Isolation,Identification And Fluorescence Quantitative RT-PCR Of H9N2 Subtype Of Avian Influenza Virus

The H9N2 subtype of avian influenza virus is lowly pathogenic,rarely causes overt diseases in birds including ducks.It is a remarkable fact that the waterfowls,especially the ducks,are the repository of the influenza viruses and they play a critical role in the transmission and evolution of AIV.Currently,H9N2 subtype AIVs have been isolated from numberous duck flocks from many farms.It raised great concerns on its economic importance to poultry industry and potential threat to public health.The study contains three parts as follows:Part I: Isolation and identification of H9N2 subtype AIVs and its whole genome sequences analysisA strain of H9N2 subtype avian influenza was separated from trachea,lung and other tissues of ducks in Jining and nomenated JN1.The pathogenicity of JN1 strain was analyzed and compared by the determination of hemagglutination properties,infected chicken embryo,the average time of death of chicken embryo and a cross-hemagglutinin-inhibition(HI)assay.The results showed that the EID50 was 10-7.54 / 0.2mL,the MDT was 72 h,the IVPI was 0.26.The HI correlation indices(R)exceeded 0.47<0.5,indicating that JN1 strain has been changed in antigenicity.According to the sequences of H9N2 genes from Genebank,11 pairs of primers were designed for amplification of whole genome sequences of 8 strains.Phylogenetic tree analysis of the eight each genes were conducted using MEGA5.1software.Meanwhile,the Eurasian classical strains(BJ94,Y280,F98 and G1)and North America strains(Wisconsin66and Y439)sequences were uesd to compare with this strains at amino acid level.Phylogenetic studies show that HA gene was located on the same branch with DK/HK/Y280/97,NA,M,NP,PA,PB1,PB2 genes had higher homology with SH/F/98,the cleavage site of HA gene was PSRSSR?GL,6 potential glycosylation were found on HA gene,the 234 site amino acid was L.The neuraminidase gene had deletions at 63,64 and 65amino acids.Part 2: Development and application of fluorescence quantitative RT-PCR assay for the rapid detection of H9N2 subtype virusSpecial primers based on H9N2 Avian influenza virus HA gene were designed and a pairof primers of house-keeping gene ?-actin was chosen for control.The producct of PCR was cloned into pMD18-T as standard products,a real-time fluorescence quantitative PCR was performed to establish the standard curve of HA and ?-actin gene.The results showed a precise linear relationship with a correlation coefficient of R>0.99.The detection limits was10 copies of DNA plasmid reaction,it was 100 times more sensitive than RT-PCR.The amplification curve showing a single peak could only been detected for H9N2 Avian influenza virus.The variation coefficient of repeatability tests was less than 1%.The developed real-time PCR assay was highly specific,sensitive,could be an available tool for diagnosis of H9N2 AIV.Part 3: Detection of H9N2 subtype virus in the tissues of artificially infected ducklingsThree weeks old ducklings were infected by intranasal inoculation,each duckling 107.84EID50.After the ducklings infected the virus,cloaca and throat swabs were collected and 3ducklings were killed at random every two days.We used the fluorescence quantitative RT-PCR assay to detect the infected ducklings' viral shedding,and diverse tissues.The results show that: serology test showed a weak HI antibody response was observed in intranasally infected ducks.Viral RNA can be detected in cloaca and throat swabs from the third day to seventeenth day groups.The content of AIV in both cloaca and throat swabs is higher on the third and thirteenth day groups.The throat swabs were higher than cloaca swabs.We used the fluorescence quantitative RT-PCR assay to detect TMUV of the infected ducklings and the results showed that: At two day postinfection(PI),the H9N2 AIV could be detected from pancreas,liver,spleen,bursa of Fabricius and lungs.The AIV reached the period of maximum quantity at seven to eleven days PI.The highest relative expression of the H9N2 subtybe AIV was the trachea,liver,pancreas and glandular stomach,with relative expression ranging from 0.6 to 0.8.The lower relative expression of the H9N2 subtybe AIV was the lung,spleen,kidney and bursa of Fabricius,with relative expression ranging from 0.2 to 0.4.The H9N2 subtype AIV isolated from ducks was low virulent strain,but had certain pathogenicity to ducks.Based on the relative fluorescence quantitative RT-PCR method,detoxification and histovirus loading of H9N2 subtype AIV were measured.Under the laboratory conditions,3-week-old ducklings infected by H9N2 subtype AIV were detoxingduring 2-17 days,tissue containing higher toxic during 7-11 days.