Effect And Mechanism Of Pdm09-derived M Gene On The Pathogenicity And Transmissibility Of H1N1 Subtype Swine Influenza Virus

Swine influenza virus(SIV)is common in pig herds in China and different subtypes are prevalent.’What’s more,genetic reassortment often occurs to form novel SIV variants.Due to pig’s tracheal epithelium containing both human-like α-2,6 receptors and avian-like α-2,3 receptors,pigs are considered as not only the intermediate hosts for avian influenza virus(AIV)adaptation to human beings but also the ’mixing vessel’ for new reassortants.Therefore,the study of SIV evolutionary dynamics has important public health significance.Relevant epidemiological investigation shows that the mainly prevalent Eurasian avian-like(EA)H1N1 SIV in China has a variety of genotypes,of which G4 is the currently dominant genotype.As compared with the previously circulating G5 genotype,the main difference in genome composition of G4 is that the M gene from EA-H1N1 source was replaced by the 2009 pandemic(Pdm09)H1N1.Given that the matrix protein M1 and ion channel protein M2 encoded by the M gene play important roles in many stages of the life cycle of influenza virus,the introduction of Pdm09-derived M gene is likely to affect the characteristics of the EA-H1N1 virus in vivo and in vitro.It is necessary to determine the pathogenicity and transmissibility of the Pdm09-like M gene reassortants,and therefore to assess the interspecies infection capacity and explore related mechanisms.1.Effects of Pdm09-derived M gene on the biological characteristics in vitro of H1N1 subtype swine influenza virusBased on the 8 reverse genetics plasmids of G5 genotype EA-H1N1 SIV A/Swine/Jiangsu/01/2016(JS01),the M gene of JS01 was replaced with that of Pdm09-H1N1 SIV A/Swine/Jiangsu/48/2010(JS48),and two model viruses rJS0l and rJS48M were rescued.After plaque purification,the HA,TCID50,EID50 and other basic biological characteristics of the two reassortant viruses were measured,then the expression levels of M1,M2 protein and autophagy levels of virus-infected cells were compared.Further,the morphology of virus particles was observed by transmission electron microscopy.The results showed that rJS01 and rJS48M had similar basic biological characteristics.There was no significant difference in the replication ability on MDCK and A549 cells at 0.1 MOI.Both rJS01 and rJS48M could induce autophagy on MDCK,but there was no significant difference on autophagy levels.However,the M2 protein expression level of rJS48M post infection on MDCK and A549 cells was significantly higher than that of rJS01 at 6h and 9h.In addition,electron microscopy results showed that although the morphology of rJS48M and rJS01 progeny virions are mostly spherical,the proportion of filamentous virions in rJS48M was significantly higher than that of rJS01.Therefore,those results suggest that the Pdm09 source M gene can promote the expression of M2 protein and affect the particle morphology of EA-H1N1 viruses.2.Effects of Pdm09-derived M gene on the pathogenicity and transmissibility of H1N1 subtype swine influenza virus and the molecular basisIn order to evaluate the pathogenicity and transmissibility of the pair of model viruses,we firstly intranasally inoculated 6-week-old BALB/c mice with rJS01 and rJS48 at 106.0EID50,respectively.Then the change of mice’s body weight was recorded for 14 days.At the same time,pathological biospay of lung tissues were observed,virus titers and relative expression levels of inflammatory factors such as IL-1β,TNF-α and IL-6 in lung were measured as well.Subsequently,different amino acids in different functional regions of M gene between JS01 and JS48 was analyzed and the prevalent proportion of differential functional sites was also analyzed according to influenza A H1N1 viruses from 2009 to 2020 in the GISAID database.Based on the above analyasis,two M gene chimeric reassortants rJ142 and r41 J2 were rescued,and their pathogenicity on mice were also evaluated.Subsequently,the transmissibility of the 4 reassortant viruses were measured in guinea pigs.Mice experiment showed that the introduction of Pdm09-derived M gene enhanced the pathogenicity and replication of EA-H1N1 virus,and rJS48M significantly increased viral loads in lung tissues of mice,resulting in stronger pathological changes and higher inflammatory expression in lung tissues.The guinea pigs experiment showed that guinea pigs experiment showed that the introduction of Pdm09-derived M gene enabled EA-H1N1 viruses to possess direct contact transmission ability among guinea pigs,and the reassortant rJ142 which had embedded the C-terminal domain of rJS48 M2 protein on the rJS01 backbone also even acquired aerosol transmission ability.3.Explore the molecular mechanism of Pdm09-derived M gene affecting the characteristics of H1N1 subtype swine influenza virus from the perspective of interaction with host proteinsAs rJS48M possessing stronger pathogenicity on mice and transmissibility on guinea pigs than rJS01,the perspective of interaction with host proteins was used to analyze the molecular mechanism of it.Firstly,JS01 and JS48 M2 proteins wereused to construct eukaryotic expression plasmids with double-labels of Flag and HA on the pCDNA3.1 vector.Then,the M2 protein expression was validated by indirect-immunofluorescence assay.Subsequently,co-immunoprecipitation(Co-IP)combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS)was applied to screen host proteins which might interact with M2 protein in 293T cells.The selected host proteins were further analyzed by Gene Ontology(GO)annotation and KEGG pathway enrichment.Lastly,the differently expressed interacting host proteins between rJS01 and rJS48M were verified by Western blot.GO enrichment analysis showed that the co-interacting host proteins are mainly enriched in nucleic acid-related proteins,and the host proteins interacting with JS01 M2 were mainly enriched in cytoskeleton-related proteins and viral processes while the situation in JS48 M2 were of cell life activities and regulation of nucleic acid replication.KEGG pathway analysis showed that the signaling pathways of co-interacting host proteins with M2 protein were mainly related to cell phagocytosis.In specific,the interacting host proteins with JS01 M2 protein were mainly focused on RNA transport,but that with JS48 M2 protein were mainly related to DNA replication.As verified by Western blot,all the three selected host proteins of Tublin,MYO6 and CAP2A2 could interact with the M2 protein.However,significant difference of interacting level with both MOY6 and CAP2A2 proteins existed between JS01 M2 and JS48 M2 proteins.