| Avian Influenza Virus(AIV) are of Orthomyxoviridae family,which, can be classified into varioussubtypes on the basis of antigenic differences between the two surface glycoproteins: hemagglutinin andneuraminidase.Serologically, 16 subtypes of HA and 9 subtypes of NA have been identified. As aconsequence, this virus had emerged inflicting major economic damage to the poultry industry. Moreover,cumulative number of confirmed human cases of avian influenza reported to WHO on 1 March 2007indicated that 277 human cases of avian influenza A (H5N1) infection had been reported, 167 of themwere fatal during 2003-2007.The studies chose seriously widespread AIV as object. The expression differences of severalsubtypes AIVs in vivo and in vitro were studied. Otherwise, we established two rapid, specific andsensitive assays using primers and special TaqMan probes corresponding to the M and H5 genes to detectAIVs and identify H5 subtype in this study, These methods were useful and suitable for large-scalescreening at times of avian influenza outbreaks.Cloning and sequencing the target genes:This study cloned and sequenced the whole genes of fourstrains different subtypes AIVs,those were A/Turkey/England/N28/73 (H5N2),A/Turkey/Wisconsin/1/66(H9N2) ,A/African Startling/983/79 (H7N1) ,A/Goose/Guangdong/1/96 (H5N1) seperately.A part ofthe results were submitted to Genebank which number was Y500365, AY724252, AY724253, AY724254,AY724255, AY724256,AY724262 respectively.The growth characteristics of different subtypes AIVs on MDCK :Four subtypes AIVs infectedMDCK in order to study the growth characteristics of different subtypes AIVs on MDCK byhemagglutination activity ,virus pathological role to MDCK and indirect immunofluorescence test. Then,the 0.1ml cell cultures of 72 hour on MDCK of four different subtypes AIVs injected 9～11 days chickenembryos.The allantoic fluid of 48～72 hour was done agglutination test by chicken erythrocytes. Theresults demonstrated that the four subtypes AIVs had pathogenicity to MDCK; hemagglutination activityof cell cultures on MDCK cells was very low. After the viruses infected chicken embryos,hemagglutination activity rised.The expression differences of different subtypes AIVs in vivo and in vitro detected by SybrGreen I real-time RT-PCR technique: Using Sybr Green I real-time RT-PCR method, the expressionlevels of different pathogenesis H5 subtypes AIVs and low pathogenesis H7 and H9 subtypes AIVs onmammalian cell in vitro and organs in vivo were detected, The results showed that the genes of H5N1strain expressed lowly at 6h,12h,24h after it infected MDCK while highly expressed at 48h and 72h.H5N2,H7N1,H9N2 strains had low expression at 6h,12h,24h,48h ,but a little higher at 72h. The experiment in vivo discovered that the H5N1 AIV widespread all the organs of SPF chickens,but theamount of expression was highest in lung and brain.The H7N1 AIV could be detected in lung,brain andpancreas,the expressions of HA, M,NS,PA in lung and brain were higher than in other organs, NA,NP, PB1and PB2 genes had the most expressions in pancreas.Rapid detection of AIVs using Taqman real-time RT-PCR technique: Four strains differentsubtypes AIVs infected MDCK cells, total RNA were extracted from supernatant fluids infected withthem and allantoic fluids from 20 strains different subtypes AIVs, real-time RT-PCR method of M genewas developed. The method could detected the AIVs in 10-5 ailantoic, which was 10～100 more sensitivethan conventional RT-PCR ,the specificity test did not detect Newcastle Disease,Infectious Bursal DiseaseVirus,Infectious Bronchitis Virus, Avian Viral Arthritis Virus, the assay was also highly reproducible.After the method was established, 350 clinical samples were detected, at the same time, the results ofreal-time RT-PCR were compared with those of virus titration. The positive ratio was 41.04% and 39.42%respectively; The real-time RT-PCR results of 264 swabs of SPF chicken experimentally infected AIVswere compared with those of RT-PCR and virus titration. The positive ratio of 3,5,7 day samples was78.52%, 81.30%, 77.50%,while the positive ratio of RT-PCR was 80.41%, 78.60%, 78.66%, the positiveratio of virus titration was 78.57%, 80.00%, 77.06%0 The results showed that the assay was sensitive,specific and rapid enough to detect AIVs in clinical and experimental samples.Rapid detection of H5 subtype AIVs using Taqman real-time RT-PCR technique: MDCK cellscultures of two strains different pathogenicity H5 subtypes AIVs and allantoic fluids from 10 strains H5subtype AIVs were used to develope the real-time RT-PCR to detect H5 subtype AIV. The results showedthat taqman real-time RT-PCR method we established could detect the AIV in 10-4 allantoic, which was10～100 more sensitive than conventional RT-PCR, the specificity test did not detect Newcastle Disease,Infectious Bursal Disease Virus,Infectious Bronchitis Virus, Avian Viral Arthritis Virus and H7,H9subtypes AIVs,reproducibility appeared very good. After the method was established, 340 clinical sampleswere detected, moreover, the results of real-time RT-PCR were compared with those of RT-PCR and virustitration. The positive ratio was 20.31%, 23.69%, 20.88% respectively: At the same time, the detectionwas done on BALB/c mice tissues infected by 10 strains different H5 subtype AIVs, Correlation withvirus titration was 89%; These results showed that the assay was sensitive, specific and rapid enough todetect H5 subtype AIVs in clinical and experimental samples. |
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