Preliminary Study On Pathogenesis And Reverse Genetic System Construction Of Avian H9N2 Influenza Virus

Influenza viruses belong to the Orthomyxoviridae family and are characterized by segmented,negative-strand RNA genomes.They are highly infectious and show great antigen diversity.Influenza virus can infect a wide variety species.Generally, there is a host range restriction,not easily cross-species transmission during influenza virus infection.However,in some cases,avian influenza virus can transmit to human directly.Until now,the known avian influenza viruses including H5N1,H9N2,H7N7 as well as H7N3 subtype viruses can infect human.Therefore,it is important to study these avian influenza viruses in the field of disease control and prevention.Since 1998, the human H9N2 infection cases were found in both the Mainland China and Hong Kong.Seroprevalence rates in human population and poultry were around 15%.The assay of receptor specificity indicated that avian H9N2 influenza virus has the ability to bind to SAα-2,6Gal receptor,all these showed it is possible to cause the influenza pandemic and we should take more attention.The main purpose of this study is to characterize the H9N2 avian influenza virus A/Guangzhou/333/99 isolated in a patient in China.Firstly,the pathogenicity of the virus was studied by using the mice in vivo model.Secondly we constructed the 8 plasmids reverse genetic system.It laid the foundation to study the pathogenicity in the future.This following topic includes:1.Infected BALB/c mice with avian H9N2 influenza virus and abserved clinical manifestations.Viral load in lung and trachea were studied. 2.Use the vector pCI-pol I as the expression vector to construct 8 plasmid rescue system.This vector is derived from the pCI vector for transcription/expression vector,in the polⅡpromoter(CMV) and termination sequence(SV40) were inserted reversely polⅠpromoter and murine polⅠtermination sequence.The eight gene segments of the cDNA were inserted into the vector,respectively, constructing pCI-PB2,pCI-PB1,pCI-PA,pCI-HA,pCI-NP,pCI-NA,pCI-M, pCI-NS eight expression plasmid.8 plasmids were co-transfected into 293T cell and rescued H9N2 virus.3.Use 7 plasmids(PB2,PB1,PA,HA,NP,NA,M) from 8 plasmids express system to match separately with the NS of H1,H3,H5 subtype viruses and rescue 7+1 reassortant viruses namely H1NS-H9N2,H3NS-H9N2,H5NS-H9N2.The validity of H9N2 virus 8 plasmids reverse system was confirmed and built the foundation for the study on the function of NS gene of different subtype viruses.The following findings were observed:1.The BALB/c mice in vivo model suggested that the H9N2 virus can infect and replicate in the lung and trachea,but cannot result in death.Significant body weight loss can be observed in the infected animals as compared with the control group.The host immune response can also be triggered by the virus.2.Using 8 plasmids reverse genetic system could rescue the virus.The biology characteristic of the rescued virus is similar to the wild virus.The receptor characteristics test also indicated that the rescued virus and wild virus both have the ability to bind to SAα-2,6Gal receptor.3.Take 8 plasmids reverse system of H9N2 avian flu virus as the backbone,the NS gene replace with H1,H3,H5 subtype NS plasmid respectively,obtains matches 7+1 reassortant virus grows well on the chicken embryo and MDCK cells,it laid the foundation to study the function of NS gene of different virus subtypes in the future.The result indicated that avian H9N2 influenza virus can infect the BALB/c mice without adaptation,death and obvious pathogenicity were not observed.The reverse genetic for avian influenza H9N2 subtype virus has been established successfully.It laid the foundation to study the pathogenesis mechanism in the future.