Construction And Virulence Mechanism Analysis Of Influenza Virus A/New Caledonia/20/1999(H1N1)in Mice

Influenza A virus continues to evolution and circulated widely among human,which becomes one of the critical pathogens that cause human morbidity and mortality.Systematic study of influenza A virus and raising awareness of the virus can strengthen our ability to prevent and control it.The most common animal model for studying human influenza virus pathogenicity in laboratory is mice.By serial passage in lung tissue,fluA viruses can acquire mouse-adapted strain.Mouse-adapted virus is not only the platform for antibody and drug evaluation,but also important for understanding the adaptation relationship between virus and host.It is also meaning for studying the cross-species transmission of viruses and the molecular mechanisms of viral virulence enhancement.H1N1 is one of the major subtypes of influenza,affecting the world and seriously endangering human health.In this study,the A/New Caledonia/20/1999(H1N1)published by the WHO from 2000 to 2007 as the vaccine strain was utilized to establish mouse-adapted virus in BALB/c mice.The research covered the aspects of clinical characteristics,viral load of lung,pathogenicity,histological analysis,comparison of replication kinetics,and amino acid substitution in the process of wild-strains to adapted strains,the adaptive process of lung adaptation strains of H1N1 mice was gradually explored and the mechanism of virus virulence change was determined,and it also provided a stable animal model for H1N1 influenza A virus-related drug evaluation.In the work of mouse-adapted virus establishment and evaluation,14 consecutive passages were performed in the mouse lung tissue to gradually increase the virulence and obtain a highly pathogenic adapted strain.Then,each generation of adapted strains was monitored.Survival status and lung viral load of mice;Secondly,virus virulence and pathological studies of lung tissue were performed on each generation of adapted strain of virus,and the replication kinetics was analyzed both in vivo and in vitro.Mechanisms of viral virulence changes were seen explore initially.In the virulence analysis work,with whole genome sequencing and analysis were performed on each generation of adapted virus,found seven non-synonymous mutations that gradually appeared,including NP-A363D,NP-A373D,NP-M440K,PA-V44I,PB2-V81M,HA-T106P,and PA-F35L Two of them are important mutations associated with the virulence change.Consequent use of reverse genetics to construct mutation-containing sites,testing the recombinant viruses polymerase activity and pathogenicity at the animal level.It was verified that the HA-T106P and PA-F35L are the key sites for the enhanced virulence of A/New Caledonia/20/1999(H1N1).In particular,PA-F35L can enhance the polymerase activity of the virus.The mouse-adapted virus was successfully applied to the evaluation of antiviral drugs,confirming the application value of A/New Caledonia/20/1999(H1N1)mouse model.In summary,this study constructed a mouse-adapted virus strain of H1N1 that can be used as a research tool for cross-species adaptation of influenza A virus.It not only elaborated the adaptation process of wild type influenza strains to new hosts,but also further studied its key virulence sites and found two fundamental mutation sites related to the enhanced virulence of viruses.Moreover,based on those researches,we have the opportunity to combine genetic modification and reverse genetics to provide ideas for the efficient establishment of more strains of other mouse-adapted influenza virus,and to establish the basis for the study of pathogenic mechanisms of other influenza A viruses.