不同感染来源间日疟原虫抗叶酸类药物相关基因的研究

Analysis of genes associated with antifolate drug resistance in Plasmodium vivax from different infection sources

目的分析云南省不同感染来源间日疟原虫抗叶酸类药物抗性相关基因二氢叶酸还原酶基因(Pvdhfr)和二氢叶酸合成酶基因(Pvdhps)的突变特点。方法收集2012年8月-2016年12月寄生虫病防治信息管理系统登记报告的云南省间日疟病例血样和流行病学史等信息。提取疟原虫DNA,巢式PCR扩增Pvdhfr和Pvdhps基因并测序,测序序列与Gen Bank中的间日疟原虫Pvdhfr(登录号为X98123)、Pvdhps基因(登录号为PVX_123230)参比序列比对。用MEGA 5.04、Arlequin 3.5.1分析Pvdhfr和Pvdhps基因的单倍型、期望杂合度(He)、遗传分化指数(Fst)等。用Network 4.6.0、Arc Gis10.1构建单倍型网络进化图和突变型分布图。结果 共收集1 203份疟疾病例血样,缅甸、非洲、云南本地感染病例血样分别为1 060、79和64份。Pvdhfr、Pvdhps基因PCR扩增产物分别测序成功272份和708份。分析结果显示,Pvdhfr基因的272条序列存在53种单倍型,He为0.243,其中云南本地分离株群体的He最高,为0.667;12个位点均为野生型的频率是8.1%(22/272),其余52种单倍型在12个位点上存在单点或双重、三重、四重、五重、六重错义突变的不同突变型。Pvdhps基因的708条序列存在35种单倍型,He为0.153,其中缅甸感染分离株群体的He最高,为0.142;5个位点均为野生型的频率是36.2%(256/708),其余34种单倍型在5个位点形成29种错义突变,产生单点、双重、三重、四重等多种突变型。Pvdhfr及Pvdhps基因均以野生型为起始,再按单重突变到多重突变的路径逐步进化。Pvdhfr基因的野生型和突变型血样中,Pvdhps基因突变型的比例分别为18.2%(4/22)和65.6%(147/224)。Pvdhps、Pvdhfr基因突变型的血样分别分布在14和9个州(市),且以缅甸感染血样占多数,分别占97.3%(440/452)和95.4%(144/151)。结论云南省不同感染来源间日疟原虫抗叶酸类药物抗性相关基因Pvdhfr、Pvdhps的突变率、突变程度均高。

Objective To analyze the mutation characteristics of dihydrofolate reductase gene（ Pvdhfr） and dihydropterroate synthase gene（ Pvdhps）, which are associated with antifolate drug resistance, in Plasmodium vivax from different infection sources in Yunnan Province. Methods Dry blood spots on filter papers were collected from vivax malaria patients reported through the Reporting System of Chinese Center for Disease Control and Prevention from August 2012 to December 2016 and the corresponding epidemiology data of the patients were collected as well.DNA was extracted from P. vivax, and the Pvdhfr and Pvdhps fragments were amplified with nest-PCR. The PCR products were sequenced, and all sequences were aligned with the reference sequences（Gen Bank ID： X98123 and PVX_123230）. The haplotype number, expected heterozygsity（He）, and genetic differentiation（Fixation index） of the two genes in different Plasmodium populations were analyzed with MEGA 5.04 and Arlequin 3.5.1 softwares. The haplotype media evolution tree was constructed with the Network 4.6.0 software. Results A total of 1 203 blood samples were collected, including 1 060 with an infection source in Myanmar, 70 in Africa and 64 from indigenous patients in Yunnan. The PCR results showed that Pvdhfr and Pvdhps were amplified and sequenced in 272 and 708 samples, respectively. There were 53 haplotyes for Pvdhfr in the 272 DNA sequences, with an He of 0.243, and the Yunnan indigenous isolates had the highest He value（0.667）. The case of all 12 loci of Pvdhfr gene being wild-type had a frequency of 8.1%（22/272）. All the other 52 haplotypes harbored various types of missense mutations at the 12 loci, including single-locus, double-loci, triple-loci, quadruple-loci, quintuple-loci and sixfold-loci mutations. There were 35 haplotyes in 708 DNA sequences of Pvdhps gene, with an He of 0.153, and the Myanmar-source isolates had the highest He（0.142）. The case of all the 5 loci of Pvdhps gene being wild-type had a frequency of 36.2%（256/708）. All the other 34 haplotypes harbored 29 types of missense mutations at the 5 loci, including single-locus, double-loci, triple-loci, and quadruple-loci mutations. The media network diagram constructed from all the haplotypes of the two P. vivax genes showed that the haplotypes occurred initially as wild-type, and then evolved from single mutation to multiple mutations. In blood samples with wild-type and mutated Pvdhfr, the percentages of Pvdhps mutations were 18.2%（4/22） and 65.6%（147/224）, respectively. The blood samples confirmed with Pvdhps mutations and Pvdhfr mutations were from 14 and 9 prefectures, respectively, and predominated by the Myanmar source samples that constituted 97.3%（442/452） and 95.4%（144/151）, respectively.Conclusion Both Pvdhfr and Pvdhps have a high mutation frequency and a high degree of mutation in P. vivax from different infection sources in Yunnan Province.