Hongxia Shao

Compatibility of H9N2 avian influenza surface genes and 2009 pandemic H1N1 internal genes for transmission in the ferret model

In 2009, a novel H1N1 influenza (pH1N1) virus caused the first influenza pandemic in 40 y. The virus was identified as a triple reassortant between avian, swine, and human influenza viruses, highlighting the importance of reassortment in the generation of viruses with pandemic potential. Previously, we showed that a reassortant virus composed of wild-type avian H9N2 surface genes in a seasonal human H3N2 backbone could gain efficient respiratory droplet transmission in the ferret model. Here we determine the ability of the H9N2 surface genes in the context of the internal genes of a pH1N1 virus to efficiently transmit via respiratory droplets in ferrets. We generated reassorted viruses carrying the HA gene alone or in combination with the NA gene of a prototypical H9N2 virus in the background of a pH1N1 virus. Four reassortant viruses were generated, with three of them showing efficient respiratory droplet transmission. Differences in replication efficiency were observed for these viruses; however, the results clearly indicate that H9N2 avian influenza viruses and pH1N1 viruses, both of which have occasionally infected pigs, have the potential to reassort and generate novel viruses with respiratory transmission potential in mammals.

Variations in the Hemagglutinin of the 2009 H1N1 Pandemic Virus: Potential for Strains with Altered Virulence Phenotype?

A novel, swine-origin influenza H1N1 virus (H1N1pdm) caused the first pandemic of the 21st century. This pandemic, although efficient in transmission, is mild in virulence. This atypical mild pandemic season has raised concerns regarding the potential of this virus to acquire additional virulence markers either through further adaptation or possibly by immune pressure in the human host. Using the mouse model we generated, within a single round of infection with A/California/04/09/H1N1 (Ca/04), a virus lethal in mice—herein referred to as mouse-adapted Ca/04 (ma-Ca/04). Five amino acid substitutions were found in the genome of ma-Ca/04: 3 in HA (D131E, S186P and A198E), 1 in PA (E298K) and 1 in NP (D101G). Reverse genetics analyses of these mutations indicate that all five mutations from ma-Ca/04 contributed to the lethal phenotype; however, the D131E and S186P mutations—which are also found in the 1918 and seasonal H1N1 viruses—in HA alone were sufficient to confer virulence of Ca/04 in mice. HI assays against H1N1pdm demonstrate that the D131E and S186P mutations caused minor antigenic changes and, likely, affected receptor binding. The rapid selection of ma-Ca/04 in mice suggests that a virus containing this constellation of amino acids might have already been present in Ca/04, likely as minor quasispecies.

Airborne Transmission of Highly Pathogenic H7N1 Influenza Virus in Ferrets

ABSTRACT Avian H7 influenza viruses are recognized as potential pandemic viruses, as personnel often become infected during poultry outbreaks. H7 infections in humans typically cause mild conjunctivitis; however, the H7N9 outbreak in the spring of 2013 has resulted in severe respiratory disease. To date, no H7 viruses have acquired the ability for sustained transmission among humans. Airborne transmission is considered a requirement for the emergence of pandemic influenza, and advanced knowledge of the molecular changes or signature required for transmission would allow early identification of pandemic vaccine seed stocks, screening and stockpiling of antiviral compounds, and eradication efforts focused on flocks harboring threatening viruses. Thus, we sought to determine if a highly pathogenic influenza A H7N1 (A/H7N1) virus with no history of human infection could become capable of airborne transmission among ferrets. We show that after 10 serial passages, A/H7N1 developed the ability to be transmitted to cohoused and airborne contact ferrets. Four amino acid mutations (PB2 T81I, NP V284M, and M1 R95K and Q211K) in the internal genes and a minimal amino acid mutation (K/R313R) in the stalk region of the hemagglutinin protein were associated with airborne transmission. Furthermore, transmission was not associated with loss of virulence. These findings highlight the importance of the internal genes in host adaptation and suggest that natural isolates carrying these mutations be further evaluated. Our results demonstrate that a highly pathogenic avian H7 virus can become capable of airborne transmission in a mammalian host, and they support ongoing surveillance and pandemic H7 vaccine development. IMPORTANCE The major findings of this report are that a highly pathogenic strain of H7N1 avian influenza virus can be adapted to become capable of airborne transmission in mammals without mutations altering receptor specificity. Changes in receptor specificity have been shown to play a role in the ability of avian influenza viruses to cross the species barrier, and these changes are assumed to be essential. The work reported here challenges this paradigm, at least for the influenza viruses of the H7 subtype, which have recently become the focus of major attention, as they have crossed to humans.

