广东省东莞市2017—2018年登革病毒E基因进化分析

Evolutionary analysis of dengue virus E gene in Dongguan, Guangdong, 2017-2018

目的测定广东省东莞市2017—2018年Ⅰ~Ⅳ型登革病毒(DENV-1~DENV-4)的E基因序列,分析其系统进化情况及流行基因亚型,从分子水平探讨病毒来源。方法收集东莞市2017—2018年140例可疑登革热患者急性期血清,用实时荧光PCR方法检测病毒核酸,阳性血清用C6/36细胞分离培养登革病毒,测定分离株E基因序列,绘制系统进化树,结合流行病学资料进行分子流行病学分析。结果140份急性期血清中DENV核酸阳性54份,阳性率38.6%,DENV-1阳性占53.7%。分离得到17株毒株。序列比对和系统进化分析显示,10株DENV-1分离株间核苷酸同源性为90.0%~99.9%,氨基酸同源性为96.6%~99.8%,分属基因Ⅰ型和基因Ⅴ型。7株基因Ⅰ型分离株与泰国2015年、缅甸2015年、中山2015年及越南2016年流行株核苷酸同源性较高(99.3%~99.9%),其中2018年本地株D2018078与2017年输入株D2017008核苷酸同源性达99.8%;3株基因Ⅴ型分离株与印度2014年、2017年株及新加坡2014年流行株核苷酸同源性较高(97.7%~99.1%)。4株DENV-2分离株间核苷酸同源性为91.0%~98.6%,推导氨基酸同源性为97.6%~99.8%,分属混合型和亚洲Ⅰ型,2株混合型分离株与马来西亚2014年、台湾2015年流行株核苷酸同源性较高(99.6%~99.7%);2株亚洲Ⅰ型分离株分别与越南2015年、泰国2011年流行株核苷酸同源性较高(99.3%~99.5%)。2株DENV-3分离株间核苷酸同源性为98.4%,氨基酸同源性为99.4%,均属基因Ⅰ型,与缅甸2017年、马来西亚2015年流行株核苷酸同源性较高(99.3%~99.7%)。1株DENV-4分离株与越南2013、2016年流行株同源性较高(99.3%~99.5%),属基因Ⅰ型。结论2017—2018年东莞市登革热的病原体主要为DENV-1,其次是DENV-2,各至少有2种基因亚型同时在流行;DENV-3和DENV-4各至少有1种基因亚型在流行。流行方式主要为南亚和东南亚国家及我国中山、台湾等地输入病例引起的本地暴发流行,存在输入引起本地感染的风险。

Objective The E gene sequence of Dongguan 2017-2018 DENV-1-4 was determined.Its phylogenetic evolution and popular genotype were analyzed,and the source of the virus was discussed in the view of the molecular level.Methods A total of 140 samples of serum of suspected dengue patients in the acute phase from 2017 to 2018 in Dongguan were collected.The viral nucleic acid was detected by real-time reverse transcription-PCR(rRT-PCR)method.The positive serum was isolated and cultured with C6/36 cells to isolate dengue virus,and the E genes of the DENV strains were determined and phylogenetic tree was drawn.Molecular epidemiological analysis was carried out according to the epidemiological data.Results In 140 samples of acute serum,54 samples were positive for dengue virus nucleic acid with the positive rate of 38.6%and the proportion of DENV-1 positive was 53.7%.17 strains were isolated.Sequence alignment and evolutionary analysis showed that the nucleotide homology among the 10 strains of DENV-1 was 90.0%-99.9%,and the amino acid homology was 96.6%-99.8%.They are genotypeⅠand genotypeⅤ.7 genotypeⅠstrains,which have higher nucleotide homology with the strains of Thailand(2015),Myanmar(2015),Zhongshan(2015)and Vietnam(2016)(99.3%-99.9%).Among them,the homology of nucleotide between the local strain D2018078 in 2018 and the imported strain D2017008 in 2017 reach 99.8%;three genotypeⅤstrains have higher nucleotide homology with the strains of India(2014 and 2017)and Singapore(2014)(97.7%-99.1%).The nucleotide homology among the 4 strains of DENV-2 was 91.0%-98.6%,and the amino acid homology was 97.6%-99.8%.They are cosmopolitan genotype and genotypeⅠ.2 cosmopolitan genotype strains have higher nucleotide homology with the strains of Malaysia(2014),Taiwan(2015)(99.6%-99.7%);two genotypeⅠstrains have higher nucleotide homology with the strains of Vietnam(2015),Thailand(2011)(99.3%-99.5%).The nucleotide homology among the two strains of DENV-3 was 98.4%,and the amino acid homology was 99.4%.They all are genotypeⅠ,and have higher nucleotide homology with the strains of Myanmar(2017),Malaysia(2015)(99.3%-99.7%).One strain of DENV-4 have higher nucleotide homology with the strains of Vietnam(2013 and 2016)(99.3%-99.5%).Its genotype is genotypeⅠ.Conclusion The pathogens of dengue fever in Dongguan during 2017-2018 are mainly DENV-1,followed by DENV-2.They each have at least two genotypes that are circulating at the same time.Also,DENV-3 and DENV-4 have at least one genotypes that are circulating at the same time.The mode of spread is Local outbreaks caused by imported cases mainly occurred in south and southeast asian countries and in Zhongshan and Taiwan.