| PA-X protein is a novel protein discovered by Jagger et al. in 2012, which is encoded by PA gene segment through +1 ribosomal frame-shifting. Previous studies of PA-X were limited to 1918 H1N1 human influenza virus, 2009 pandemic H1N1 human influenza virus, highly pathogenic avian influenza(HPAI) H5N1, and H9N2 avian influenza viruses. However, the roles of PA-X on the replication and virulence of swine influenza virus are still unknown. To evaluate the effects of PA-X protein on the replication ability and virulence of swine influenza virus, the PA-X deficient plasmid was constructed on the basis of reverse genetic system of Swine/Guangdong/1/2011. Swine/Guangdong/1/2011(wild type G11) virus and its PA-X deficient virus were rescued, and then their replication ability and virulence were compared in vitro and in vivo(mice), moreover, their polymerase activity in 293 T cells were compared. The results showed that, the deficiency of PA-X could significantly enhance the replication efficiency of the virus in MDCK, A549 and PK-15 cells, the difference between their replication ability was not so apparent but the PA-X deficient virus could replicate a little more efficiently than the wild type G11 virus; the deletion of PA-X increased viral replication and pathogenicity, which were related to high viral loads in lungs, severe pathological damage, more weight loss and higher mortality; the polymerase activity was improved while PA-X protein unexpressed.Generally, the C-terminal of PA-X protein is composed of 61 amino acids expressed by X-ORF. However, in some viruses, the X-ORF can naturally express a peptide with only 41 amino acids infronted because of a TGG(Trp) to TAG(stop codon) mutation at codon 42 in X-ORF. The wild type G11 virus in our study naturally encodes X with only 41 amino acids. To explore the effects of the truncated 20 amino acids on the replication and virulence of the virus, we rescued the Swine/Guangdong/1/2011(wild type G11) virus and its mutant virus with full length PA-X via reverse genetics. We then compared their replication ability and virulence in vitro and in vivo(mice). The results showed that, compared to wild type G11, the mutant virus with full length PA-X can significantly enhance the replication efficiency of the virus in MDCK cells and A549 cells, the difference between their replication ability in PK-15 cells is not so apparent but the mutant virus can replicate a little more efficiently than the wild type G11 virus; virus with full-length PA-X increased viral replication and pathogenicity which was related to high viral loads in lungs, severe pathological damage, more weight loss and higher mortality; the polymerase activity was improved when the premature termination codon was deleted.PB2-627 K plays an important role in the influenza host adaptation and can contribute to the virulence of avian, human, classical H1N1 swine and triple reassortant swine influenza virus in mouse model. According to the phylogenetic analysis, the PB2 gene of human-origin H3N2 swine influenza virus is very different from the PB2 gene of classical and triple reassortant swine influenza virus. And the effect of PB2-627 amino acid in human-origin H3N2 swine influenza virus is still unknown. In our studies, we firstly constructed the reverse genetic system of a human-origin H3N2 swine influenza virus through reverse genetics. Then the PB2-627 amino acid was mutated from K to E, the PB2-627 K wild type virus and PB2-627 E mutant virus were rescued. We compared their replication ability and virulence in cultured cells and mice. As shown in the results, the replication ability decreased while the PB2-627 postion was mutated from K to E; the plaques formed in MDCK cells also demonstrated that PB2-627 K virus could replicate more efficiently than PB2-627 E virus; the polymerase activity also decreased while PB2-627 position was mutated from K to E; viral virulence and replication ability in lungs were also decreased because of the PB2 K627 E mutation. |
PDF Full Text Request