The Effect Of PA Gene On The Adapation And Pathogenicity Of Influenza Viruses In Mammal

Animal influenza virus not only caused serious economic losses to livestock, but also crossed species barrier to infected people, posing a serious threat to public health. Therefore, it is important to understand mechanisms of adaptation and pathogenesis for prevention and controlling of influenza viruses. PA gene is one gene segment of genomes of type A influenza virus, which encodes a variety of viral proteins including PA protein, PA-X protein, PA-N155 protein and PA-N182 protein. It has been demonstrated that these viral proteins encoded by PA gene were closely related to the host specificity and adaptation of influenza virus. However, the key residues in PA protein influencing the adaptation and pathogenic of influenza virus has not been fully revealed.Through alignment of PA protein sequences of H9N2, H7N9 and H10N8 viruses available in the National Center for Biotechnology Information (NCBI) Influenza Viruses Resource, we found that mutation PA-K356R has become predominant since 2014 in avian H9N2 viruses in China. The same mutation is also found in most human isolates of emergent avian H7N9 and H10N8 viruses, which contains six internal gene segments from the H9N2 virus. PA-K356R alone, or coupled with the well known mammalian adaptive mutation PB2-E627K, exhibit higher prevalence in human-originated H7N9 and H10N8 influenza viruses instead of avian-originated strains. Therefore, we hypothesize that the PA-K356R or/and PB2-E627K facilitate interspecies transmission of H7N9 and H10N8 virus from avian to humans.To verity this hypothesis, we rescued viruses possessing single mutation of PA-K.356R, PB2-E627K and combined mutations (co-mutant) of PA-K356R and PB2-E627K, which were designated as PA-K356R, PB2-E627K and PA-K356R/PB2-E627K respectively. And we further demonstrated the mammalian adaptive functionality of PA-K356R mutation. Avian H9N2 virus harboring mutation PA-K356R in human A549 cells showed increased nuclear accumulation of PA and raised viral polymerase activity that resulted in elevated viral transcription and virus output. The same mutant virus in mice also enhanced virus replication and caused lethal infection. In addition, combined mutations of PA-K356R with PB2-E627K, a well-known mammalian adaptive marker, in H9N2 virus showed further cooperative increase in virus production and severity of infection in vitro and in vivo. In summary, PA-K356R behaves as a novel mammalian tropism mutation which along with other mutations such as PB2-E627K could render avian H9N2 viruses adapted for human infection.PA-X is a fusion protein in influenza A virus encoded in part from a+1 frameshift open reading frame (X-ORF) in segment 3 which is conserved in influenza viruses, suggests important functional roles. However, the importance of PA-X protein in current circulating swine influenza virus (SIV) in pigs is still unknown. In present study, we constructed PA-X deficient virus in the background of a "triple-reassortment" (TR) H1N2 SIV, and evaluated the biological characteristics in vitro and in vivo. Compared with PA-X deficient virus, wild type H1N2 SIV with PA-X had increased viral replication in porcine cells, along with enhanced pathogenicity, replication and transmissibility in pigs. Furthermore, we found that SIV with PA-X improved the inhibition for non-viral protein synthesis and innate immune response. Our study highlights the importance of PA-X on the moderation of viral pathogenesis and pathogenicity of SIV in pigs, which indicated that PA-X is a pro-virulence factor in SIV.As we all known, the X-ORF of PA-X exists in either full length or a truncated form (either 61-or 41-condons). Genetic evolution analysis indicates that all swine influenza viruses (SIVs) possessed full-length PA-X prior to 1985, but since then SIVs with truncated PA-X have gradually increased and become dominant, implying that truncation of this protein may contribute to the adaptation of influenza virus in pigs. To verify this hypothesis, we constructed PA-X extended viruses in the background of a "triple-reassortment" H1N2 SIV with truncated PA-X, and evaluated their biological characteristics in vitro and in vivo. Compared with full-length PA-X, SIV with truncated PA-X had increased viral replication in porcine cells and swine respiratory tissues, along with enhanced pathogenicity, replication and transmissibility in pigs. Furthermore, we found that truncation of PA-X improved the inhibition of IFN mRNA expression. Hereby, our results implied that truncation of PA-X may contribute to the adaptation of SIV in pigs.In conclusion, this study demonstrated that PA-K356R is a novel mammalian adaptive marker and revealed the potential molecular mechanisms, which contributed to the adaptation and pathogenicity of H9N2 virus in mammals. We also found that H1N2 SIV with PA-X inhibited innate immune response, which consisted with the enhanced replication, pathogenicity and transmissibility of PA-X mutants in pigs. Furthermore, compared with that of full length PA-X, SIV with truncated PA-X conferred increased adaptation and pathogenicity in pigs, which was consistent with the increasing occurrence of SIV with truncated PA-X. Therefore, our study provides the theoretical foundation for the assessment of epidemic potential and development of prevention and control strategies of influenza A viruses in mammals and humans.