Identification And Characteristcs Of Avian Influenza Virus Isolated From Tibet

Avian Influenza(AI)is one of fatal infectious disease caused by influenza A virus in avian,it is hard to control AIVs as the virus continue changing.The gene reassortment process is an important evolutionary mechanism of influenza virus.The three influenza virus pandemics of the 20th century and the H1N1 pandemic in 2009 as well as the recently H7N9 virus outbreak are resulted from reassortment events of different virus subtypes.Tibet located on the central Asia-India migratory routes.Meanwhile,Tibet is on the Qinghai-Tibet Plateau,containing the unique ecological environment,numerous lakes and plenty of birds,and is an important gathering place for wild and migratory birds.The unique Tibetan high altitude and barometric pressure are beneficial for reassortment of influenza virus.In addition,the poultry breeding of the Tibetan is mainly in the backyard and closed to the wild bird habitat,offering the two-way transmission of influenza virus between poultry and wild birds as well as increasing the opportunity of the reassortant of influenza virus.In order to investigate the genetic evolution of the influenza virus in Tibet,the epidemiology of influenza virus between 2010 and 2012 was carried out.Five H5 subtype influenza viruses were isolated from sick chickens,and the whole genome and the biological properties of the isolates were performed.The study was divided into the following sections:1.The epidemiology and phylogenetic analysis of the Tibet influenza virusThe epidemiology of influenza virus in Tibet was carried out.One H5N2 subt ype avian influenza virus(AIV)and four H5N1 AIVs were isolated from Linzhi and Shannan during years 2010 to 2012,they were named as A/chicken/Tibet/LZO 1/2010(H5N2),A/chicken/Tibet/LZ02/2010(H5N1),A/chicken/Tibet/LZ03/2010(H5N 1),A/chicken/Tibet/SN03/2011(H5N1)and A/chicken/Tibet/LZ04/2012(H5N1),respe ctively.The viral genome phylogenetic analysis revealed that all five isolates were products of reassortment between H5N1 and H9N2 subtype influenza viruses.The PB1,PA,NP and NA genes of LZ01(H5N2)were originated from H9N2 virus,and the other four genes from H5N1 influenza virus.The PB1,PA and NP gene s of LZ02(H5N1)were originated from H9N2 AIV and the other five genes wer e from H5N1 AIV.The PB1 gene of LZ03(H5N1)and SN03(H5N1)was origin ated from H9N2 AIV and the other seven segments from H5N1 AIV.The M gen e of LZ04(H5N1)was originated from H9N2 virus and the other seven segments from H5N1 AIV.Phylogenetic analysis also revealed that the LZ01,LZ02,LZ03,SN03 and LZ04 were located in different evolutional branch,indicating that these two genotypes were produced by different reassortant events of different viruses.2.The pathogenicity analysis of the AIV isolated in TibetThe further biological properties of the LZ01(H5N2)and LZ02(H5N1)were carried out.The results indicated that both isolates were highly pathogenic to chick embryos and men death time(MDT)of the embryo infected by the lowest lethal dose of the viruses was 42h.And the two isolates could form plaques in the passaged chicken embryo fibroblasts DF1 cells which were similar to the highly pathogenic AIV.Although the HA protein of the two isolates contained the multiple basic amino acids in the cleavage sites,belonging to the molecular property of highly pathogenic AIV,the two isolates showed distinct pathogenicity in animal model.The results indicated that the IVPI and ICPI values of LZ02(H5N1)was 3 and 1.7,respectively,and the MLD50 value was lower than 103 TCID50,which is high pathogenic to chicken and mouse.The IVPI and ICPI values of LZ01(H5N2)were 0.68 and 0.27,respectively,and the MLD50 value was higher than 105 TCID50,which is low pathogenic to chicken and mouse.3.The antigenic and vaccine protection analysis of the Tibet isolatesWe further analyze the antigenic properties of the AIVs isolated in Tibet.The cross HI assays revealed that,the hyperimmune serum raised by LZ01(clade 2.3.4)showed highly HI efficiency to LZ02,LZ03 and SN03,but basically showed no HI effect to LZ04,Re-5 and other AIV belonging to clade 2.3.4 and clade 2.3.2.The hyperimmune serum raised by LZ04(clade 2.3.2.1)showed no HI effect to LZ01,LZ02,LZ03 and SN03.the antigenic variation index R value also showed thatthe Tibet isolates and Re-5 virus contained different antigenic property.We further evaluated the protective efficiency of the commercial Re-5 vaccine to LZ01 and LZ02.The results indicated the Re-5 vaccine could protect the chickens infected by LZ04 and 80%of the chickens infected by LZ02.The deduced amino acids displayed that the HA protein of LZ01,LZ02,LZ03 and SNO 3 were identical.However,when compared with Re-5,plenty of amino acids of th e four isolates located in the A,B and C antigenic epitopes were mutated.The m utated amino acids on LZ04 were less than that of LZ02 that may be the reason that Re-5 provides higher protection to LZ04 isolate.