| High pathogenic Avian Influenza virus,belonged to type A Influenza virus,is mainly transmitted between poultry.In recent years,a lot of cases have been reported that human died due to the infection of high pathogenic avian influenza virus,as well as swine,canine,tiger,leopard and so on.The ability of avian influenza virus to transport between different hosts has drawn peoples' attentions.In this study,a H5N1 avian influenza virus was isolated from tiger and its biological characteristics and source were studied.1.Isolation of a H5N1 avian influenza virus from tigerA Bengal tiger of a zoo died without a harbinger.Being confirmed that the tiger was infected with H5N1 subtype avian influenza virus by real-time PCR,a virus strain,named as A/Tiger/Jiangsu/1/2013,has been identified from lung tissue using SPF chicken embryos.The results of histology showed the focal necrosis in the central veins of liver,the decrease of lymphocytes in the peripheral lymphatic sheath of splenic artery and lymphonoduli splenici,and blockage of lumen in the small bronchus of the lung by epithelial cells,which would cause the breathing problems.Then the presence of H5N1 virus antigen in the lung was confirmed by immunohistochemistry,which provides the evidence for the infection of the Bengal tiger with highly pathogenic avian influenza virus.2.Determination of sequence and pathogenicity of H5N1 avian influenza virus from tiger The strain,A/Tiger/Jiangsu/1/2013,was purified with MDCK cells and sequenced.The nucleotide sequence was aligned with the database of influenza virus in NCBI.The result showed that it had the highest homology with the strain,A/muscow duck/Vietnam/LBM/227/2012(H5N1).The 50%egg infectious dose(EID50)of purified virus was determined and Balb/c mice were inoculated with virus at a series of dilution from 101 to 106 EID50 to detect the virus titers of tissues.The results showed that lung tissue has the highest virus titer.Median lethal dose 50(MLD50)was calculated by Reed-Muench methods,and all the mice inoculated with different dose died at the eighth day after inoculation,implying the high pathogenicity of virus to mice.3 Tracking the virus from tigerIt has been speculated that the tiger would be infected with avian influenza virus through its food--chicken.However,due to the lack of more detailed information of the chicken,it's impossible to find out the source of it.Epidemiological investigation on samples from live-bird markets and poultry farms of 12 cities around Nanjing was carried out by the cooperation teams.1168 samples were collected during one year,including rectal swabs,throat swabs,wash sewage,aquaculture water,excrement,cages and chopping board swabs and so on.All the samples were detected by Real-time PCR in our lab.Then the viruses of the samples with positive results were isolated,purified and sequenced.A total of 25 samples showed positive results by PCR,among which 6 samples were from Changzhou and Zhenjiang respectively.The positive rate were 3.8%and 2.99%individually.None of the samples from Huaian nor Taizhou has been identified avian influenza virus.The average positive rate of the whole samples was 1.8%.The positive rate of chopping board swabs(3.53%)was the highest,followed by swabs from cages(3.18%)and wash sewage(3.09%).Only one positive sample was from excrement(0.62%).A total of 27 viruses were isolated from 25 samples,among which one is H3N2,H5N1 and H6N6 individually,2 belonged to H5N6,3 are H5N8 and the rest are H7N9(10 strains)and H9N2(9 strains).4.Analysis of biological characteristics of H5 subtype avian influenza virusThe phylogenetic trees of eight gene segments in H5 subtype avian influenza virus were constructed.Through the analysis of biological characteristics of amino acids,it has been showed that seven gene segments of A/Tiger/Jiangsu/1/2013(H5N1)were in the same branch with H5N1 subtype avian influenza virus isolated from Zhenjiang in 2014.And the cleavage sites of HA1 and HA2 in these two viruses were same and showed the characteristics of high pathogenic avian influenza virus.What's more,not only in mutation sites of mammalian receptors in HA,but also in glycosylation sites of NA and HA and mutation sites related to antimicrobial resistance or host specific in NA,as well as in the key point mutation sites of M1?M2?NS1?NS2?NP?PB1?PB2?PA,these two viruses were highly consistent.All of the results have confirmed that avian influenza virus subtype H5N1 from tiger and the avian influenza virus subtype H5N1 isolated from Zhenjiang had a highly close evolutional relationship,implying the two viruses were may from the same parents.The phylogenetic trees of H5 subtype still showed PB2 segment of A/Tiger/Jiangsu/1/2013(H5N1)was in the same branch with EV/Nantong/1029/2014(H5N8)and EV/Zhenjiang/4342/2014(H5N8),rather than the virus isolated from Zhenjiang in 2014,implying that the PB2 segment of A/Tiger/Jiangsu/1/2013(H5N1)would from H5N8 subtype avian influenza virus.5.The comparison of capsule internal segments in H7N9 and H9N2 subtype avian influenza virus to H5 subtype.Among the isolated strains in this study,more than one third were H7N9 subtype,followed by H9N2 subtype(33.3%).It's said that the internal segments of H7N9 subtype are all from H9N2 subtype.Because these viruses were identified nearly in the same time with the virus isolated from tiger,the phylogenetic trees of gene segments of H7N9,H9N2 and H5 subtype has been analyzed to find out the evolutional relationships of influenza virus subtype.The phenomenon that the gene segments from different subtype were in the same branch had been showed in the phylogenetic trees of six gene segments,confirming that that the internal segments of H7N9 subtype are from H9N2 subtype.What's more,there is a genetic relationship of PB2 fragment of these three subtypes. |
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