我国Colti病毒基因分型的研究

Genotyping of the Chinese isolates of coltivirus

目的 对我国分离的Colti病毒进行基因分型。方法 采用针对Colti病毒B2亚组第9和12片段和B1亚组第12片段的3对引物,对我国近年来分离的Colti病毒利用逆转录聚合酶链反应(RT-PCR)方法进行基因分型。同时对北京分离株BJ95-75和云南分离株YN-6的PCR产物进行了克隆测序及同源性分析。结果 Colti病毒北京分离株BJ95-75、云南分离株YN-6、-67-1、-68-1、-69、-70-1、-70-2、-90、-92-2、-93病毒用B2亚组特异引物12-854-S/12-B2-R均能得到相对分子质量为850 bp的特异条带,用针对于第9片段的B2亚组引物9-JKT-S/9-JKT-R均得到了相对分子质量为492 bp的特异条带。而Colti病毒东北分离株NE97-12、NE97-31及对照病毒如环状病毒YN-99、乙脑病毒YN-151-1未能扩出特异条带。所有Colti病毒分离株及病毒对照用B1亚组特异引物12-B1-S/12-B1-R均未见特异条带。BJ95-75、YN-6这2株病毒第12片段核苷酸序列与B2亚组其他毒株间的同源性达到89.4％以上,特别是与中国早期分离株Banna病毒,其同源性达到98.9％以上;2株病毒第9片段的核苷酸序列与属于B2a型的Banna病毒的同源性可达到99.2％和96.5％,和JKT6423病毒的同源性也能达到84.0％,而与属于B2b型的JKT6969和JKT7043相应片段核苷酸序列的同源性只有53.0％左右。结论

Objective To classify the Chinese isolates of Coltiviruses. Methods Three sets of primers were selected, among them two were specific to the 9th and 12th segments of subgroup B2, and one was for the 12th segment of subgroup B1. All the Chinese isolates of Coltivirus selected in the experiment were classified according to the lengths of different amplicons of the reverse transcriptase-polymerase Chain reaction (RT-PCR) . The homogenicity of the nucleic acids of the isolates BJ95-75 and YN-6 was also compared with other Coltivirus strains belonging to subgroup B2. Results With the primers 12-854-S/12-B2-R, which were specific to the 12th segment of Coltivirus subgroup B2. 850 bp amplicons were obtained from Beijing isolate BJ95-7S and all the Yunnan isolates such as YN-6,-67-1 ,-68-1,-69,-70-1,-70-2,-90,-92-2,-93 of Coltivirus. 492 bp DNA fragments were also amplified from all of them with the segment 9th specific primers 9-JKT-S/9-JKT-R. However no positive results were obtained from Northeast isolates NE97-12,NE97-31 and control viruses YN-99 ( Orbivirus) ,YN-151-1(JEV) with the same two sets of primers. With 12-B1-S/12-B1-R primers specific to the 12th segment of subgroup B1, no amplicons of right length were obtained from any of the Chinese isolates of Coltivirus and the control viruses. When compared the nucleic acid sequences of BJ95-75 and YN-6 with other Coltivirus strains such as Bannavirus, JKT6423, JKT6969, JKT7043, the amplicons from segment 12th of these two strains had more than 89.4% homology with the other strains, especially to the earlier Chinese isolate Bannavirus, the homolog was more then 98.9% . Nearly 96.5% and 99.2% of the nucleic acids of the amplicons from segment 9th of the two strains were being homologousto Bannavirus and about 84.0% to JKT6423, which had been classified into type B2a. But the maximal homogenicity was about 53% when compared withthe other two coltivirus strains JKT6969 and JKT7043 which had been classified into type B2b. Conclusion Genotyping the recent Chinese isolates of coltivirus for the first time in our country. Most of the Chinese isolates belong to subgroup B2, more exactly type B2a. The Northeast isolates NE97-12 and NE97-31 were not correctly grouped with the available primers.