大连和丹东地区甲型肝炎病毒流行株基因型分析

Genotype Analysis of Epidemic Strains of Hepatitis A Virus in Dalian and Dandong

本研究共采集大连和丹东地区2016—2020年急性甲肝患者血清标本129份,提取甲型肝炎病毒(Hepatitis A virus,HAV)的RNA后,以VP1和VP3区序列作为特异性基因片段进行巢氏PCR扩增反应,将阳性PCR产物进行基因测序,并与Genbank中下载的26条参考序列比对构建系统进化树,分析不同编码区样本序列核苷酸和氨基酸的一致性,并确定基因型。实验结果显示,30岁以上HAV阳性患者占比86.7%(104/120),男性患者(63.3%)多于女性患者(36.7%)。从血清标本中成功获得72条VP1区序列及84条VP3区序列,序列均与日本HAV序列的核苷酸序列一致性最高(VP1区:97.45%~99.79%;VP3区:97.27%~99.76%),系统进化树显示甲型肝炎病毒流行株均属于基因ⅠA亚型;序列在VP1区和VP3区氨基酸一致性分别为99.36%~100%和99.29%~100%,均无氨基酸变化。结果表明,大连和丹东地区2016—2020年HAV流行株基因型均为ⅠA型,与日本HAV流行株属于同一基因型。本研究为预防控制大连和丹东地区的甲型肝炎流行提供了分子流行病学依据,并为辽宁地区HAV溯源研究奠定基础。

This study collected a total of 129 serum samples from acute hepatitis A patients in Dalian and Dandong from 2016 to 2020.After the RNA of hepatitis A virus(HAV)being extracted,the VP1 and VP3 region sequences were used as specific gene targets for Nest PCR amplification reaction.The positive PCR products were sequenced and compared with 26 reference sequences downloaded from Genbank to construct a phylogenetic tree.The homology of nucleotide and amino acid sequences in samples with different coding regions was analyzed for determining the genotype.The experimental results showed that 86.7%(104/120)of HAV-positive patients were over 30 years old,and the proportion of male patients(63.3%)were higher than that of female patients(36.7%).72 VP1 region sequences and 84 VP3 region sequences were successfully obtained from serum samples,all of which had the highest nucleotide identity with the Japanese HAV sequence(VP1 region:97.45%-99.79%;VP3 region:97.27%-99.76%).The phylogenetic tree showed that the prevalent strains of hepatitis A virus all belonged to gene subtype IA.The obtained sequences have amino acid identity of 99.36%to 100%and 99.29%to 100%in the VP1 and VP3 regions,respectively,with no amino acid changes.The research results indicated that the HAV prevalent strains in Dalian and Dandong from 2016 to 2020 were genotype IA,which belonged to the same genotype as the prevalent strains of HAV in Japan.This study provides molecular epidemiological evidence for the prevention and control of the spread of hepatitis A in Dalian and Dandong,and lays the foundation for the tracing research of HAV in the region.