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Genetic and phylogenetic analyses suggest that the pandemic H1N1/2009 virus was derived from well-established swine influenza lineages; however, there is no convincing evidence that the pandemic virus was generated from a direct precursor in pigs. Furthermore, the evolutionary dynamics of influenza virus in pigs have not been well documented.
Here, we subjected a recombinant virus (rH1N1) with the same constellation makeup as the pandemic H1N1/2009 virus to nine serial passages in pigs. Severity of infection sequentially increased with each passage. Deep sequencing of viral quasi-species from the ninth passage found five consensus amino acid mutations: PB1 A469T, PA 1129T, NA N329D, NS1 N205K and NEP T48N. Mutations in the HA protein, however, differed greatly between the upper and lower respiratory tracts. Three representative viral clones with the five consensus mutations were selected for functional evaluation. Relative to the parental virus, the three viral clones showed enhanced replication and polymerase activity in vitro, and enhanced replication, pathogenicity and transmissibility in pigs, guinea pigs and ferrets in vivo. Specifically, two mutants of rH1N1 (PB1 A469T, and combined NS1 N205K and NEP T48N) were identified as determinants of transmissibility in guinea pigs. Crucially, one mutant viral clone with the five consensus mutations, which also carried D187E, K211E and S289N mutations in its hemagglutinin (HA), was additionally able to infect ferrets by airborne transmission as effectively the pandemic virus.
Our findings demonstrate that influenza virus can acquire viral characteristics that are similar to those of the pandemic virus after limited serial passages in pigs.
Importance
We demonstrated here an engineered reassortant swine influenza virus, with the same gene constellation pattern as the pandemic H1N1/2009 virus, subjected to only nine serial passages in pigs acquired greatly enhanced virulence and transmissibility. In particular, one representative pathogenic passaged virus clone, which carried three mutations in the HA gene and five consensus mutations in PB1, PA, NA, NS1, and NEP genes, was additionally able to confer respiratory droplet transmission as effectively as the pandemic H1N1/2009 virus. Our findings suggest that pigs can readily induce adaptive mutational changes to a precursor pandemic-like virus to transform it into a highly virulent and infectious form akin to that of the pandemic H1N1/2009 virus which underlines the potential direct role of pigs in promoting influenza A virus pathogenicity and transmissibility.
K Wei, H Sun, Z Sun, Y Sun, W Kong, J Pu, G Ma, Y Yin, H Yang, X Guo, RG Webster, K-C Chang and J Liu. Influenza A virus Acquires Enhanced Pathogenicity and Transmissibility After Serial Passages in Swine. J Virol. 2014 Aug 6. doi: 10.1128/JVI.01679-14