Effect Of M Gene Derived From Cs And Pdm09 On Pathogenicity Of H1N1 Subtype Swine Influenza Virus

Swine influenza virus(SIV)is a negative-stranded RNA virus with eight segments,belonging to the family Orthomyxoviridae.The infection of pigs with SIV will lead to the decline of pig production performance,affect the health of the pig population and product quality,and cause huge economic losses.As the pig body simultaneously contains an avian influenza receptor and an abortion receptor,swine,poultry and human influenza can all infect the pig,and various influenza viruses can mutually carry out gene rearrangement in the pig body,thereby promoting the appearance of pandemic influenza virus strains.The M gene of the virus encodes matrix protein M1 and ion channel protein M2,which play important roles in multiple stages of the life cycle of the influenza virus.Some studies have claimed that M genes of different lineages can affect the pathogenicity of the recombinant virus.At present,there is no research on the influence of M gene from the 2009 pandemic source(pdm09)and classical source(CS)on the pathogenicity of H1N1 SIV.Therefore,it is necessary to study the related mechanisms of their effects on pathogenicity to provide new ideas for the prevention and control of swine influenza virus.Two representative strains of H1N1 SIV were isolated in the early stage of our laboratory,which were respectively A/swine/Henan/NY361/2013(H1N1),or NY361 for short.The M gene of each strain was derived from Pdm09 H1N1 SIV.The other strain is A/swine/Hubei/HG394/2018(H1N1),abbreviated as HG394,with the M gene derived from CS H1N1 SIV.The pathogenicity of the two viruses to mice was verified in an in vivo test,and the results showed that the lethal rate of HG394 to mice was much higher than that of NY361.Through sequence alignment,we found that the amino acid homology of NY361 and HG394 was very close,and they were significantly different only in the M gene,with the homology of the remaining seven genes ranging from 99.9% to 100%,suggesting that the M gene affected their pathogenicity to mice.In order to further analyze the molecular pathogenic mechanism of the virus,in this study,the parent strains of H1N1 swine influenza viruses NY361 and HG394 were used as the skeleton,and the recombinant virus was rescued by reverse genetic manipulation for in vitro and in vivo experiments.The results showed that the replication abilities of the rescue strain and the wild strain in A549 cells were consistent,and the pathogenicity of the recombinant virus was not changed by reverse genetic manipulation.The pathogenicity of HG394 to mice and its ability to replicate in cells were higher than those of NY361,and the replication level of HG394 at low temperature was higher than that of NY361.In the next step,the recombinant virus was rescued and identified by replacing M1,M2,and M gene fragments of NY361 and HG394,and the effect of M1,M2,and M genes derived from pdm09 and CS on the pathogenicity of H1N1 subtype swine influenza virus was further studied.Data from in vitro and in vivo studies showed that CS-derived M1 gene and M2 gene enhanced the pathogenicity of the recombinant virus to mice and the level of replication in cells,and CS-derived M2 gene enhanced the replication of the recombinant virus in mouse turbinate and in cells at low temperature.The recombinant virus was rescued and identified by further substitution of M1 and M2 gene fragments against NY361 strain.In vitro experiments showed that the 207AA-232 AA regions of CS-derived M1 gene and the 43AA–49AA regions of M2 gene had key amino acid sites that affected the pathogenicity and replication ability of the recombinant virus in cells,while the 43AA–49AA regions of M2 gene had key amino acid sites that affected the replication ability of the recombinant virus at low temperature.To sum up,in this study,we analyzed the effects of the M gene derived from CS and pdm09 on the pathogenicity of H1N1 SIV and found the amino acid regions that affected the pathogenicity of the recombinant virus and its replication ability at low temperature,which will help us to understand the effects of the M gene derived from CS and pdm09 on the biological characteristics of H1N1 SIV,further improve our understanding of the molecular mechanism of influenza virus pathogenicity,and grasp the potential risk of influenza virus to humans,so as to provide a reliable theoretical basis for the next influenza pandemic.