厦门市11株柯萨奇病毒A4型分离株全基因组序列分析

Whole genome analysis of 11 Coxsackievirus A4 strains isolated in Xiamen

目的通过分析11株CVA4病毒毒株的全基因序列,揭示其基因进化及变异特征。方法将PCR检测阳性且Ct值<25的样本进行病毒分离培养,获得的毒株提取核酸(RNA)后采用高通量宏基因组测序方法进行序列测定,将质控合格的测序数据进行基因组组装;获得的基因组序列提交至肠道病毒分型网站获得毒株血清型,同时与公共数据库下载的其他国家或地区的CVA4毒株序列通过MegAlign、Mega X、Simplot3.5.1软件分别进行相似性分析、系统进化树分析和重组分析。结果11株毒株全基因组序列长度在7434~7419 bp之间,血清型均为CVA4,核苷酸与氨基酸序列相似性分别为91.7%~99.3%和98%~100%;与原型株序列相比,核苷酸序列何氨基酸序列相似性分别在84.3%~84.7%和97.1%~97.3%之间;与其他参考序列相比,核苷酸与氨基酸序列相似性分别为80.8%~98.8%和96.9%~99.6%;全基因组遗传进化分析表明,其与中国其他地区CVA4毒株序列属同一遗传分枝;根据VP1基因进化树分析,本研究的毒株与中国其他地区毒株属于C基因型中的C2亚型;重组分析结果显示,11株CVA4与其他毒株无明显重组特征,但其中1株的部分区段序列与CVA2毒株序列一致性可达97.2%,剩余毒株与既往重组株相似度可高达98.6%。结论11株CVA4毒株为中国主要流行基因型,虽然与其他毒株无重组现象,但是与其他型别及以往重组株相似性较高,故在工作中需加强毒株重组监测,为手足口病的预防控制工作提供数据支撑。

Objective To analyze the whole genome of 11 strains of CVA4 to reveal its gene evolution and mutation characteristics.Methods The samples with PCR positive results and Ct value<25 were cultured.Viral RNA was extracted from the isolates,then the qualified sequencing data were assembled for genome assembly after sequences were acquired by high-throughput metagenomic sequencing.The obtained genome sequences were submitted to the enterovirus typing website to determine strains'serotypes.Meanwhile,the 11 CVA4 strains sequences were compared with other whole genomes downloaded from the public database by MegAlign,Mega X and Simplot 3.5.1 software to obtain results of similarity analysis,phylogenetic tree and recombination analysis.Results The whole genome sizes of the 11 strains identified as CVA4 ranged from 7434 bp to 7419 bp,and the nucleotide and amino acid sequence similarities were 91.7%-99.3%and 98%-100%,respectively;Compared with prototype strain sequence,the consistency of nucleotide and amino acid sequences was 84.3%-84.7%and 97.1%-97.3%;Compared with other reference sequences,the nucleotide and amino acid sequence similarities were 80.8%-98.8%and 96.9%-99.6%;11 CVA4 stains and other strains from China were clustered into the same lineage based on Genome-wide genetic evolution analysis;According to VP1 gene phylogenetic tree,the strains in our study and strains from other areas of China belong to C2 subtype of C genotype;The results of recombination analysis showed that 11 CVA4 strains had no apparent recombination characteristics with other strains.However,part gene region of one strain was 97.2%consistent with the sequence of CVA2 strain,and the similarity of the remaining strains with the previous recombinant strain was 98.6%.Conclusion There were not prominent differences between 11 CVA4 strains and epidemic genotypes in China.Although there was no recombination with other strains,they were highly similar to the previous recombination strain.Therefore,it is necessary to strengthen the monitoring of strain recombination in routine work to provide data support for the prevention and control of HFMD.