocirculation of different subtypes poses concern for potential reassortment. Avian influenza continues to threaten public and animal health in Egypt, and continuous surveillance for avian influenza virus is needed.
    Emerging Infectious Diseases 04/2014; 20(4):542-51. · 6.79 Impact Factor
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    ABSTRACT: The cleavage motif in the hemagglutinin (HA) protein of highly pathogenic H5 and H7 subtypes of avian influenza viruses is characterized by a peptide insertion or a multibasic cleavage site (MBCS). Here, we isolated an H4N2 virus from quails (Quail/CA12) with two additional arginines in the HA cleavage site, PEKRRTR/G, forming an MBCS-like motif. Quail/CA12 is a reassortant virus with the HA and neuraminidase (NA) gene most similar to a duck-isolated H4N2 virus, PD/CA06 with a monobasic HA cleavage site. Quail/CA12 required exogenous trypsin for efficient growth in culture and caused no clinical illness in infected chickens. Quail/CA12 had high binding preference for α2,6-linked sialic acids and showed higher replication and transmission ability in chickens and quails than PD/CA06. Although the H4N2 virus remained low pathogenic, these data suggests that the acquisition of MBCS in the field is not restricted to H5 or H7 subtypes.
    Virology. 01/2014; s 468–470:72–80.
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    ABSTRACT: nfluenza A (H9N2) viruses are a genetically diverse population that infects wild and domestic avian species and mammals and contributed the internal gene segments to the A/H5N1 and A/H7N9 viruses associated with lethal human infections. Here we comprehensively assess the potential risk to mammals of a diverse panel of A/H9N2 viruses, representing the major H9N2 clades, using a combination of in vitro assays (e.g., antiviral susceptibility and virus growth in primary differentiated human airway cells) and in vivo assays (e.g., replication, transmission and/or pathogenicity of viruses in ducks, pigs, mice and ferrets). We observed that viruses isolated from humans, A/Hong Kong/1073/1999 and A/Hong Kong/33982/2009, had the highest risk potential. However, the A/swine/ Hong Kong/9A-1/1998 and A/chicken/Hong Kong/G9/1997 viruses also displayed several features suggesting a fitness profile adapted to human infection and transmission. The North American avian H9N2 clade virus had the lowest risk profile, and the other viruses tested displayed various levels of fitness across individual assays. In many cases, the known genotypic polymorphisms alone were not sufficient to accurately predict the virus’ phenotype. Therefore, we conclude that comprehensive risk analyses based on surveillance of circulating influenza virus strains are necessary to assess the potential for human infection by emerging influenza A viruses.
    Emerging Microbes and Infections. 11/2013;
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    ABSTRACT: Immune recognition of protein antigens relies on the combined interaction of multiple antibody loops, which provide a fairly large footprint and constrain the size and shape of protein surfaces that can be targeted. Single protein loops can mediate extremely high-affinity binding, but it is unclear whether such a mechanism is available to antibodies. Here we report the isolation and characterization of an antibody called C05, which neutralizes strains from multiple subtypes of influenza A virus, including H1, H2 and H3. X-ray and electron microscopy structures show that C05 recognizes conserved elements of the receptor-binding site on the haemagglutinin surface glycoprotein. Recognition of the haemagglutinin receptor-binding site is dominated by a single heavy-chain complementarity-determining region 3 loop, with minor contacts from heavy-chain complementarity-determining region 1, and is sufficient to achieve nanomolar binding with a minimal footprint. Thus, binding predominantly with a single loop can allow antibodies to target small, conserved functional sites on otherwise hypervariable antigens.
    Nature 09/2012; 489(7417):526-32. · 38.60 Impact Factor
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    ABSTRACT: Avian influenza viruses (H5N1) of clades 2.3.4.1, 2.3.4.2, and 2.3.2.1 were introduced into Laos in 2009-2010. To investigate these viruses, we conducted active surveillance of poultry during March 2010. We detected viruses throughout Laos, including several interclade reassortants and 2 subgroups of clade 2.3.4, one of which caused an outbreak in May 2010.
