2014年广东省登革热大流行的病原体来源及分子进化特点

Origin and Phylogenetic Characteristics of Dengue Virus Isolated from the Large Outbreak of Dengue in Guangdong Province in 2014

【目的】分离培养与鉴定2014年广东省登革热暴发流行的病原体,从分子进化方向分析其来源和遗传进化特点,为登革热的监测和防控提供科学依据。【方法】在2014年登革热暴发流行期间,采集广东省不同地区登革热患者的血清标本,从中分离培养登革病毒并进行型别鉴定。采用RT-PCR和测序技术获取病毒的E基因和全基因组序列,并对其进行系统进化分析、贝叶斯进化分析和遗传变异分析。【结果】分离培养与鉴定了40株登革病毒1型(DENV-1)病毒,获得了这40株分离株的完整E基因序列和其中6株分离株的全基因组序列。E基因进化分析结果显示,40株分离株中有16株为基因Ⅰ型,24株为基因Ⅴ型。16株基因Ⅰ型毒株中有14株与2013年广州市和2013年新加坡流行的毒株高度同源,核苷酸相似度为99.6%~99.9%,另2株基因Ⅰ型毒株则与2013年马来西亚流行的毒株亲缘关系最近,核苷酸相似度为99.7%。24株基因Ⅴ型毒株均与2009年孟加拉、广州市和2013年不丹流行的毒株同源性高,核苷酸相似度为99.0%~99.9%。对6株病毒全基因组进化分析结果显示,其中5株病毒与2013年广州市和中山市流行的毒株高度同源,核苷酸相似度为99.6%~99.8%,另一株病毒则与2002年的缅甸流行株亲缘关系最近,核苷酸相似度为98.8%。对E基因的遗传变异分析结果显示,与2013年广东省的流行代表株比较,2014年广东省分离株有5个氨基酸位点发生了变异,其中3个(S88V,E203G,T275R)位于EⅡ区的毒力位点区域,1个(S305P)位于EⅢ区的毒力位点区域。【结论】2014年广东省登革热的病原体主要为DENV-1,且至少有2种基因型同时在流行;其可能来源于2013年广东省流行的毒株,也可能来源于新加坡、马来西亚、缅甸等东南亚国家的毒株,其中2013年广东省流行的毒株可能是重要来源之一,提示须加强对本地毒株及传播媒介的监测和研究,以便了解登革热是否在广东省本地化的问题;在流行过程中,病毒有些氨基酸位点已发生变异,其中有4个变异位点位于登革病毒的毒力位点区域,提示有必要对这些变异的生物学意义开展进一步研究。

【Objects】To isolate and identify the pathogen of the large outbreak of dengue in Guangdong province in 2014. To understand the origin and the phylogenetic characteristics of the isolates,and provide scientific foundation for the surveillance and prevention of dengue fever.【Methods】Collected the patient serum samples over all the Guangdong province during the 2014 outbreak period,isolated and identified the virus from these samples. Amplified complete E gene and complete genome with certain primers and sequenced all the products. Then the Phylogenetic,Bayesian phylogeography and mutations analysis were carried.【Results】40DENV-1 strains were isolated and identified. 40 complete E gene sequences and 6 complete genome sequences of DENV-1 were obtained. Phylogenetic analysis with E gene sequences revealed that the 40 isolates were classified into two genotypes including 16genotypeⅠ（Asia）and 24 genotypeⅤ（America/Africa）. 14 genotypeⅠisolates were clustered closest with isolates from Guangdong province（2013）and Sigapore（2013）which share the nucletide identities of 99.6% - 99.9%,other two genotypeⅠisolates were clustered with strains from Malaysia（2013）and both share the nucletide identities of 99.7%;24 genotype Ⅴ isolates were all classified in one clade with striains from Bangladesh（2009）,China（2009）and Bhutan（2013）which share nucletide identities of99.0%- 99.9%. Further analysis with six complete genome sequences showed that five isolates were clustered closest with strains isolated from Guangdong province（2013）share the nucletide identities of 99.6%-99.8% while the sixth stains closest with strains isolated from Myanmar（2002）share the nucletide identities of 98.8%. The isolates have five amino acid mutations compared with strains epidemic in Guangdong province in 2013,three mutations（S88V,E203 G,T275R）are in the EⅡ domain and one mutation（S305P）is in the EⅢ domain which associated with virulence.【Conclusions】During the outbreak in Guangdong province in 2014,DENV-1 is the predominant causative serotype,and there are at least two different kinds of genotypes of DENV-1 largely epidemiced in the whole province. Evolution analysis reveals the multiple origins of the isolates which may origin from Guangdong province,Sigapore,Malaysia,Myanmar so that we should enhance the study and surveillance of autochthonous and vectors in order to understand the epidemic way of dengue in Guangdong province. The isolates have had four mutations in the domain associated with virulence which remain further study to know their biological effects.