Influenza A Virus Acquires Enhanced Pathogenicity and Transmissibility after Serial Passages in Swine

Abstract

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. The severity of infection sequentially increased with each passage. Deep sequencing of viral quasispecies from the ninth passage found five consensus amino acid mutations: PB1 A469T, PA 1129T, NA N329D, NS1 N205K, and NEP T48N. Mutations in the hemagglutinin (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 a combination of 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 HA, additionally was able to infect ferrets by airborne transmission as effectively as 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. IMPORTANCEWe demonstrate here that an engineered reassortant swine influenza virus, with the same gene constellation pattern as the pandemic H1N1/2009 virus and 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, additionally was 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.

Pathogenesis of Influenza D Virus in Cattle

“…It is also possible that the virus gained host adaptations upon transmission that could have facilitated more efficient replication. Phenotypic changes in influenza viruses due to adaptation to the host have been shown in IAV studies in feral swine, domestic swine, and mice and have also been demonstrated for IDV in the guinea pig transmission model (11)(12)(13)(14). Future studies will characterize the pathogenesis in the contact calves and potentially identify mutations in the isolates recovered from the contact calves.…”

Genome plasticity of triple-reassortant H1N1 influenza A virus during infection of vaccinated pigs

“…Multiple IAV subtypes can co-circulate in swine herds and persist at the population level for prolonged periods of time (Corzo et al, 2013;Diaz et al, 2015). Additionally, multiple alleles (sequence variants of the same virus) can coexist during IAV infection of pigs (Murcia et al, 2012), and the same virus can evolve differently in the upper and lower respiratory tract of pigs (Wei et al, 2014). Furthermore, nucleotide substitutions within the HA antigenic sites can occur shortly after infection of pigs with no significant differences noticed between pigs with or without immunity to the virus (Diaz et al, 2013;Murcia et al, 2012).…”

Swine Influenza Virus PA and Neuraminidase Gene Reassortment into Human H1N1 Influenza Virus Is Associated with an Altered Pathogenic Phenotype Linked to Increased MIP-2 Expression

“…It has been previously shown that IAV reassortants having genetic exchange of regulatory genes such as NS, or replication machinery such as PA and PB2, can alter the viral kinetics and immune modulatory phenotype of IAVs (26)(27)(28)(29)(30). Thus, replication kinetics were evaluated for reassortants having genetic exchange between structural and nonstructural genes among the parental viruses, swH1N2 and huH1N1.…”

“…The polymerases from the 1957, 1968, and 2009 pandemics were derived from reassortants (5). Protein components of the RNP complex have been implicated in various aspects of influenza virus virulence and pathogenicity in a variety of mammalian models, including mice, ferrets, and swine (25)(26)(27)(28)(29). For example, the well-described amino acid K627 in the PB2 protein enhances polymerase activity, leading to increased transmission, replication, and pathogenicity in mammals (30).…”