    Emerging Infectious Diseases 07/2012; 18(7):1139-43. · 6.79 Impact Factor
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    ABSTRACT: We describe virus isolation, full genome sequence analysis, and clinical pathology in ferrets experimentally inoculated with pandemic (H1N1) 2009 virus recovered from a clinically ill captive cheetah that had minimal human contact. Evidence of reverse zoonotic transmission by fomites underscores the substantial animal and human health implications of this virus.
    Emerging Infectious Diseases 02/2012; 18(2):315-7. · 6.79 Impact Factor
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    ABSTRACT: As a result of human-to-pig transmission, pandemic influenza A (H1N1) 2009 virus was detected in pigs soon after it emerged in humans. In the United States, this transmission was quickly followed by multiple reassortment between the pandemic virus and endemic swine viruses. Nine reassortant viruses representing 7 genotypes were detected in commercial pig farms in the United States. Field observations suggested that the newly described reassortant viruses did not differ substantially from pandemic (H1N1) 2009 or endemic strains in their ability to cause disease. Comparable growth properties of reassortant and endemic viruses in vitro supported these observations; similarly, a representative reassortant virus replicated in ferrets to the same extent as did pandemic (H1N1) 2009 and endemic swine virus. These novel reassortant viruses highlight the increasing complexity of influenza viruses within pig populations and the frequency at which viral diversification occurs in this ecologically important viral reservoir.
    Emerging Infectious Diseases 09/2011; 17(9):1624-9. · 6.79 Impact Factor
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    ABSTRACT: Because of continuous circulation in different animal species and humans, influenza viruses have host-specific phenotypic and genetic features. Reassortment of the genome segments can significantly change virus phenotype, potentially generating virus with pandemic potential. In 2009, a new pandemic influenza virus emerged. In this study, we attempted to find precursor viruses or genes of pandemic H1N1 influenza 2009 among 25 swine influenza viruses, isolated in the West Central region of the United States of America (USA), between 2007 and 2009. The Phylogenetically Similar Triple-Reassortant Internal Genes (PSTRIG) cassette of all the viruses studied here as well as the PSTRIG cassette of pandemic H1N1 viruses have close but equidistant phylogenetic relationships to the early triple-reassortant swine H3N2 influenza A isolated in the USA in 1998. Samples (nasal swabs and lung tissue lavage) were taken from swine with or without clinical signs of respiratory disease via farmer-funded syndromic surveillance. All studied viruses were isolated in Madin-Darby Canine Kidney cell cultures from the above-mentioned samples according to standard protocols recommended for influenza virus isolation. Sequences were obtained using BigDye Terminator v3.1 Cycle Sequencing kit. Phylogenetic trees were built with MEGA 4.0 software using maximum composite likelihood algorithm and neighbor-joining method for tree topology reconstruction. Among the 25 viruses studied, we have not found any gene segments of Eurasian origin. Our results suggest that pandemic H1N1 viruses diverged and are not directly descended from swine viruses that have been circulating in USA since 1998.
    Influenza and Other Respiratory Viruses 05/2011; 5(3):188-97. · 1.47 Impact Factor
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    ABSTRACT: Early epidemiologic and serologic studies have suggested pre-existing immunity to the pandemic A (H1N1) 2009 influenza virus (H1N1pdm) may be altering its morbidity and mortality in humans. To determine the role that contemporary seasonal H1N1 virus infection or trivalent inactivated vaccine (TIV) might be playing in this immunity we conducted a vaccination-challenge study in ferrets. Vaccination with TIV was unable to alter subsequent morbidity or contact transmission in ferrets following challenge with H1N1pdm. Conversely, prior infection with the contemporary seasonal H1N1 strain altered morbidity, but not transmission, of H1N1pdm despite the detection of only minimal levels of cross reactive antibodies.