Influenza Viruses with Rearranged Genomes as Live-Attenuated Vaccines

ABSTRACT H5N1 and H9N2 avian influenza virus subtypes top the World Health Organizations list for the greatest pandemic potential. Inactivated H5N1 vaccines induce limited immune responses and, in the case of live-attenuated influenza virus vaccines (LAIV), there are safety concerns regarding the possibility of reassortment between the H5 gene segment and circulating influenza viruses. In order to overcome these drawbacks, we rearranged the genome of an avian H9N2 influenza virus and expressed the entire H5 hemagglutinin open reading frame (ORF) from the segment 8 viral RNA. These vectors had reduced polymerase activities as well as viral replication in vitro and excellent safety profiles in vivo. Immunization with the dual H9-H5 influenza virus resulted in protection against lethal H5N1 challenge in mice and ferrets, and also against a potentially pandemic H9 virus. Our studies demonstrate that rearranging the influenza virus genome has great potential for the development of improved vaccines against influenza virus as well as other pathogens.

Evidence for avian H9N2 influenza virus infections among rural villagers in Cambodia

BACKGROUND Southeast Asia remains a critical region for the emergence of novel and/or zoonotic influenza, underscoring the importance of extensive sampling in rural areas where early transmission is most likely to occur. METHODS In 2008, 800 adult participants from eight sites were enrolled in a prospective population-based study of avian influenza (AI) virus transmission where highly pathogenic avian influenza (HPAI) H5N1 virus had been reported in humans and poultry from 2006 to 2008. From their enrollment sera and questionnaires, we report risk factor findings for serologic evidence of previous infection with 18 AI virus strains. RESULTS Serologic assays revealed no evidence of previous infection with 13 different low-pathogenic AI viruses or with HPAI avian-like A/Cambodia/R0404050/2007(H5N1). However, 21 participants had elevated antibodies against avian-like A/Hong Kong/1073/1999(H9N2), validated with a monoclonal antibody blocking ELISA assay specific for avian H9. CONCLUSIONS Although cross-reaction from antibodies against human influenza viruses cannot be completely excluded, the study data suggest that a number of participants were previously infected with the avian-like A/Hong Kong/1073/1999(H9N2) virus, likely due to as yet unidentified environmental exposures. Prospective data from this cohort will help us better understand the serology of zoonotic influenza infection in a rural cohort in SE Asia.

Outbreaks of serotype 4 fowl adenovirus with novel genotype, China

Emerging Microbes and Infections (2016) 5, e50; doi:10.1038/emi.2016.50; published online 25 May 2016

Phylogenetic Analysis of H6 Influenza Viruses Isolated from Rosy-Billed Pochards (Netta peposaca) in Argentina Reveals the Presence of Different HA Gene Clusters

ABSTRACT Until recently, influenza A viruses from wild waterfowl in South America were rarely isolated and/or characterized. To explore the ecology of influenza A viruses in this region, a long-term surveillance program was established in 2006 for resident and migratory water birds in Argentina. We report the characterization of 5 avian influenza viruses of the H6 hemagglutinin (HA) subtype isolated from rosy-billed pochards (Netta peposaca). Three of these viruses were paired to an N2 NA subtype, while the other two were of the N8 subtype. Genetic and phylogenetic analyses of the internal gene segments revealed a close relationship with influenza viruses from South America, forming a unique clade and supporting the notion of independent evolution from influenza A viruses in other latitudes. The presence of NS alleles A and B was also identified. The HA and NA genes formed unique clades separate from North American and Eurasian viruses, with the exception of the HA gene of one isolate, which was more closely related to the North American lineage, suggesting possible interactions between viruses of North American and South American lineages. Animal studies suggested that these Argentine H6 viruses could replicate and transmit inefficiently in chickens, indicating limited adaptation to poultry. Our results highlight the importance of continued influenza virus surveillance in wild birds of South America, especially considering the unique evolution of these viruses.

