Pathogenicity Enhancement Of Reasortant H1N1 Swine Influenza Virus Containing The H9N2 Avian Influenza Virus Internal Gene Cassete In Mice

In recent years,the predominant swine influenza viruses in China are Eurasian avian-like H1N1 viruses and the subtype viruses frequently reassort with other influenza viruses,constantly infecting swine herds to cause economic losses,and even leading to human cases to threaten public health.In addition,pigs play the role of "mixing vessel" in the ecology of influenza viruses.Types of ?-2,3 and ?-2,6 sialic acid receptors were both found in the respiratory epithelium of pigs,which means that pigs can be infected with both mammalian and avian influenza viruses.Studies have shown that since 1998,pigs infected with H9N2 avian influenza viruses have been repeatedly reported in several regions of China.And,the currently dominant H9N2 viruses in domestic poultry flocks of the country are the genotype S H9N2 viruses,which could regularly provide the whole internal gene cassette for novel avian influenza viruses that can infect human such as H7N9,H10N8 and H5N6.Therefore,when the Eurasian avian-like H1N1 and genotype S H9N2 viruses coexist in the swine herds,it is highly likely that the two may exchange gene segments to generate the novel reassortant H1N1 virus with internal genes entirely from H9N2.It is necessary to evaluate the basic biological characteristics and potential risk of human infection of the H1N1 swine influenza reassortant,and to explore the related molecular mechanisms.1.Construction of a reassortant H1N1 swine influenza virus containing the H9N2 avian influenza virus internal gene cassette and its pathogenicity in miceThe Eurasian avian-like H1N1 virus A/swine/Jiangsu/01/2016(H1N1)[JS/01]and the genotype S H9N2 virus A/chicken/Jiangsu/CZ73/2014(H9N2)[CZ/73]were selected to construct their reverse genetic plasmids,respectively.And the six internal genes involving PB2,PB1,PA,NP,M and NS of CZ/73(H9N2)were fixed with the HA and NA genes of JS/01(H1N1)to rescue the reassortant virus rH1N1/H9N2.Experiments in vitro showed that the reassortant rH1N1/H9N2 caused a higher percentage of early apoptosis in MDCK cells and therefore exhibited a lower TCID50 and weaker replication in cells,and formed plaques in MDCK cells with a larger diameter to indicate a stronger invasion ability,as compared with its two parental viruses.Infectivity experiments in vivo performed on BALB/c mice by intranasally inoculation showed that rH1N1/H9N2 virus was moderately pathogenic to mice with the MLD50 of 105.0EID50,while the two parental viruses were both low pathogenic with the MLD50 greater than 106.5EID50.Challenging at the dosage of 106.0EID50,rH1N1/H9N2 virus induced 80%mice mortality,whereas the two parental viruses did not cause any death of infected mice.On 1,3 and 5 days post inoculation(dpi),virus titers in the mice lung of rH1N1/H9N2 group were dramatically higher than that of the JS/01(H1N1)group,but with no significant difference from the CZ/73(H9N2)group.Analysis of pathological sections in the lung revealed that rH1N1/H9N2 generated evidently severe pathological changes than the two parental viruses on 5 dpi.Furthermore,transcriptome sequencing and fluorescence quantitative PCR verification revealed that the expression levels of inflammation-related genes including GlyCAM1,CR2,CD19,CD22,CXCR5,CD79a,H2-Ob,and H2-Oa were significantly higher in the lungs of mice infected with rH1N1/H9N2 virus.Therefore,the introduction of the internal gene cassette of genotype S H9N2 virus significantly enhanced the pathogenicity of the reassortant H1N1 swine influenza virus in mice.2.Molecular basis on the pathogenicity enhancement of reassortant H1N1 swine influenza virus containing H9N2 avian influenza virus internal gene cassette in miceIn order to further analyze the molecular basis of reassortant rH1N1/H9N2 virus with enhanced virulence in mice,we conducted the following two parts of experiments.On one hand,the internal genes from CZ/73(H9N2)virus were gradually reduced on the basis of rH1N1/H9N2 virus backbone to construct reassortant viruses,and to identify gene combinations leading to increased virulence.On the other hand,reassortant viruses containing only one internal gene from CZ/73(H9N2)were constructed to clarify whether single gene replacement has an impact on the viral pathogenicity.Serial reassortant viruses were successively rescued,indicating that the Eurasian avian-like H1N1 viruses and genotype S H9N2 viruses had good genome compatibility.The pathogenicity experiments in mice showed that the six reassortants at least containing the joint PB1 and PA genes from CZ/73(H9N2)virus,including rJS/01-RNP+M,rJS/01-RNP+NS,rJS/01-RNP,rJS/01-PB2+PB1+PA,rJS/01-PB1+PA+NP and rJS/01-PB1+PA,as well as the reassortant rJS/01-NP just containing single NP gene from CZ/73(H9N2)virus,could lead to significant body weight loss in infected mice.Among them,rJS/01-PB1+PA and rJS/01-NP viruses could even lead to mice death,and the former was more pathogenic than the latter,but both were weaker than rH1N1/H9N2 virus.Experiments in vitro of the above seven reassortants showed that rJS/01-PB1+PA and rJS/01-NP possessed higher or equivalent replication ability in MDCK,CEF,A549 cells than the others.However,the results of polymerase activity determination showed that only rJS/01-PB1+PA+NP virus increased significantly while the other 6 strains including rJS/01-PB1+PA and rJS/01-NP all decreased significantly,which might be related to the reduced expression of RNP-related proteins.The above results indicated that the combination of PB1 and PA genes,or the single NP gene played important roles in the process of enhancing the mice pathogenicity of reassortant H1N1 swine influenza virus containing complete set of internal genes from genotype S H9N2 virus.And there may be certain synergistic effect among the six internal genes,but the specific mechanism still needs to be further explored.In summary,we evaluated the pathogenicity to mammals of the reassortant H1N1 swine influenza virus with HA and NA genes derived from an Eurasian avian-like H1N1 influenza virus but all the internal genes derived from a genotype S H9N2 virus,and also identified the relevant molecular basis in the present study.The results will provide theoretical reference for the early warning of human infection with swine influenza viruses and the mechanism analysis of genotype S H9N2 as internal gene donor for novel influenza viruses.