Genetic Compatibility and Virulence of Reassortants Derived from Contemporary Avian H5N1 and Human H3N2 Influenza A Viruses

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Abstract
The segmented structure of the influenza virus genome plays a pivotal role in its adaptation to new hosts and the emergence of pandemics. Despite concerns about the pandemic threat posed by highly pathogenic avian influenza H5N1 viruses, little is known about the biological properties of H5N1 viruses that may emerge following reassortment with contemporary human influenza viruses. In this study, we used reverse genetics to generate the 63 possible virus reassortants derived from H5N1 and H3N2 viruses, containing the H5N1 surface protein genes, and analyzed their viability, replication efficiency, and mouse virulence. Specific constellations of avian–human viral genes proved deleterious for viral replication in cell culture, possibly due to disruption of molecular interaction networks. In particular, striking phenotypes were noted with heterologous polymerase subunits, as well as NP and M, or NS. However, nearly one-half of the reassortants replicated with high efficiency in vitro, revealing a high degree of compatibility between avian and human virus genes. Thirteen reassortants displayed virulent phenotypes in mice and may pose the greatest threat for mammalian hosts. Interestingly, one of the most pathogenic reassortants contained avian PB1, resembling the 1957 and 1968 pandemic viruses. Our results reveal the broad spectrum of phenotypes associated with H5N1/H3N2 reassortment and a possible role for the avian PB1 in the emergence of pandemic influenza. These observations have important implications for risk assessment of H5N1 reassortant viruses detected in surveillance programs. The influenza pandemics of 1957 and 1968 were caused by hybrid viruses consisting of a mixture of human and avian influenza genes. The introduction of avian genes resulted in a sudden change of the virus surface antigens, allowing its worldwide spread due to lack of immunity in the population. The highly pathogenic avian influenza H5N1 virus has continued its spread in domestic and wild birds in Asia, Europe, and Africa. Although H5N1 infection in humans is rare and person-to-person transmission is very inefficient, the steady accumulation of human cases has raised concern over the possible reassortment between H5N1 and human seasonal influenza resulting in a virus with new surface antigens and pandemic potential. In this study, we used recombinant DNA technology to generate a systematic collection of hybrid viruses (with genes from human and avian viruses) bearing H5N1 surface antigens and analyzed their properties in cell culture and in mice. The H5N1 hybrid viruses revealed a broad range of viability and multiplication capacity in cell cultures. In addition, several H5N1 hybrid viruses were highly virulent in mice. Results from this systematic analysis provide important insight to support risk assessment of reassortant H5N1 avian influenza viruses.