The Etiological Investigation Of Improved Canine H5N2Influenza Virus And Gene Sequence Analysis Of HA, NP, NA

In2009for the first time, a new strain of H5N2subtype influenza virus was isolatedfrom dogs by Zhan Guangjian et al. in our laboratory. Then Song Qianqian et al. proved thatthe subtype of the virus can cause horizontal transmission in dogs through the pathogenicexperiments, which further reminded that the influenza virus may become popular quickly. Toreveal the epidemic of the subtype of virus in the natural dogs as well as its variation innatural conditions, this study collected a large number of samples of the influenza virus to doetiology investigation and did the further analysis in the level of molecular biology with threeimportant genes of HA, NA, NP.This study collected96nasal swabs from sickness dogs to do pathogen separation.We dealed with them in normal processing and inoculated chicken embryos of9-11age. Finally we got three strains of influenza virus. Through a series of experiments such as the HA assay, HI assay, RT-PCR and gene sequence analysis, we finally succeeded to get three strains of H5N2subtype influenza virus. We named them A/Canine/Shan Dong/01/2012(H5N2), A/Canine/Shan dong/02/2012(H5N2), A/Canine/Shan dong/03/2012(H5N2) respectively. And those viruses were named CA/SD/01/12(H5N2), CA/SD/02/12(H5N2), CA/SD/03/12(H5N2) for short.There were many receptor binding sites in HA protein of influenza virus and the aminoacids of receptor binding sites of the HA protein in the three isolated strains and otherreference strains were conservative. Other mutations were not happened in them. In the aspectof amino acid homology, CA/SD/01/12(H5N2),CA/SD/02/12(H5N2), CA/SD/01/12(H5N2)is above90.0%each other. The three isolated strains had the highest amino acid homology(99.4%,99.3%,99.6%) with A/Dog/Shandong/JT01/2009respectively. In terms of proteinlocus, there was one mutation respectively in the three isolated strains comparingA/Dog/Shandong/JT01/2009. The mutations of CA/SD/01/12(H5N2), CA/SD/02/12(H5N2),CA/SD/03/12(H5N2) were87th,91th,169th respectively. And there were some mutations in this three isolated strains. In the two strains of CA/SD/02/12(H5N2) andCA/SD/03/12(H5N2), two mutations were91th and169th. The amino acid sequence at HA0cleavage site in the three strains was PQ↓REIRRK, which was representative of a non-highlypathogenic. There were3glycosylation sites in the isolated segment of the HA protein ofCA/SD/01/12(H5N2). They were169th-NQTD-,310th-NSSM-,478th–NGTY-. There were4glycosylation sites in the isolated segment of the HA proteins of CA/SD/02/12(H5N2).They were169th-NQTD-,310th-NSSM-,460th–NGTY-,478th-NSSY-. And there were3glycosylation sites in the isolated segment of the HA proteins of CA/SD/01/12(H5N2). Theywere169th-NQTD-,310th-NSSM-,460th–NSSY-. The change of sugar chain had veryimportant influence on the strains. That caused the enhancement of the HA sensitivity inprotease easily and leaded to be high pathogenicity.NA gene’ length of influenza virus was1309basic group encoding401amino acids afterPCR amplification of three isolated strains. CA/SD/01/12(H5N2) had high amino acidhomology(99.1%) with CA/SD/02/12(H5N2), CA/SD/01/12(H5N2) had the amino acidhomology(99.4%) with CA/SD/03/12(H5N2), CA/SD/02/12(H5N2) had the amino acidhomology(99.5%) with CA/SD/03/12(H5N2). And the three strains had the amino acidhomology (98.1%,98.3%,98.4%) with A/Dog/Shandong/JT01/2009(H5N2) respectively.Those analyses proved that the NA of isolated three H5N2influenza strains had the recentgenetic relationship with A/Dog/Shandong/JT01/2009(H5N2). The neck of NA gene and theimportant amino acid residues such as118,119,151,152and the7major antigen sites in thehead of NA gene did’t occurred amino acid mutation and loss. But there were two mutationsin other parts of the CA/SD/01/12(H5N2) NA gene comparing the strain ofA/Dog/Shandong/JT01/2009(H5N2). They were62th and340th. And there were threemutations in the isolated strain of CA/SD/02/12(H5N2) and CA/SD/03/12(H5N2). They were62th,233th,340th. Then it can be speculated that the two mutations would produce certaineffect on the replication and host range of the virus.NP gene’ length was1592basic group encoding508amino acids after PCRamplification of three isolated strains. In terms of amino acid homology, In terms ofnucleotide homology, CA/SD/01/12(H5N2),CA/SD/02/12(H5N2), CA/SD/01/12(H5N2)is above90.0%each other. this three isolated strains had high nucleotide homology with A/Dog/Shandong/JT01/2009(H5N2) and A/Swine/Fujian/F1/2001(H5N1)(99.5%). But thethree strains of the NP protein had the highest amino acid homology withA/Swine/Fujian/F1/2001(H5N1)(99.1%,99.5%,99.3%) respectively. Then in terms ofprotein locus, there were four mutations, five mutations, four mutations in the three isolatedstrains of CA/SD/01/12(H5N2), CA/SD/02/12(H5N2), CA/SD/03/12(H5N2)respectivelycomparing the reference strain of A/Dog/Shandong/JT01/2009(H5N2). And there was onemutation in102th in CA/SD/01/12(H5N2)comparing the strain of CA/SD/02/12(H5N2).There were two mutations in153th and154th in CA/SD/01/12(H5N2)comparing the strain ofCA/SD/03/12(H5N2). There were three mutations in102th,153th and154th in CA/SD/02/12(H5N2)comparing the strain of CA/SD/03/12(H5N2).We succeed to separate three strains of influenza viruses after2009once again. Itfurther proves that the new type of H5N2subtype of canine influenza virus in dogs. Becauseof the contact in dogs and beasts and birds, especially the dogs as pets, increasing breeding,they can cause potential threats in the health and life of human. So the study providesepidemiological data of H5N2canine influenza virus prevention. It has important publichealth significance.