Sequence Analysis Of Avian Influenza Virus Epidemic Strains And Preparation Of A Monoclonal Antibody Against PA Protein

Avian Influenza(AI)caused by avian influenza virus(AIV)is an acute and highly contagious infectious disease.Avian influenza not only seriously harms the poultry industry,but also pose a threat to public health.1.In the present study,5192 samples collected from Anhui,Shandong and Zhejiang provinces and other regions of China from May 2018 to February 2020.They were tested for AIV by RT-PCR.The results showed that the overall positive rate of AIV was 1.25%.Six H9N2 isolates and two H5N6 isolates were identified from RT-PCR AIV-positive samples by chicken embryonated egg inoculation.Genome sequence analysis showed that the HA cleavage site of six H9N2 isolates was PSRSSRGLF,which matched the sequence characteristics of a low pathogenic AIV.The amino acid at position 234 of the HA receptor binding site was Leu,which had the activity to bind to mammalian sialic acid receptors.All six H9N2 isolates had potential glycosylation site at position 313 of HA protein.S3 IN mutation was also found in the M2 protein of these H9N2 isolates.The HA cleavage site of the two H5N6 isolates was LRERRRKRGLF,which matched the sequence characteristics of a highly pathogenic AIV.Q226L and G228S mutations were not found in HA protein of two H5N6 isolates,indicating that their receptor binding characteristics remain of avian origin.Phylogenetic analysis showed that the HA and NA genes of six H9N2 isolates were classified into the branch of Y280-like,PB2 and M genes belonged to the branch of G1-like,and NP,NS,PA and PB1 genes belonged to the branch of F/98-like.The HA genes of the two H5N6 isolates were classified into clade 2.3.4.4 of the H5 subtype AIV,and the NA genes belonged to the branch of the Eurasian Lineage.This study provided reference for the prevention and control of AIV through the sequence analysis of AIV epidemic strains in some regions from the year 2018 to 2020.2.The prokaryotic expression vector of pET-28a(+)-H9N2-sPA encoding PA truncated protein of H9N2 subtype AIV was constructed,and the His tagged sPA protein was expressed and purified.The purified protein His-sPA was used to immunize BALB/c mice,and then spleen cells of the immunized mice were fused with SP2/0 cells.Three hybridoma cell strains(1F5,4B9 and 8G3)secreted mAbs against PA and were successfully propagated.Antibody characteristics showed that the ELISA titer of the three mAbs of 1F5,4B9 and 8G3 were above 105,and the results of WB also showed that the three mAbs of 1F5,4B9 and 8G3 could specifically react with the PA protein of H9N2 subtype AIV.The results of IFA showed that 8G3 could recognize H9N2-infected MDCK cells,while 1F5 and 4B9 had weak reactivity with H9N2-infected MDCK cells.The light chains of the three mAbs were kappa chains.The heavy chains of 4B9 and 8G3 were IgG1,and the heavy chains of 1F5 was IgG2b.The antigen region recognized by 1F5 and 4B9 were located in the C-terminal 505-558aa of the PA protein.The antigen region recognized by 8G3 were located in the C-terminal 607-660aa of the PA protein.The universal identification of the mAbs showed that the 8G3 mAb could specifically react with the PA protein of H1-H13 subtype IAV.The preparation of mAbs against the PA Protein of H9N2 subtype AIV had laid a foundation for further research of the structure and biological function of PA protein,virus replication and transcription.