Establishment Of Triple Real-time Fluorescence Quantitative RT-PCR Assay For AIV H5,H7 And H9 Subtypes And CELISA Assay For H7 Subtype And Preparation Of Monoclonal Antibody Against Ha Protein Of H5 Subtype

Avian influenza is an acute,highly contagious infectious disease caused by the avian influenza virus（AIV）in the genus of influenza viruses belonging to the Orthomyxoviridae family.AIVs can be classified as multiple subtypes accompanied by frequent antigenic variations,with no cross-protection between different subtypes.Highly pathogenic AIV represented by H5 and H7 subtypes spread quickly and their outbreaks impose huge damage to social economy.As the most widespread low pathogenic AIV is in the world,the H9 subtype continues to provide internal genes for other subtypes of AIV,which seriously endangers the production efficiency of poultry.Through long-term evolution,H5,H7,and H9 subtypes AIV acquire the ability to directly infect people across the interspecies barrier,which seriously threatens human life and health.In order to achieve effective monitoring and prevention of avian influenza,it is necessary to apply fast and accurate diagnostic techniques.Currently,the real-time fluorescent quantitative RT-PCR assay is fast,sensitive,and accurate,rendering it an important tool for AIV identification,subtyping and epidemiological surveillance.And the ELISA assay is easy to operate,highly sensitive,specific and fast with high-throughput,which promotes avian influenza early diagnosis and monitoring of antibody levels after immunization.In this study,a one-step triple real-time fluorescence quantitative RT-PCR detection method was constructed for detection of H5,H7,and H9 subtypes AIV.Besides,the H7 subtype AIV competition ELISA antibody detection assay was established;finally,monoclonal antibodies（Mc Abs）against H5subtype AIV were successfully prepared by using lymphocyte hybridoma technology.The specific research is as follows:1.Development of one-step triple real-time fluorescent quantitative RT-PCR rapid detection assay for H5,H7 and H9 AIVsIn this study,by comparing and analyzing the HA gene sequences of all H5,H7,and H9 subtypes AIV retrieved from Genbank,we designed and synthesized specific probes and primer pairs.After adjusting and optimizing the reaction system and reaction procedures,we established the one-step triple real-time quantitative RT-PCR assay for differential detection of H5,H7 and H9 subtype AIVs within 70 minutes.The minimum detection limits of this assay for H5,H7 and H9 subtype AIV titers were 10³,10²and 10²EID₅₀/100μL,respectively.For H5 subtype,the viral titers were linear in the range of 10⁷-10³EID₅₀/100μL;For H7 and H9 subtypes,the viral titers were within a linear range of quantification between 10⁶-10²EID₅₀/100μL.This assay only showed specific amplification curves for H5,H7,and H9 subtypes AIV without false positive results with other subtypes AIV and a variety of other avian viruses.The variation coefficients of intra-and inter-assay repeatability tests of this method fell less than 4%,showing good repeatability.It was proved that the assay could accurately detect mixed infections of H5,H7,and H9 subtype avian influenza viruses with different virus titers in the co-infection model.After analyzing the efficacy test results of clinical swab samples,the coincidence rate of this assay with virus isolation and identification was extremely high.In summary,this assay owns the advantages of high throughput,rapidity,accuracy and sensitivity,thus providing a new tool for the rapid clinical early differential diagnosis of H5,H7 and H9subtype AIV.2.Development of a c ELISA antibody detection assay for H7 subtype AIVWith H7 inactivated whole virus particles purified by sucrose density gradient centrifugation as the coating antigen,and a purified Mc Ab against HA protein of H7subtype AIV as competitive antibody,a c ELISA antibody detection assay for H7 subtype AIV was established.The optimal reaction conditions were optimized and determined at the beginning.When the antigen coating concentration was 4μg/m L,the concentration of the Mc Ab was 0.625μg/m L,and the dilution ratio of the test serum was 1:5,the positive monospecific serum inhibition rate reached its peak.The ROC curve analysis was used to define the detection thresholds of the method.The cut-off values of the chicken,duck and peacock serum detection inhibition rates were determined to be 30.11%,26.85%and45.66%,respectively.The performance evaluation test results showed that the assay could specifically detect anti-H7 antibodies in serum samples with no cross-reaction with other subtypes of AIV and common avian respiratory virus positive sera;With the hemagglutination inhibition test（HI）as a control,the minimum detection limits for chicken,duck and peacock positive serum reached 2⁰,2¹and 2^-1HI titers respectively;a total of 400clinical serum samples for chicken,duck and peacock were tested in parallel by using the established assay and HI test.Compared to the HI test,the compliance rates reached 100%,98.6%,and 99.3%,respectively.The above results showed that the H7 antibody rapid detection assay established in this study was highly specific,extremely sensitive,convenient for large-scale detection,and highly correlated with the HI test with practical value for the serological diagnosis of H7 subtype AIV and the monitoring and evaluation of vaccine immunization efficacy.3.Preparation and Identification of Monoclonal Antibody against HA Protein of H5 Subtype AIVIn this study,the purified prokaryotic expressed recombinant protein p Cold I-H5-HA1were used as immunogen to immunize Balb/c female mice,and splenocytes from mice with high serum antibody titers and sp2/0 cells in logarithmic growth phase were used for cell fusion.HAT selection culture was used to screen positive hybridomas.After the cells were subcloned by four rounds of limiting dilution,two monoclonal antibody hybridoma cell lines capable of stably secreting anti-H5-HA1 protein antibodies were obtained,named11H4 and 12G4 in sequence.The Mc Abs 11H4 and 12G4 belonged to Ig G1 subclass and the light chains wereκchains.The antibody titers of the two Mc Ab cellular supernatants tested by the indirect ELISA method reached 1:10,000 and 1:10,respectively.Western blot analysis showed that the two Mc Abs could specifically react with purified H5-HA1recombinant protein;IFA analysis showed that 11H4 and 12G4 could specifically react with MDCK cells infected with H5 subtype AIV virus,but did not cross-react with MDCK cells infected with other subtype AIV.In summary,the above two Mc Abs 11H4 and 12G4 are expected to be useful in the establishment of early diagnostic methods for H5 subtype AIV.