Establishment Of A Mouse-adapted Seasonal H3N2 Influenza Virus In Mice And Its Animal Model

IntroductionInfluenza viruses can cause infections in many animals and seriously endanger human health and public health.In recent years,influenza A and B viruses are reported as the seasonal influenza activities worldwide.The continuous recombination and reassortment of surface neuraminidase（NA）and hemagglutinin（HA）of influenza A virus are the main factors leading to the uncontrollable viral infection.Recently,the H3N2 virus has been mutated,resulting in "off-target" vaccines,drug resistance,and the prevention and treatment of influenza A.Therefore,establishment of a suitable animal model is the key of pathogenesis of H3N2 and the evaluation of therapentic efficacy.At present,H3N2 strains have been constructed using reverse genetics techniques and related animal models have been established.However,there is still a lack of relevant resources in China.In order to develop corresponding therapeutic drugs,screen new vaccine strains,and evaluate the availability of drug resources,this study established H3N2 mouse adaptation strains and established a mouse model of H3N2 adaptation strains.ObjectiveTo establish adapted strain of seasonal H3N2 influenza virus in mice and its animal model and investigate the potential molecular mechanism of adaptation.Methods1.Establishment of a mouse-adapted seasonal H3N2 Influenza Virus in Mice1)BALB/c mice were intranasally infected with seasonal influenza virus of A/Aichi/2/68（H3N2）（WT）,and lung tissue suspension was prepared at the peak of virus replication.The mouse-adapted strain was obtained by continuous passage the A/Aichi/2/68（H3N2）virus in the mice lungs.2)The clinical signs and histopathology of the mouse-adapted strain and WT stain were analyzed.3)Sequences of different generations were obtained by sequencing,and the mutation sites of the sequences of different generations were compared and analyzed by MEGA.4)Analyze and compare the expression levels of inflammatory cytokines in serum and BALF of mice infected with the mouse-adapted strain and WT stain by CB A.5)Analyze and compare the the changes of immune cell types in lung tissues of mice infected with the mouse-adapted strain and WT stain by the TS A multiplex IHC workflow.2.Establishment of a mouse-adapted seasonal H3N2 virus-infected murine modelThe mouse-adapted strain（MA-7）was obtained by continuous passage the A/Aichi/2/68（H3N2）virus in the mice lungs,and infected the BALB/c mice intranasally to establish the mice model.The clinical signs,body weight loss rate,virus loads in the tissues and histopathology was observed.ResultsAfter 7 passages in the lung tissue of BALB/c,virulence of the seasonal influenza virus A/Aichi/2/68（H3N2）（WT）was increased to a high level.1.Keep losing weight in mice infected with the mouse-adapted virus and all died within 14 d.p.i.Meanwhile,there was no significant change in symptoms was observed in mice infected with the WT.2.The lung tissue of mice infected with the mouse-adapted virus showed obvious viral interstitial pneumonia.3.Five mutations from genome sequencing and alignment.Genetic analysis indicated that the increase of virulence of the mouse-passaged virus was attributed to the incremental acquisition of mutations in the HA（P162Q,N483D）,NA（V398I）,NP（D290N）,and PA（G631S）.4.The infection with the mouse-passaged virus（MA-7）virus,induced high levels of cytokine and chemokine in BALF and serum,such as IL-1α,IL-1β,TNF-α,IL-6,IFN-γ,IL-5,MCP-1,GM-CSF.5.Mice infected with mouse-passaged viruses（MA-7）recruited amount of immune cells,such as CD4⁺T,CD8⁺T,Treg,DCs,NK cells in the pulmonary tissue and the percentage was increased along with the increase in passaged-generations.And the number of CD4⁺T,CD8⁺T,Treg,NK cells gathered around H3N2 virus within 100μm was obviously enhanced,however,there are no dendritic cells was detected around the mouse-passaged H3N2 virions within 100um in the lungs.Mice infected with A/Aichi/2/68（H3N2）showed no obvious changes in symptoms and no virus replication was detected.However,all mice infected with MA-7 died within 9 d.p.i,the weight loss rate was more than 30%.The virus replication was detected in multiple tissues,especially in lung tissues(10^5.5TCID₅₀),which caused interstitial pneumonia.According to the observation results of symptoms after infection in mice and virus loads in the tissues and histopathology,the mouse-adapted seasonal H3N2 virus-infected murine model was successfully established.Conclusion1.The mouse-adapted strain was obtained;2.The mouse-adapted seasonal H3N2 virus-infected murine model was successfully established;3.The increased virulence of adaptive virus strain may be related to the five mutations in the virus genes;4.The infection by the mouse-adapted seasonal H3N2 virus can induce a strong immune response,such as cytokine storm,a large number of inflammatory cell infiltration.