A Novel Multiplex RT-PCR Assay For The Detection Of Influenza A Virus And Differentiation Of Four Hemagglutinin Subtypes And Pathogenic Characterization Of Two H5N1Isolates

The seasonal influenza to human is caused by H1and H3influenza, and they often co-circulate together. Outbreaks of avian influenza viruses H5N1and H9N2on different district have shown that this two subtypes can jump the species barrier from poultry to human directly, causing high mortality. Thorefore, to develop a multiplex RT-PCR method for detecting H1,H3,H5,H9subtype in one tube is significant for the early diagnosis, preservation and treatment of influenza. This thesis focused on development of the H1,H3,H5,H9subtyping and detecting assays, and also studied the biological characteristics of two H5N1influenza virus isolates. This thesis obtain the following results.First, the four subtype viruses were propagated using specefic pathogen-free (SPF) embryonating chicken eggs, and virus titers in the allantoic fluid were determined by the hemagglutination assay,and the EID50of every subtype were deteted.Second, the PCR primers were designed based on the analysis of H1, H3, H5and H9subtype HA and M gene sequences from NCBI (National Center for Biotechnology Information) database by the CLUSTALX software. By optimizing the experiment condition, we set up the multiplex RT-PCR assay for the detection of influenza A virus and differentiation of the H1,H3,H5,H9subtypes in one reaction. The detection product of influenza A Virus is239bp, the H1, H3, H5, H9subtypes is612bp,189bp,338bp and289bp.Third, the HA1,HA3,HA5,HA9fragments were amplified by RT-PCR by using RNAs of the H1N1,H3N2,H5N1,H9N2viruses as templates, and then cloned into pMD18-T vector separately. The recombinant plasmids were confirmed by enzyme digestion and DNA sequencing. The recombinant plasmids were called pMD18-HAl, pMD18-HA3, pMD18-HA5and pMD18-HA9. The recombinant plasmids were used for analyzing sensitibility of the multi-RT-PCR assays.Forth, The sensitivity and specificity of the multi-RT-PCR assay are analyzed,The sensitivity analysis result shows that the detection limit of the viral RNA of H1,H3,H5subtypes is lng,H9subtype is O.lng. The result of this assay is compared with the result of the Viral isolation and identification and the chicken embryo inoculation. The virus positive samples from multiplex RT-PCR assasy were confirmed by virus isolation and gene sequencing. The results of multiplex RT-PCR assay were good consistent with virus isolation and gene sequencing, which show that this assay has good specificity and sensitivity. Fifth, from H5N1positive avian tissues by the muliplex RT-PCR method, two H5N1isolates were obtained and confirmed, A/Quail/Nanchang/02/2013(H5N1) and A/Duck/Nanchang/01/2013(H5N1). By limiting dilution assay, the two H5N1isolates were further purified. The HA and NA genes of the two H5N1isolates were amplified, cloned and sequenced. The phylogenetic relationships were analysed. The relationship between this two strains with the other strains have been reported was determinated.Sixth, both growth characteristic in cell and pathogenicity of A/Quail/Nanchang/02/2013(H5N1) and A/Duck/Nanchang/01/2013(H5N1) in mice were investigated. Our data revealed the different pathogenicity of the two H5N1isolates in mice.The above results could be contribute to the development of rapid detection kit for the detecting and subtyping of the H1, H3, H5, H9subtypes and the understanding of the cross species transmission of the H5N1influenza virus, it has important significance for the level of the control of human avian influenza.