The Isolation,and Replication Mechanism Study Of Eurasian Avian-like H1N1 Swine Influenza Virus

Swine influenza is a common respiratory disease in pigs,often accompanied by cough,sneezing,runny nose,fever and dyspnea.Swine flu causes not only a great loss to the pig industry,but also a serious threat to human health.So it has important public health significance.In this study,an H1N1 subtype swine influenza virus was isolated from a diseased pig farm.Further analysis showed that the virus belonged to Eurasian Avian-like swine influenza virus.By using RT-PCR technology,8 gene fragments were amplified and cloned into the vector of influenza virus reverse genetic system,and 8 plasmids were co-transfected into 293 T cells,and the H1N1 virus was successfully rescued.Sequencing showed that the rescued H1N1 virus is consistent with the complete sequence of parental isolate.Compared with sequences,we found that there are two start codons in PB2 gene,the second ORF encodes a protein with a length of 71 amino acids,named PB2-X.By site directed mutagenesis,the PB2 gene second start codon was mutated(ATG-ATT),and a mutant H1N1 that did not express PB2-X was rescued.By comparing the growth characteristics of two viruses on MDCK and ST respectively,the results showed that all two viruses had strong replication ability in ST.The growth curve test and polymerase activity test showed that the replication ability of the mutant H1N1 that did not express PB2-X protein was superior to the parental H1N1 virus,which indicates that the presence of PB2-X affects the replication ability of virus in vitro.By comparing the virulence of two H1N1 strains of virus in mice,the results showed that the weight of two groups of infected mice did not decrease obviously,which indicated that the pathogenicity of the isolated Eurasian avian-like H1N1 swine influenza virus was weak to the mice,and the influenza of PB2-X on pathogenicity of H1N1 is limited.The reverse genetic system of Eurasian avian-like H1N1 established in this study lays the foundation for the study of its replication and pathogenic mechanism,and for the first time the PB2 gene of this Eurasian avian-like H1N1 is found to express a new PB2-X protein.