Establishment Of Real-time RT-PCR?Digital PCR For H5 Subtype Avian Influenza Virus And Recombinant Expression MAb Against HA Protein

H5 subtype avian influenza viruses(AIVs)can be classified into two groups inlcuding highly pathogenic and low pathogenic viruses.Highly pathogenitic viruses can infect wild birds and cause continuous disease outbreaks in chicken farms.In recent years,genetic replacement of N1 gene by N2,N3,N5,N6,N8,or N9 gene occured in the H5 AIVs further diversified their genetical and antigenical charateristics.The highly pathogenic H5N6 AIVs emerged in poultry in Asia,especially in Southeast Asia,in 2013.Since then these viruses gradually replaced the H5N1 viruses and became the prodominant strains.More imporotant,sporadic infections and death of human cases casued by H5N6 influenza viruses circulating in poultry highlight their potential threats to public health.Therefore,it is essential to establish a fast and accurate detection method for constantly mutated H5 subtype AIVs.Firstly,we carried out studyies on H5N6 influenza virus duplex TaqMan Real-time RT-PCR.The primers and probes including three groups of HA and four groups of NA for H5N6 influenza virus were evaluated.Then a set of ideal primer and probes were selected to further optimize the reaction system and established a double RT-PCR assay.The specificity of the method was determined by using H1~H16 subtype AIV.The results showed that fluorescence signals were obtained for H5 virus in FAM channel and N6 virus in VIC channel,and no fluorescent signal was observed in the other subtype AIVs.The detection limit of the assay was 69 copies for H5 and 83 copies for N6 gene.And,the variability tests of intra-and inter-assay showed excellent reproducibility.Moreover,this assay showed 100%agreement with virus isolation method in detecting samples from poultry.The duplex RT-PCR assay presented here has high specificity,sensitivity and reproducibility,and can be used for laboratory surveillance and rapid diagnosis of the newly emerging H5N6 subtype AIVs.Secondly,we developed a detection method of H5 subtype AIVs digital PCR(d PCR).Based on the HA specific primers and probes of H5 subtype influenza viruses screened previously and combined with the working principle of d PCR,we established a method of d PCR assay.The specificity of this method was determined by using H1~H16 subtype AIVs.The results showed that fluorescence signals were obtained for H5 virus in FAM channel,and no fluorescent signal was observed in the other subtype AIVs Compared with q RT-PCR,d PCR could detect limited 7.19 copies.The result showed the sensitivity of dPCR was ten times higher than qRT-PCR.H5 subtype influenza virus intra-and inter-coefficients of variation(CV)between 2.08%-12.41%.The result indicated that the detection method of H5 virus had good reproducibility.Meantime,the field samples detected 24.2%,which showed 100% agreement with qRT-PCR.In conclusion,this assay can be used for quantification of H5 subtype influenza virus nuclear detection and nuclear standard materials,and provided a new method for the absolute quantification of influenza viruses.Finally,we carried out monoclonal antibodies(Mabs)preparation,sequencing and recombinant expression of H5 subtype influenza virus HA protein.In this study,we screened 35 strains of Mabs of H5N1 AIVs,in which,the heavy chains of 21 strains belonged to IgG,thoes of 14 strains belonged toIgM,all of their light chains belonged to ? chain.The results of competitive ELISA showed that 20B3,21D2 and 22G3 had good competitive inhibition effects.Therefore,these three Mabs were further sequenced.The results of IFA showed that these three antibodies were successfully expressed in vitro,and the HI test showed that two Mabs including 20B3 and 22G3 had HI activities.Above these results will lay a foundation for the development of the subsequent research on monoclonal antibody based diagnostic methods.