The Multibasic Cleavage Site of the Hemagglutinin of Highly Pathogenic A/Vietnam/1203/2004 (H5N1) Avian Influenza Virus Acts as a Virulence Factor in a Host-Specific Manner in Mammals

Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Journal of Virology (Impact Factor: 4.44). 12/2011; 86(5):2706-14. DOI: 10.1128/JVI.05546-11
Source: PubMed


Highly pathogenic avian influenza (HPAI) viruses of the H5 and H7 subtypes typically possess multiple basic amino acids around
the cleavage site (MBS) of their hemagglutinin (HA) protein, a recognized virulence motif in poultry. To determine the importance
of the H5 HA MBS as a virulence factor in mammals, recombinant wild-type HPAI A/Vietnam/1203/2004 (H5N1) viruses that possessed
(H5N1) or lacked (ΔH5N1) the H5 HA MBS were generated and evaluated for their virulence in BALB/c mice, ferrets, and African
green monkeys (AGMs) (Chlorocebus aethiops). The presence of the H5 HA MBS was associated with lethality, significantly higher virus titers in the respiratory tract,
virus dissemination to extrapulmonary organs, lymphopenia, significantly elevated levels of proinflammatory cytokines and
chemokines, and inflammation in the lungs of mice and ferrets. In AGMs, neither H5N1 nor ΔH5N1 virus was lethal and neither
caused clinical symptoms. The H5 HA MBS was associated with mild enhancement of replication and delayed virus clearance. Thus,
the contribution of H5 HA MBS to the virulence of the HPAI H5N1 virus varies among mammalian hosts and is most significant
in mice and ferrets and less remarkable in nonhuman primates.

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    • "The lack of extrapulmonary viral dissemination is compatible with our recent clinical study showing lack of viremia and absence of viruses in tissues from patients [5]. This is in contrast to H5N1 infection, in which extrapulmonary spread is commonly seen in mouse models [38]. One major characteristic of H5N1 virus is the presence of multibasic amino acid at the cleavage site of HA, which renders it susceptible to cleavage by a wide range of tissue proteases [7], whereas H7N9 virus with only one arginine at this cleavage site may have limited its tropism to the respiratory tissues [1], [2]. "
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    ABSTRACT: Background Human infection caused by the avian influenza A H7N9 virus has a case-fatality rate of over 30%. Systematic study of the pathogenesis of avian H7N9 isolate and effective therapeutic strategies are needed. Methods BALB/c mice were inoculated intranasally with an H7N9 virus isolated from a chicken in a wet market epidemiologically linked to a fatal human case, (A/chicken/Zhejiang/DTID-ZJU01/2013 [CK1]), and with an H7N9 virus isolated from a human (A/Anhui/01/2013 [AH1]). The pulmonary viral loads, cytokine/chemokine profiles and histopathological changes of the infected mice were compared. The therapeutic efficacy of a non-steroidal anti-inflammatory drug (NSAID), celecoxib, was assessed. Results Without prior adaptation, intranasal inoculation of 106 plaque forming units (PFUs) of CK1 caused a mortality rate of 82% (14/17) in mice. Viral nucleoprotein and RNA expression were limited to the respiratory system and no viral RNA could be detected from brain, liver and kidney tissues. CK1 caused heavy alveolar inflammatory exudation and pulmonary hemorrhage, associated with high pulmonary levels of proinflammatory cytokines. In the mouse lung cell line LA-4, CK1 also induced high levels of interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) mRNA. Administration of the antiviral zanamivir did not significantly improve survival in mice infected with CK1, but co-administration of the non-steroidal anti-inflammatory drug (NSAID) celecoxib in combination with zanamivir improved survival and lung pathology. Conclusions Our findings suggested that H7N9 viruses isolated from chicken without preceding trans-species adaptation can cause lethal mammalian pulmonary infection. The severe proinflammatory responses might be a factor contributing to the mortality. Treatment with combination of antiviral and NSAID could ameliorate pulmonary inflammation and may improve survival.
    Full-text · Article · Sep 2014 · PLoS ONE
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    • "TaqMan chemistry incorporates target-specific fluorescent labeled probes enabling multiple genes can be assessed in a single real time PCR reaction (Giulietti et al., 2001). To date, TaqMan real time RT-PCR assays have only been developed for a smaller number of ferretspecific gene targets (Nakata et al., 2009; Suguitan et al., 2012). To enable a broader characterization of the immune response in the ferret model, we developed a panel of TaqMan assays to detect mRNA of fifteen ferret cytokines, chemokines and immune mediators (IFN␣, IFN␤, IFN␥, IL1␣, IL1␤, IL2, IL4, IL6, IL8, IL10, IL12p40, IL17, granzyme A, MCP-1, TNF␣) and four housekeeping genes (ATF4, GAPDH, L32 and HPRT). "
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    ABSTRACT: The ferret is an excellent model for many human infectious diseases including influenza, SARS-CoV, henipavirus and pneumococcal infections. The ferret is also used to study cystic fibrosis and various cancers, as well as reproductive biology and physiology. However, the range of reagents available to measure the ferret immune response is very limited. To address this deficiency, high-throughput real time RT-PCR TaqMan assays were developed to measure the expression of fifteen immune mediators associated with the innate and adaptive immune responses (IFNα, IFNβ, IFNγ, IL1α, IL1β, IL2, IL4, IL6, IL8, IL10, IL12p40, IL17, Granzyme A, MCP1, TNFα), as well as four endogenous housekeeping genes (ATF4, HPRT, GAPDH, L32). These assays have been optimized to maximize reaction efficiency, reduce the amount of sample required (down to 1ng RNA per real time RT-PCR reaction) and to select the most appropriate housekeeping genes. Using these assays, the expression of each of the tested genes could be detected in ferret lymph node cells stimulated with mitogens or infected with influenza virus in vitro. These new tools will allow a more comprehensive analysis of the ferret immune responses following infection or in other disease states.
    Full-text · Article · May 2014 · Journal of virological methods
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    • "However, experimental infection of non-human primates with HPAI H5N1 virus caused respiratory tract disease only with no evidence of systemic virus replication.53 Recent studies showed that the MBCS in an HPAI H5N1 virus was important for systemic spread in ferrets, since deletion of the MBCS from HPAI H5N1 virus resulted in replication that was restricted to the respiratory tract.54,55 However, insertion of an MBCS in a human H3N2 virus did not result in systemic spread in ferrets.56 "
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    ABSTRACT: A wide range of influenza A viruses of pigs and birds have infected humans in the last decade, sometimes with severe clinical consequences. Each of these so-called zoonotic infections provides an opportunity for virus adaptation to the new host. Fortunately, most of these human infections do not yield viruses with the ability of sustained human-to-human transmission. However, animal influenza viruses have acquired the ability of sustained transmission between humans to cause pandemics on rare occasions in the past, and therefore, influenza virus zoonoses continue to represent threats to public health. Numerous recent studies have shed new light on the mechanisms of adaptation and transmission of avian and swine influenza A viruses in mammals. In particular, several studies provided insights into the genetic and phenotypic traits of influenza A viruses that may determine airborne transmission. Here, we summarize recent studies on molecular determinants of virulence and adaptation of animal influenza A virus and discuss the phenotypic traits associated with airborne transmission of newly emerging influenza A viruses. Increased understanding of the determinants and mechanisms of virulence and transmission may aid in assessing the risks posed by animal influenza viruses to human health, and preparedness for such risks.
    Full-text · Article · Feb 2014 · Emerging Microbes and Infections
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