| Avian influenza (AI), also known as true roup, is an infectious disease mainly in birds caused by avian influenza type A virus. In history there have been many times of avian flu outbreak in flocks and humans, not only caused the death of a large number of birds that causing huge economic losses, but also can break the species barrier and infect humans. Over the past decade, highly pathogenic avian influenza virus(HPAIV) and low pathogenic avian influenza virus(LPAIV), such as H5N1, H1N1, H7N9, H10N8, break out in humans alternately, which threaten human health and life enormously. Therefore, the research in influenza virus have great value on economy and public healthy.Wild birds, especially wild waterbirds of the orders Anseriformes (ducks, geese, swans) and Charadriiformes (gulls, terns,shore birds), are the natural hosts of avian influenza virus (AIV), and all the 16 HA subtypes and 9 NA subtypes of AIV can be isolated in wild birds. Wild birds is the most important and abundant storage and gene library of avian influenza virus. AIV and it’s genes may be spread around the world by birds infected by AIV as migrating. Therefore, the molecular epidemiological surveillance on AIV in wild birds, especially the orders Anseriformes (ducks, geese, swans) and Charadriiformes (gulls, terns,shore birds), has important ecological significance on the spread of avian flu and important implications for the early warning of flu outbreak.一、Influenza virus surveillance in wild birds of the orders Charadriiformes in Qin huangdao3153 feces samples of wild birds mainly in the orders Charadriiformes, of which including 12 orders,16 families and 36 species, were collected in Qinhuangdao of China from October 2012 to November 2014. After virus isolation and subtype identification,33 samples were positive for AIV by PCR, as a result, virus isolation rate is 1.05%(33/3153).Virus isolation results show that different virus isolation rates differ in different years, and the overall isolation rate is lower; different virus isolation rates differ in different sampling seasons, and the isolation rate is higher in spring, but virus subtypes are more abundant in autumn and winter; different virus isolation rates differ in different hosts, and the isolation rate is higher in Charadriiformes and Anseriformes; different virus isolation rates differ in different monitoring points, and the isolation rate is higher in Qilihai Wetland Park.Results of subtype identification on 32 AIVs, while one isolate failed to be subtyped, showed that there were isolates subtyped of 1 H2,2 H4,27 H10,1 HI3 and 1 HI6, and there were isolates subtyped of 2 N3,2 N6,27 N7 and 1 N8. Therefore, H10N7 is the dominant subtype of the Qinhuangdao, and it’s isolation rates was 81.82%(27/33).The two-year epidemiological survey of avian influenza in wild birds of the orders Charadriiformes in Qinhuangdao is the first time in surveillance in Qinhuangdao, and the first time in long-term systematic influenza surveillance on wild birds of the orders Charadriiformes in Chinese mainland. Also it is the first isolations of H13 and H16 influenza virus in China.二、Influenza virus surveillance in wild birds of the orders Anseriformes in Xianghai in Jilin1576 samples of wild birds mainly in the orders Anseriformes, of which including 4 orders, 4 families and 23 species, were collected in XiangHai of JinLin province in China from September 2010 to October 2013. After virus isolation and subtype identification,172 samples were positive for AIV by PCR, as a result, virus isolation rate is 10.91%(172/1576).Virus isolation results show that different virus isolation rates differ in different years, and the overall isolation rate is higher; different virus isolation rates differ in different sampling seasons, and the isolation rates is higher in autumn; different virus isolation rates differ in different hosts, the number of isolates and subtypes of virus are most abundant in wild birds of the orders Anseriformes. There is no significant differences in virus isolation rates between Throat swabs and Cloacal swabs.Results of subtype identification on 135 AIVs showed that there were isolates subtyped of 3 H1,3 H2,25 H3,1 H4,68 H5,28 H6,5 H7,1 H9 and 1 H10, and there were isolates subtyped of 13 N1,100 N2,12 N3,1 N7,8 N8 and 1 N9. Thus, H5, H6 and H3 are the dominant HA subtypes of XiangHai in JinLin, while N2 is the dominant NA subtype. There are 60 strains of H5N2 subtype influenza virus, and the number of H5N2 subtype accounted for 44.44%(60/135) of 135 subtyped strains, so H5N2 is the dominant subtype of XiangHai in JinLin.The three-year epidemiological survey of avian influenza in wild birds of the orders Anseriformes in XiangHai is the first time in surveillance in JinLin province.三、Gene cloning and phylogenetic analysis on representative isolatesIn order to understand the genetic evolution and variation of AIV isolates in wild birds of the orders Anseriformes and Charadriiformes in Qinhuangdao and XiangHai of JinLin, and to understand the role of wild birds in influenza epidemic on poultry, domestic animals and human, gene cloning and sequence were carried out on 45 representative strains selected from the 205 isolates. As a result, total 344 genes were sequenced successfully including 40 HA,38 NA,43 PB2,45 PB1,44 PA,44 NP,45 M and 45 NS.The results of viral genes phylogenetic analysis showed that there was a complex and huge influenza virus gene pool combinations of 11 HA and 7 NA in wild birds in Qinhuangdao and XiangHai of JinLin. and there were different evolutionary sources of genes between the different viruses of the same subtypes or of different subtypes. Conclusions are as follows:(1) It is the first time for isolating and successfully sequencing genome of HI3 and HI6 subtypes AI Vs in China.(2) Part of the HA and NA genes and all internal genes of the isolates exist in different genotypes.(3) Extensive gene rearrangements existed in influenza virus from wild birds in Qinhuangdao and XiangHai of JinLin; Compared with the external genes, internal genes rearrangement are More universal, and compared with the HA genes, NA genes rearrangement are More extensive.(4) The results of phylogenetic analysis showed that gene exchange exist in some external genes of AIVs between the North American and Eurasia, illustrating that migratory birds, especially wild birds of the orders Charadriiformes, could do long-distance migration with LPAIV.(5) The results of phylogenetic analysis showed that gene exchange exist in some external genes of AIVs between the North American and Africa, and phylogenetic trees of AIVs should be divided into two braches, one cluster is Eurasia and Africa, the other cluster is North American.(6) The results of homology comparison and phylogenetic analysis showed that the HA genes, from which H10N8 human influenza viruses in JiangXi and H10N7 influenza viruses isolated from Qinhuangdao and XiangHai, may probably have a common evolutionary ancestors. |
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