Analysis Of Molecular Pathogenic Mechanism Of Novel Triple-reassortant H1N1 Subtype Swine Influenza Virus

Swine influenza virus(SIV)is composed of eight single-stranded,negative-strand RNAs and belongs to the Orthomyxoviridae family.At present,in domestic pigs the main epidemic subtypes of SIV are H1N1,H1N2 and H3N2.Among them,triple-recombinant H1N1 subtype SIV is one of the significant epidemic strains.The preliminary research data shows that,compared with European avian-like H1N1 subtype SIV(A/swine/Tianjin/6/2011(H1N1),TJ6 for short),the pathogenicity of new triple-recombinant SIV(A/swine/Tianjin/10/2013(H1N1),TJ10 for short)in mammalian models has significantly enhanced.The NS gene and ribonucleoprotein complex genes(RNP)of TJ10 synergistically promote the replication and pathogenic ability of the virus.This study will further explore the molecular pathogenic mechanism of the triple-recombinant H1N1 subtype SIV,which will provide strong data and theoretical support for the next influenza pandemic.Non-structural protein 1(NS1)is a pathogenic factor of influenza virus,it interacts extensively with cells and viral proteins to promote efficient virus replication and inhibit host antiviral reactions.When influenza virus infects the host,the host will produce a natural immune response against the virus.The pattern recognition receptors of the host can mediate antiviral signaling pathway and it can induce the expression of interferon and other related cytokines under the control of various mechanisms.RIG-I exists in the cytoplasm of infected cells and it serves as the main pattern recognition receptor that initiates the innate immune response against IAV.The NS gene of TJ10 plays an important role in the process of TJ6 infection,and the recombinant virus TJ6-TJ10 NS is more pathogenic in mice.There was certain difference of interferon beta induced by two viruses when infecting cells.We speculate that different abilities of NS1 to inhibit the host's antiviral responses affect the virulence of viruses,hence we respectively constructed recombinant plasmid NS1 of TJ6 and TJ10,then overexpressed NS1 of TJ6 and TJ10,induced with poly(I:C),and results showed that inhibition ability for ? interferon of TJ10 is stronger compared to TJ6.Then we overexpressed NS1 and IRF3,induced with poly(I:C),tested the expression of ? interferon,in the end we drew a preliminary conclusion that IRF3 and its downstream were found to be the reason for the difference in the expression of ? interferon induced by the two strains.In order to deeply explore whether NS gene and RNP of TJ10 play an important role in H3N2 subtype of swine influenza viruses,so we rescued recombinant viruses with H3N2 subtype swine influenza virus(A/Swine/Heilongjiang/1/2005,HL for short).The pathogenicity of recombinant viruses depends on their ability to replicate in cells and animals.The data showed that NS gene and RNP of TJ10 play an important role in the process of infection of H3N2 swine influenza virus(HL).HL-TJ10(NS RNP)has a much higher replication capacity on MDCK cells compared to other recombinant viruses,the pathological damage of the lungs was more serious,and the titers of the lungs and turbinate bones were higher,which triggered a huge cytokine storm in the body.Data in vivo and in vitro showed that HL-TJ10(NS RNP)presented higher pathogenicity,suggesting that NS and RNP synergisticallypromoted viral transcription and replication to enhance virulence.At the same time,we also explore whether NS gene and RNP of TJ10 play an important role in pathogenicity of H1N1 subtype classical SIVs(A/Swine/Guangdomg/1/2011,G11 for short),so we rescued recombinant viruses with H1N1 subtype classical SIVs.Data in vivo and in vitro showed that NS gene of TJ10 played a key role in the process of infection of G11.Survival rate of experimental mice inoculated with G11-TJ10(NS)is only 20%,while there is no death in experimental mice inoculated with G11 and other recombinant viruses.G11-TJ10(NS)presented higher replication capacity in MDCK cells,presented higher pathogenicity in mice,the more serious pathologic damage,higher titers of lungs and turbinate bones and triggered a more serious cytokine storm compared with other recombinant viruses.Therefore,we speculate that the NS gene of TJ10 could inhibit the innate immune response of the host,and indirectly promote the replication of the virus and enhance the virulence of the virus.