Virulence and Genetic Compatibility of Polymerase Reassortant Viruses Derived from the Pandemic (H1N1) 2009 Influenza Virus and Circulating Influenza A Viruses

Abstract

Gene mutations and reassortment are key mechanisms by which influenza A virus acquires virulence factors. To evaluate the role of the viral polymerase replication machinery in producing virulent pandemic (H1N1) 2009 influenza viruses, we generated various polymerase point mutants (PB2, 627K/701N; PB1, expression of PB1-F2 protein; and PA, 97I) and reassortant viruses with various sources of influenza viruses by reverse genetics. Although the point mutations produced no significant change in pathogenicity, reassortment between the pandemic A/California/04/09 (CA04, H1N1) and current human and animal influenza viruses produced variants possessing a broad spectrum of pathogenicity in the mouse model. Although most polymerase reassortants had attenuated pathogenicity (including those containing seasonal human H3N2 and high-pathogenicity H5N1 virus segments) compared to that of the parental CA04 (H1N1) virus, some recombinants had significantly enhanced virulence. Unexpectedly, one of the five highly virulent reassortants contained a A/Swine/Korea/JNS06/04(H3N2)-like PB2 gene with no known virulence factors; the other four had mammalian-passaged avian-like genes encoding PB2 featuring 627K, PA featuring 97I, or both. Overall, the reassorted polymerase complexes were only moderately compatible for virus rescue, probably because of disrupted molecular interactions involving viral or host proteins. Although we observed close cooperation between PB2 and PB1 from similar virus origins, we found that PA appears to be crucial in maintaining viral gene functions in the context of the CA04 (H1N1) virus. These observations provide helpful insights into the pathogenic potential of reassortant influenza viruses composed of the pandemic (H1N1) 2009 influenza virus and prevailing human or animal influenza viruses that could emerge in the future.

Influenza A Virus Infections in Swine

“…Compatibility of the polymerases with each other as well as with the other viral proteins appeared to be critical. 17,88,133 Surprisingly, the pandemic 2009 H1N1 virus, which was more virulent than most seasonal influenza viruses for humans and in experimental mammalian hosts, did not have the reputed virulence marker mutations in its PB2 protein. 45 When the molecular mutations in PB2 suggested to be virulence markers for avian viruses were introduced individually into the avianorigin PB2 of the pandemic 2009 H1N1 virus, virulence was not altered.…”

“…12,83,177,178 All 3 polymerases have some effect on virulence in various studies, including PA 47,133,178 and PB1, 133 but PB2 protein seems to most significantly alter pathogenicity. 88,133,177 The amino acid at position 627 of the PB2 protein influences virulence and host preference.…”

“…88,133,177 The amino acid at position 627 of the PB2 protein influences virulence and host preference. A glutamic acid at this site (627E) favors replication in avian hosts, possibly through temperature sensitivity (ie, increased replication rates at the warmer body temperatures of birds).…”

Virulence and transmissibility of H1N2 influenza virus in ferrets imply the continuing threat of triple-reassortant swine viruses

“…Although most pH1N1 infections were mild, the virus was highly transmissible and could replicate deep in the lungs (5). Introduction of known virulence factors into the pH1N1 virus did not significantly alter its disease phenotype (6,7), suggesting the presence of unknown molecular determinants of pathogenicity and transmissibility.…”

Virulence of pandemic (H1N1) 2009 influenza A polymerase reassortant viruses

“…17 Therefore to address this, we generated various polymerase point mutant pandemic viruses (PB2, 627K/701N; PB1, expression of PB1-F2 protein; PA, 97I) by an established reverse genetics system. 16 We found in our work that expression of the 627K or 701N mutation in PB2 or driving the PB1-F2 protein expression enhanced reporter gene activity in vitro compared with that of the wild-type (WT) CA04 polymerase complex.…”