淮南市手足口病病毒分离株VP1基因分子特征分析

Molecular characterization of the VP1 gene of HFM disease virus isolated from the City of Huainan

目的了解淮南市手足口病病毒分离株VP1基因遗传特征及进化关系。方法用人横纹肌肉瘤(rhabdomyosarcoma,RD)细胞对2014-2016年荧光PCR检测为EV71型、CA16型及CA10型阳性标本进行病毒分离,经2代接种出现细胞病变(CPE)后取培养物上清,提取病毒RNA,RT-PCR法扩增不同基因型VP1基因并测序,采用Bioedit5.0.6、DNAstar7.1和Mega 6.06生物软件对测序结果进行比对及进化分析。结果 11株EV71型分离株VP1编码区的核苷酸片段长度为891nt,同源性为93.5%-100.0%,氨基酸同源性为98.3%-100.0%;7株CVA16型分离株VP1编码区的核苷酸片段长度为891nt,同源性为95.1%-97.0%,氨基酸同源性为98.7%-100.0%;3株CVA10型分离株VP1编码区的核苷酸片段长度为894nt,同源性为96.5%-100.0%,氨基酸同源性则为98.7%-100.0%。系统进化分析EV71型淮南株与安徽阜阳、山东、河南、北京、云南分离毒株亲缘性较近,处于进化分支C4a,核苷酸同源性为93.5%-100.0%,氨基酸同源性为98.7%-100.0%;CVA16型淮南株为B1b亚型,与山东株、河南株、深圳株、南京株亲缘关系较近,核苷酸序列同源性为95.1%-99.1%,氨基酸序列同源性98.7%-100.0%;CVA10淮南株处于D分支内,与云南、河南、河北株亲缘关系较近,核苷酸序列同源性为96.5%-100.0%,氨基酸序列同源性98.3%-100.0%。结论淮南市2014-2016年流行的EV71为C4a亚型,CVA16为B1b亚型,CVA10处于D聚类分支,与国内其他地区流行的手足口病毒亚型一致。

Objective To investigate the characteristics of the VP1 gene of hand,foot,and mouth disease virus isolated from Huainan and its evolutionary relationships. Methods Fluorescence PCR was used to isolate EV71,CA16,and CA10 from positive samples from 2014-2016.EV71,CA16,and CA10 were passaged twice in rhabdomyosarcoma cells until a cytopathic effect（CPE）was evident.The culture supernatant was collected,viral RNA was extracted,different genotypes of VP1 were amplified with RT-PCR,and then the products of amplification were sequenced.The biological software Bioedit5.0.6,DNAstar7.1,and Mega 6.06 were used to align sequences,and strains were subjected to evolutionary analysis. Results The nucleotide fragment of the VP1 coding region of 11 EV71 strains was 891 nt in length,the nucleotide similarity was 93.5%-100.0%,and the amino acid similarity was 98.3%-100.0%.The nucleotide fragment of the VP1 coding region of 7 CVA16 strains was 891 nt in length,the nucleotide similarity was 95.1%-97.0%,and the amino acid similarity was 98.7%-100.0%.The nucleotide fragment of the VP1 coding region of 3 CVA10 strains was 894 nt in length,the nucleotide similarity was 96.5%-100.0%,and the amino acid similarity was 98.7%-100.0%.Phylogenetic analysis indicated that EV71 isolates from Huainan were closely related to isolates from Fuyang（Anhui）,Shandong,Henan,Beijing,and Yunnan and were in clade C4 a.The nucleotide similarity was 93.5%-100.0% and the amino acid similarity was 98.7%-100.0%.CVA16 strains from Huainan belonging to subgenotype B1 b were closely related to isolates from Shandong,Henan,Shenzhen,and Nanjing.The nucleotide similarity was 95.1%-99.1 and the amino acid similarity was 98.7%-100.0%.CVA10 strains from Huainan in the D clade were closely related to isolates from Yunnan,Henan,and Hebei.The nucleotide similarity was 96.5%-100.0%and the amino acid similarity was 98.3%-100.0%. Conclusion The prevalent subgenotype of EV71 in Huainan from 2014-2016 was C4 a.The prevalent subgenotype of CVA16was B1b.CVA10is in the D clade.These findings are consistent with the subtypes of HFM virus prevalent in China.