| Genetic reassortment was the major way to generate new pandemic viruses. Pigs are thought to be a mixing vessel of influenza A viruses (IAVs) and play a significant role for the evolution of pandemic viruses by genetic reassortment because of their capacity to be infected with both avian and human influenza viruses. Currently pdm/09(H1N1/2009) virus and EA (Eurasian Avian-like, EA) swine influenza viruses are co-circulating in pigs. Co-circulation of these different virus lineages will naturally increase virus interaction and reassortment, and the genetic diversity in swine influenza viruses. Due to there is no comprehensive and systematic investigation on the swine influenza virus gene pool, it is difficult to analysis systematically these reassortant virus between pdm/09and EA influenza virus. To better understand the genetic diversity and biological properties of novel ressortants between pdm/09and EA influenza virus, we used the swine model to explore the generation of reassortant viruses in a host coinfected EA swine H11and human pdm/09viruses and the extent to which such reassortants replicate and transmit from animal to animal.Reassortant viruses were isolated from the nasal secretions and lung of all coinfected pigs (55reassortants from a total of300virus plaques), suggesting an ecologically significant and reproducible yield (18%) of reassortant viruses from coinfected animals. Importantly, at least60.0%of the recovered reassortant viruses possessed the EA H1HA (33of the55reassortant viruses), which comprised10distinct genotypes. The rest of22reassortant viruses belong to pdm/09-origin HA reassortant swine influenza virus, which contained7distinct genotypes. RNPs of pdm/09origin were frequently isolated in reassortant viruses, which appear to have an advantage over segments of EA origin. HA and NS of EA H1Nl virus showed high genetic compatibility with pdm/09virus. Viruses of a pdm/09genotype were isolated at high frequency during105days of coinfection. Interestingly, viruses with EA H1N1genotype could not be isolated in coinfected animals. The reassortant virus isolation rate was8%(19/240) in upper respiratory tract. Most of the reassortants beared the pdm/09-origin HA exhibited high frequency84%(16/19). A total of36reassortant isolates were from lungs and contained14different genotypes. And the reassortant virus isolation rate was60%(36/60) in lower respiratory tract. In contrary, most of them carried the EA-origin HA were the predominant virus (78%,28/36). These findings indicated that the lung is a major place of reassortant between EA and pdm/09, and different genotypes reassortants had preference in the upper and lower respiratory tract.To assess the pathogenicity and transmissibility of the reassortant virus, BALB/c mice and guinea pig were chosen to proceed the experiment. The results indicated that the reassortant viruses can efficiently infect mice and some viruses showed high virulence. It is noteworthy that six reassortant viruses belonging to high virulent viruses bearing HA/NS from EA H1N1virus, suggesting that HA/NS contributed to the high-pathogenicity phenotype. Thirteen reassortants exhibited efficient transmissibility among guinea pigs,5of13reassortants showed high transmissibility. And those high virulent reassortant viruses also showed high transmissibility, which carrying RNPs gene originating from the pdm/09virus and HA/NS from EA H1N1virus. To further explain the molecular mechanism of the pathogenesis of reassortants, the receptor binding specificity, polymerase activity and expression of IFN-β mRNA were measured to determine the effect of EA-origin HA/NS on pathogenesis. Previous studies showed that avian-type receptor-binding ability can increase influenza virus pathogenicity in macaques and mice. These results suggested that receptor binding specificity of IAVs may contribute to virus adaptation in mice. We also demonstrated that HA G222E affected the influenza virus virulence in mice due to loss of the binding to avian-type receptors. The HA-222G substitution appears to increase the pathogenicity of influenza viruses in mice under certain conditions and, as such, could be considered a virulence maker. Our results showed NS of EA virus enhanced significantly the polymerase activity when combined with RNP complex of H1N1/2009compared to others. This may be the reason that NS of EA virus enhanced high viulence in mice.Our previous results showed that HA222G enhanced the virulence in mice. The D222N HA mutation within the receptor-binding site was detected with higher frequencies in severe cases of2009pandemic H1N1infections. The impact of this mutation was investigated in vitro and in vivo using recombinant viruses. The recombinant D222N virus grew to significantly lower viral titers than the wild-type virus (WT) in A549but not in MDCK cells. A dose-dependent glycan array analysis with the D222N virus showed a modest increase in the binding avidity to human-like (a-2,6sialylated glycan) receptors and avian-like (a-2,3sialylated glycan) receptors than the WT virus. The D222N HA mutation resulted in slight weight loss, lower lung titers, inflammatory cytokines and alveolar inflammation in mice than the WT virus. This may or may not be associated with severe clinical outcomes reported in humans.In summary, our study revealed that coinfection of pigs reproducibly generated novel predominant genotypes reassortant viruses and bias of reassortant viues was present between the upper and lower respiratory tract,polymerase activity played a pivotal role in selecting reassortant viruses. The reassortant viruses bearing HA/NS form EA virus and RNP origined from pdm/09not only have a high frequency but also have an advantage in high-pathogenicity and high-transmission phenotype. Our findings provided theoretical basis for the next influenza pandemic warning forecast. |
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