Intranasal Delivery of an IgA Monoclonal Antibody Effective against Sublethal H5N1 Influenza Virus Infection in Mice

ABSTRACT Highly pathogenic avian H5N1 influenza viruses are endemic in poultry in Asia and pose a pandemic threat to humans. Since the deployment of vaccines against a pandemic strain may take several months, adequate antiviral alternatives are needed to minimize the effects and the spread of the disease. Passive immunotherapy is regarded as a viable alternative. Here, we show the development of an IgA monoclonal antibody (DPJY01 MAb) specific to H5 hemagglutinin. The DPJY01 MAb showed a broad hemagglutination inhibition (HI) profile against Asian H5N1 viruses of clades 0, 1.0, 2.1, 2.2, and 2.3 and also against H5 wild bird influenza viruses of the North American and Eurasian lineages. DPJY01 MAb displayed also high neutralization activity in vitro and in vivo. In mice, DPJY01 MAb provided protection via a single dose administered intranasally before or after inoculation with a sublethal dose of H5N1 viruses of clades 1.0 and 2.2. Pretreatment with 50 mg of DPJY01 MAb kg of body weight at either 24, 48, or 72 h before highly pathogenic H5N1 virus (A/Vietnam/1203/2004 [H5N1]) inoculation resulted in complete protection. Treatment with 50 mg/kg at either at 24, 48, or 72 h after H5N1 inoculation provided 100%, 80%, and 60% protection, respectively. These studies highlight the potential use of DPJY01 MAb as an intranasal antiviral treatment for H5N1 influenza virus infections.

Improved hatchability and efficient protection after in ovo vaccination with live-attenuated H7N2 and H9N2 avian influenza viruses

Mass in ovo vaccination with live attenuated viruses is widely used in the poultry industry to protect against various infectious diseases. The worldwide outbreaks of low pathogenic and highly pathogenic avian influenza highlight the pressing need for the development of similar mass vaccination strategies against avian influenza viruses. We have previously shown that a genetically modified live attenuated avian influenza virus (LAIV) was amenable for in ovo vaccination and provided optimal protection against H5 HPAI viruses. However, in ovo vaccination against other subtypes resulted in poor hatchability and, therefore, seemed impractical. In this study, we modified the H7 and H9 hemagglutinin (HA) proteins by substituting the amino acids at the cleavage site for those found in the H6 HA subtype. We found that with this modification, a single dose in ovo vaccination of 18-day old eggs provided complete protection against homologous challenge with low pathogenic virus in ≥70% of chickens at 2 or 6 weeks post-hatching. Further, inoculation of 19-day old egg embryos with 106 EID50 of LAIVs improved hatchability to ≥90% (equivalent to unvaccinated controls) with similar levels of protection. Our findings indicate that the strategy of modifying the HA cleavage site combined with the LAIV backbone could be used for in ovo vaccination against avian influenza. Importantly, with protection conferred as early as 2 weeks post-hatching, with this strategy birds would be protected prior to or at the time of delivery to a farm or commercial operation.

Antigenic Mapping of the Hemagglutinin of an H9N2 Avian Influenza Virus Reveals Novel Critical Amino Acid Positions in Antigenic Sites

ABSTRACT H9N2 influenza virus is undergoing extensive genetic and antigenic evolution, warranting detailed antigenic mapping of its hemagglutinin (HA). Through examining antibody escape mutants of an Asian avian H9N2 virus, we identified 9 critical amino acid positions in H9 antigenic sites. Five of these positions, 164, 167, 168, 196, and 207, have not been reported previously and, thus, represent novel molecular markers for monitoring the antigenic change of H9N2 virus.