    Vaccine 04/2011; 29(17):3335-9. · 3.77 Impact Factor
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    ABSTRACT: Swine outbreaks of pandemic influenza A (pH1N1) suggest human introduction of the virus into herds. This study investigates a pH1N1 outbreak occurring on a swine research farm with 37 humans and 1300 swine in Alberta, Canada, from 12 June through 4 July 2009. The staff was surveyed about symptoms, vaccinations, and livestock exposures. Clinical findings were recorded, and viral testing and molecular characterization of isolates from humans and swine were performed. Human serological testing and performance of the human influenza-like illness (ILI) case definition were also studied. Humans were infected before swine. Seven of 37 humans developed ILI, and 2 (including the index case) were positive for pH1N1 by reverse-transcriptase polymerase chain reaction (RT-PCR). Swine were positive for pH1N1 by RT-PCR 6 days after contact with the human index case and developed symptoms within 24 h of their positive viral test results. Molecular characterization of the entire viral genomes from both species showed minor nucleotide heterogeneity, with 1 amino acid change each in the hemagglutinin and nucleoprotein genes. Sixty-seven percent of humans with positive serological test results and 94% of swine with positive swab specimens had few or no symptoms. Compared with serological testing, the human ILI case definition had a specificity of 100% and sensitivity of 33.3%. The only factor associated with seropositivity was working in the swine nursery. Epidemiologic data support human-to-swine transmission, and molecular characterization confirms that virtually identical viruses infected humans and swine in this outbreak. Both species had mild illness and recovered without sequelae.
    Clinical Infectious Diseases 01/2011; 52(1):10-8. · 9.37 Impact Factor
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    ABSTRACT: Exposure to contemporary seasonal influenza A viruses affords partial immunity to pandemic H1N1 2009 influenza A virus (pH1N1) infection. The impact of antibodies to the neuraminidase (NA) of seasonal influenza A viruses to cross-immunity against pH1N1 infection is unknown. Antibodies to the NA of different seasonal H1N1 influenza strains were tested for cross-reactivity against A/California/04/09 (pH1N1). A panel of reverse genetic (rg) recombinant viruses was generated containing 7 genes of the H1N1 influenza strain A/Puerto Rico/08/34 (PR8) and the NA gene of either the pandemic H1N1 2009 strain (pH1N1) or one of the following contemporary seasonal H1N1 strains: A/Solomon/03/06 (rg Solomon) or A/Brisbane/59/07 (rg Brisbane). Convalescent sera collected from mice infected with recombinant viruses were measured for cross-reactive antibodies to pH1N1 via Hemagglutinin Inhibition (HI) or Enzyme-Linked Immunosorbent Assay (ELISA). The ectodomain of a recombinant NA protein from the pH1N1 strain (pNA-ecto) was expressed, purified and used in ELISA to measure cross-reactive antibodies. Analysis of sera from elderly humans immunized with trivalent split-inactivated influenza (TIV) seasonal vaccines prior to 2009 revealed considerable cross-reactivity to pNA-ecto. High titers of cross-reactive antibodies were detected in mice inoculated with either rg Solomon or rg Brisbane. Convalescent sera from mice inoculated with recombinant viruses were used to immunize naïve recipient Balb/c mice by passive transfer prior to challenge with pH1N1. Mice receiving rg California sera were better protected than animals receiving rg Solomon or rg Brisbane sera. The NA of contemporary seasonal H1N1 influenza strains induces a cross-reactive antibody response to pH1N1 that correlates with reduced lethality from pH1N1 challenge, albeit less efficiently than anti-pH1N1 NA antibodies. These findings demonstrate that seasonal NA antibodies contribute to but are not sufficient for cross-reactive immunity to pH1N1.
    PLoS ONE 01/2011; 6(10):e26335. · 3.53 Impact Factor
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    ABSTRACT: Influenza poses a persistent worldwide threat to the human population. As evidenced by the 2009 H1N1 pandemic, current vaccine technologies are unable to respond rapidly to this constantly diverging pathogen. We tested the utility of adenovirus (Ad) vaccines expressing centralized consensus influenza antigens. Ad vaccines were produced within 2 months and protected against influenza in mice within 3 days of vaccination. Ad vaccines were able to protect at doses as low as 10(7) virus particles/kg indicating that approximately 1,000 human doses could be rapidly generated from standard Ad preparations. To generate broadly cross-reactive immune responses, centralized consensus antigens were constructed against H1 influenza and against H1 through H5 influenza. Twenty full-length H1 HA sequences representing the main branches of the H1 HA phylogenetic tree were used to create a synthetic centralized gene, HA1-con. HA1-con minimizes the degree of sequence dissimilarity between the vaccine and existing circulating viruses. The centralized H1 gene, HA1-con, induced stronger immune responses and better protection against mismatched virus challenges as compared to two wildtype H1 genes. HA1-con protected against three genetically diverse lethal influenza challenges. When mice were challenged with 1934 influenza A/PR/8/34, HA1-con protected 100% of mice while vaccine generated from 2009 A/TX/05/09 only protected 40%. Vaccination with 1934 A/PR/8/34 and 2009 A/TX/05/09 protected 60% and 20% against 1947 influenza A/FM/1/47, respectively, whereas 80% of mice vaccinated with HA1-con were protected. Notably, 80% of mice challenged with 2009 swine flu isolate A/California/4/09 were protected by HA1-con vaccination. These data show that HA1-con in Ad has potential as a rapid and universal vaccine for H1N1 influenza viruses.
