虎源犬瘟热病毒的分离鉴定及H基因的序列分析
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摘要
对分离自国内虎源的犬瘟热病毒进行细胞培养及PCR鉴定,并对H蛋白进行序列及遗传演化分析。结果显示:本分离株经传代培养后可以引起Vero细胞脱落和死亡,且扩增出580 bp的特异性目的片段。H蛋白作为犬瘟热病毒表面膜蛋白,在病毒感染过程中担负着重要的作用,H蛋白识别并吸附于细胞表面受体,决定病毒细胞嗜性,进而决定病毒的宿主特异性。对本研究中H蛋白进行分析发现:全长共1 824个碱基,共计编码608个氨基酸,H蛋白具有9个N-糖基化位点,具有549Y的特点。在Genbank中进行Blast分析表明,分离株的H序列与登录号为AF178038的小熊猫源、登录号为KM386683的东北虎源犬瘟热分离株的核苷酸序列相似性最高,均为99%。下载犬瘟热相关序列进行遗传演化、氨基酸序列比对分析,发现本分离株的H基因与标准强毒株A75/17的氨基酸同源性为96.2%,与疫苗Onderstepoort株的氨基酸同源性为89.8%。遗传演化分析显示本分离株基因型属于Asia-1型,与我国流行的野毒株相似。
We cultured the canine distemper virus isolated from siberian tiger in China and identified by PCR,and analyzed the sequence and genetic evolution of H protein. The results showed that the subcultured isolate could cause Vero cells to shed and die,and the specific fragment of 580 bp sequence could be amplified. H protein,as the surface membrane protein of canine distemper virus,plays important role in the process of virus infection. H protein recognizes and is bound to the receptor on the cell surface. It determines the preferendum of virus to cell,therefore determines the host specificity of the virus. The analysis of H protein gene showed that the total length was1 824 bases,encoding 608 amino acids,and H protein had 9 N-glycosylation sites,which had thecharacteristics of 549 Y. Blast analysis in GenbankTMshowed that the nucleotide sequence of the isolate H protein was most similar between isolates from red panda( accession number AF178038)and isolates from siberian tiger( accession number KM386683). The similarities of those sequences were both 99%. Analysis of genetic evolution and amino acid sequence with the related sequences of canine distemper virus showed that the amino acid homology of the H gene from this isolate was 96. 2% with that of the standard strain A75/17,and was 89. 8% with the Strain Onderstepoort. Genetic evolution analysis showed that the isolate belonged to Type Asia-1,similar to those of wild strains in China.
We cultured the canine distemper virus isolated from siberian tiger in China and identified by PCR,and analyzed the sequence and genetic evolution of H protein. The results showed that the subcultured isolate could cause Vero cells to shed and die,and the specific fragment of 580 bp sequence could be amplified. H protein,as the surface membrane protein of canine distemper virus,plays important role in the process of virus infection. H protein recognizes and is bound to the receptor on the cell surface. It determines the preferendum of virus to cell,therefore determines the host specificity of the virus. The analysis of H protein gene showed that the total length was1 824 bases,encoding 608 amino acids,and H protein had 9 N-glycosylation sites,which had thecharacteristics of 549 Y. Blast analysis in GenbankTMshowed that the nucleotide sequence of the isolate H protein was most similar between isolates from red panda( accession number AF178038)and isolates from siberian tiger( accession number KM386683). The similarities of those sequences were both 99%. Analysis of genetic evolution and amino acid sequence with the related sequences of canine distemper virus showed that the amino acid homology of the H gene from this isolate was 96. 2% with that of the standard strain A75/17,and was 89. 8% with the Strain Onderstepoort. Genetic evolution analysis showed that the isolate belonged to Type Asia-1,similar to those of wild strains in China.
引文
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[2]Seimon T A,Miquelle D G,Chang T Y,et al.Canine distemper virus:an emerging disease in wild endangered Amur tigers(Panthera tigris altaica)[J].m Bio,2013,4(4):e00410-e00413.
[3]Guo Ling,Yang Shaolin,Wang Chengdong,et al.Phylogenetic analysis of the haemagglutinin gene of canine distemper virus strains detected from giant panda and raccoon dogs in China[J].Virology Journal,2013,10(1):109.
[4]Kapil S,Allison R W,Johnston L,et al.Canine distemper virus strains circulating among North American dogs[J].Clinical and Vaccine Immunology,2008,15(4):707-712.
[5]Origgi F C,Plattet P,Sattler U,et al.Emergence of canine distemper virus strains with modified molecular signature and enhanced neuronal tropism leading to high mortality in wild carnivores[J].Veterinary Pathology,2012,49(6):913-929.
[6]Maes R K,Wise A G,Fitzgerald S D,et al.A canine distemper outbreak in Alaska:diagnosis and strain characterization using sequence analysis[J].Journal of Veterinary Diagnostic Investigation,2003,15(3):213-220.
[7]Terio K A,Craft M E.Canine distemper virus(CDV)in another big cat:should CDV be renamed carnivore distemper virus?[J].m Bio,2013,4(5):e00702-e00713.
[8]Mochizuki M,Hashimoto M,Hagiwara S,et al.Genotypes of canine distemper virus determined by analysis of the hemagglutinin genes of recent isolates from dogs in Japan[J].Journal of Clinical Microbiology,1999,37(9):2936-2942.
[9]Stanton J B,Givens L,Evermann J F,et al.Immunohistochemical analysis of two strains of lion(Panthera leo)-adapted canine distemper virus in ferrets(Mustela putorius furo)[J].Veterinary Pathology,2003,40(4):464-467.
[10]张晓明,单芬,陈武,等.小熊猫犬瘟热病毒的分离鉴定及分子特点[J].畜牧与兽医,2013,45(10):60-63.