小熊猫源犬瘟热病毒株H、F基因的克隆及序列分析

Cloning and Sequence Analysis of H and F Genes of Canine Distemper Virus Strain from Red Panda

为了解1株圈养小熊猫源犬瘟热病毒(CDV)GD-1的遗传变异情况,通过RT-PCR方法对该株CDV进行H、F基因的克隆、测序及序列分析。结果显示:该分离株的H基因序列与GenBank中丹麦报道的登录号为GU266280的犬源CDV毒株的核苷酸序列相似性最高,为96%;F基因序列与巴西报道的登录号为KY057355的犬源CDV的核苷酸序列相似性最高,为95.7%。下载CDV代表毒株序列进行遗传演化、氨基酸序列比对及分子特征分析。结果显示:H蛋白共有8个潜在的N-糖基化位点,分别位于19、149、309、391、422、456、587、603位点;H蛋白的SLAM受体结合位点氨基酸序列与欧亚野生型毒株一致,与疫苗株相比,530、549位氨基酸不同,与其他CDV参考毒株H蛋白相比还存在24、41等9处氨基酸位点发生明显变异,与标准强毒株A75/17的氨基酸相似性为95.2%,与Onderstepoort、Convac等5株疫苗株的氨基酸序列相似性为88.2%~89.3%;F蛋白共有6个N-糖基化位点,分别位于62、108、141、173、179、517位,与Onderstepoort等疫苗株氨基酸相似性为89.1%~89.7%;与其他参考毒株相比还存在115、130等11处氨基酸发生变异;构建基于H、F基因的遗传进化树,结果显示:该毒株位于Asia-4型的一个小的进化分支,这与目前我国流行毒株主要位于Asia-1型存在明显不同。本研究首次报道了小熊猫源的Asia-4基因型CDV野毒株,并对毒株的H及F基因进行了序列分析,对于了解我国CDV流行株的遗传变异情况、流行病学调查、疾病防控及疫苗研发等具有重要意义。

To understand the genetic variation of a strain(GD-1)of canine distemper virus(CDV)from captive red panda,the H and F genes of the CDV were cloned by RT-PCR,then were sequenced and analyzed.The results showed that the H gene sequence of this isolate had the highest nucleotide similarity(96%)with the CDV strain(accession number:GU266280)reported from Denmark in GenBank,and the F gene sequence had the highest nucleotide similarity with the CDV strain(accession number:KY057355)reported from Brazil.The sequences of CDV representative strains were downloaded for genetic evolution,amino acid sequence alignment and molecular characterization analysis.The results showed that there were 8 potential N-glycation sites in H protein,which were located at sites 19,149,309,391,422,456,587 and 603,respectively.The amino acid sequence of the SLAM receptor binding site of H protein was the same as that of Eurasian wild type virulent strain.Compared with the vaccine strains,the amino acid sequences at positions 530,549 were different.Compared with other CDV reference strains,there were significant variations in H protein at 24 and 41 sites.The amino acid homology between H protein and standard virulent strain A75/17 was 95.2%.The amino acid sequence similarity with five vaccine strains such as Onderstepoort and Convac was 88.2%-89.3%.There were six N-glycosylation sites in F protein,which were located at 62,108,141,173,179 and 517 sites,respectively.The amino acid similarity between F protein and other vaccine strains ranged from 89.1%to 89.7%.Compared with other reference strains,there were eleven amino acid mutations in 115,130 and so on.The genetic evolution tree based on H and F genes was constructed.The results showed that the strain was located in a small evolutionary branch of Asia-4 type,which was different from the current epidemic strains mainly located in Asia-1 type in China.In this study,the Asia-4 genotype CDV wild strain from red panda was reported for the first time,and the H and F genes of the strain were sequenced,which is of great significance for the study of genetic variation,epidemiological investigation,disease prevention and control and vaccine development of CDV epidemic strains in China.