    PLoS ONE 01/2011; 6(3):e18314. · 3.53 Impact Factor
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    ABSTRACT: Our ability to rapidly respond to an emerging influenza pandemic is hampered somewhat by the lack of a susceptible small-animal model. To develop a more sensitive model, we pathotyped 18 low-pathogenic non-mouse-adapted influenza A viruses of human and avian origin in DBA/2 and C57BL/6 mice. The majority of the isolates (13/18) induced severe morbidity and mortality in DBA/2 mice upon intranasal challenge with 1 million infectious doses. Also, at a 100-fold-lower dose, more than 50% of the viruses induced severe weight loss, and mice succumbed to the infection. In contrast, only two virus strains were pathogenic for C57BL/6 mice upon high-dose inoculation. Therefore, DBA/2 mice are a suitable model to validate influenza A virus vaccines and antiviral therapies without the need for extensive viral adaptation. Correspondingly, we used the DBA/2 model to assess the level of protection afforded by preexisting pandemic H1N1 2009 virus (H1N1pdm) cross-reactive human antibodies detected by a hemagglutination inhibition assay. Passive transfer of these antibodies prior to infection protected mice from H1N1pdm-induced pathogenicity, demonstrating the effectiveness of these cross-reactive neutralizing antibodies in vivo.
    Journal of Virology 08/2010; 84(15):7662-7. · 5.08 Impact Factor
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    ABSTRACT: In Egypt, efforts to control highly pathogenic H5N1 avian influenza virus in poultry and in humans have failed despite increased biosecurity, quarantine, and vaccination at poultry farms. The ongoing circulation of HP H5N1 avian influenza in Egypt has caused >100 human infections and remains an unresolved threat to veterinary and public health. Here, we describe that the failure of commercially available H5 poultry vaccines in Egypt may be caused in part by the passive transfer of maternal H5N1 antibodies to chicks, inhibiting their immune response to vaccination. We propose that the induction of a protective immune response to H5N1 is suppressed for an extended period in young chickens. This issue, among others, must be resolved and additional steps must be taken before the outbreaks in Egypt can be controlled.
    Proceedings of the National Academy of Sciences 06/2010; 107(24):11044-9. · 9.81 Impact Factor
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    ABSTRACT: Influenza viruses elude immune responses and antiviral chemotherapeutics through genetic drift and reassortment. As a result, the development of new strategies that attack a highly conserved viral function to prevent and/or treat influenza infection is being pursued. Such novel broadly acting antiviral therapies would be less susceptible to virus escape and provide a long lasting solution to the evolving virus challenge. Here we report the in vitro and in vivo activity of a human monoclonal antibody (A06) against two isolates of the 2009 H1N1 pandemic influenza virus. This antibody, which was obtained from a combinatorial library derived from a survivor of highly pathogenic H5N1 infection, neutralizes H5N1, seasonal H1N1 and 2009 "Swine" H1N1 pandemic influenza in vitro with similar potency and is capable of preventing and treating 2009 H1N1 influenza infection in murine models of disease. These results demonstrate broad activity of the A06 antibody and its utility as an anti-influenza treatment option, even against newly evolved influenza strains to which there is limited immunity in the general population.
    PLoS Pathogens 01/2010; 6(7):e1000990. · 8.14 Impact Factor