云南省不同感染来源间日疟原虫环子孢子蛋白基因多态和种群结构分析

Polymorphisms of circumsporozoite protein gene and population structure analysis of Plasmodium vivax with different infection sources in Yunnan Province

目的分析云南省不同感染来源间日疟原虫环子孢子蛋白(Pvcsp)基因序列,揭示当地Pvcsp基因的种群结构和遗传多样性。方法收集中国疾病预防控制中心传染病网络直报系统中2014-2017年报告的云南省本地和输入性间日疟病例流行病学资料及血样。提取血样中疟原虫基因组DNA,巢式PCR扩增并测序。采用MEGA 5.04、Arlequin 3.5.2.2软件进行单倍型、期望杂合度(He)分析,DnaSP 5.10计算核苷酸多样性(TT)、同义置换率(Ks)、错义置换率(Ka)及群体间遗传分化指数(Fst)。结果共检测间日疟病例血样969份,扩增获得650~750 bp大小的目的条带759份,包括云南本地感染者血样39份、非洲16份、缅甸688份、老挝13份、柬埔寨2份、巴基斯坦1份。单倍型分析结果显示,759条Pvcsp基因序列存在90个单倍型,其中29个为PV-I型温带族(类似VK210型),50个为PV-Ⅰ型热带族(类似VK210型),11个为PV-Ⅱ型(类似VK247型);3种基因型所占比例分别为51.4%(390/759)、41.1%(312/759)、7.5%(57/759),分布于云南本地感染、缅甸输入性病例中,但非洲、老挝及其他地区的输入性病例只表现为PV-Ⅰ型。PV-Ⅰ型、PV-Ⅱ型氨基酸序列突变分别发生在29、10个位点。759份病例血样的He为0.224,π为0.075,Ka/Ks为0.48。除去柬埔寨和巴基斯坦,Pvcsp基因在老挝输入性病例中的遗传多样性最高(He=0.422,π=0.03),云南本地与非洲输入性病例中的遗传分化最高(Fst=0.082),与缅甸输入性病例的遗传分化最低(Fst=0.002);Pvcsp基因在非洲与东南亚地区输入病例中属中等程度的遗传分化,在东南亚地区输入病例中遗传分化很小。结论云南省不同感染来源间日疟原虫环子孢子蛋白基因存在3种基因型,以PV-Ⅰ型温带族为优势虫株,不同基因型的种群结构和遗传分化不同。

Objective To analyze the gene sequences of circumsporozoite protein of Plasmodium vivax(Pvcsp) collected from different infection sources in Yunnan Province,in order to understand the population structure and genetic diversity of Pvcsp gene in the Province.Methods Epidemiological data and blood samples were collected from the local and imported P.vivax malaria cases in Yunnan Province reported in the China Infectious Disease Network Direct Reporting System from 2014 to 2017.Genomic DNA of Plasmodium was extracted from blood samples,amplified by nested PCR and sequenced.MEGA 5.04 and Arlequin 3.5.2.2 were used to analyze the haploid type and the expected heterozygosity(He),respectively,and DnaSP 5.10 was used to calculate nucleotide diversity(π),synonymous replacement rate(Ks),missense replacement rate(Ka) and interpopulation genetic differentiation index(Fst).Results A total of 969 blood samples of P.vivax malaria cases were analyzed,and 759 samples were successfully amplified to produce target bands of 650-750 bp,including 39 cases in Yunnan,16 cases from Africa,688 cases from Myanmar,13 cases from Laos,2 cases from Cambodia and 1 case from Pakistan.The haploid type analysis showed the presence of 90 haploid types among the 759 Pvcsp gene sequences,of which 29 belonged to the PV-Ⅰ temperate family(similar to the VK210 type),50 to the PV-Ⅰ tropical family(similar to the VK210 type),and 11 were of the PV-Ⅱ type(similar to the VK247 type).The proportions of three genotypes to the total examinees were 51.4%(390/759),41.1%(312/759),and 7.5%(57/759),respectively,distributed in Yunnan local and Myanmar imported cases,while imported cases from Africa,Laos and other places only exhibited the PV-Ⅰtype.The amino acid sequence mutations of the PV-Ⅰ and PV-Ⅱ types occurred at 29 and 10 loci,respectively.The He,π and Ka/Ks of the 759 samples were 0.224,0.075,and 0.480,respectively.Among the infection sources other than Cambodia/Pakistan,Pvcsp had the highest genetic diversity within imported cases from Laos(He,0.422;π,0.03).Pvcsp in Yunnan local cases and the imported cases from Africa showed the highest genetic differentiation(Fst,0.082),while the lowest genetic differentiation was seen in the imported cases from Myanmar(Fst,0.002);the Pvcsp gene displayed moderate genetic differentiation in the imported cases from Africa and Southeast Asia;and minor genetic differentiation was deteted in the imported cases from Southeast Asia.Conclusion There are three genotypes of Pvcsp of P.vivax from different sources of infection detected in Yunnan Province,with PV-Ⅰtemperate family as the dominant strain.The population genetic structure and differentiation varies with different genp types.