卵形疟原虫非洲分离株K13基因的多态性分析

Polymorphism analysis of K13 gene of Plasmodium ovale isolates from Africa

目的分析卵形疟原虫柯氏亚种(Plasmodium ovale curtisi)和沃氏亚种(P. ovale wallikeri)非洲分离株K13蛋白编码基因的多态性,为卵形疟原虫青蒿素抗性监测提供科学依据。方法收集江苏省2012-2016年卵形疟患者血样,经流行病学调查确认为非洲输入性卵形疟病例。提取血样DNA,通过实时荧光PCR探针法鉴别卵形疟原虫柯氏亚种和沃氏亚种。采用巢式PCR扩增K13基因的结构域并送测序,以卵形疟原虫柯氏亚种参考序列(PlasmoDB登录号:PocGH01_12019400)和沃氏亚种参考序列(GenBank登录号:LT594516.1)为模板,应用BioEdit软件对测序序列进行比对,应用DNAstar软件分析序列的突变情况,用DnaSP软件分析序列多态性,用MEGA软件构建系统进化树。结果 2012-2016年共收集168例卵形疟患者的血样,感染来源地为中非(95例)、南非(37例)、西非(34例)、东非(1例)和北非(1例);卵形疟原虫柯氏亚种和沃氏亚种各84例。168例血样K13基因巢式PCR均扩增获得1 500 bp的条带且均测序成功。卵形疟原虫K13基因核苷酸多样性指数π为0.000 02,单体型多样性指数Hd为0.024, 2个亚种K13基因分别含有2个单体型,分别发现1个单核苷酸多态性位点,各有1例样本检出卵形疟原虫核苷酸碱基突变,柯氏亚种在核苷酸717 bp (氨基酸239E)位点处存在A/G突变,沃氏亚种在核苷酸1998 bp (氨基酸666P)位点处存在T/A突变,均为同义突变,与卵形疟原虫青蒿素耐药性无关。本研究患者血样卵形疟原虫2个亚种K13基因序列中性检验统计结果无明显差异,符合中性进化模型。系统进化树显示,卵形疟原虫柯氏亚种2个单体型聚为一支,沃氏亚种2个单体型聚为另一支。结论卵形疟原虫非洲分离株柯氏亚种和沃氏亚种K13基因未发现非同义突变,其基因多态性水平较低,无青蒿素耐药性相关的基因突变。

Objective To analyze the polymorphism of the K13 protein-encoding gene of Plasmodium ovale curtisi and wallikeri isolates from Africa so as to provide molecular basis for monitoring artemisinin resistance of P.ovale in Africa.Methods The blood samples were collected from imported malaria patients with P.ovale identified by microscopic and epidemiological investigation from 2012 to 2016 in Jiangsu Province.DNA was extracted from the blood samples and the P.ovale K13 gene was amplified by nested PCR.The subspecies of P.ovale were further determined by Real-time TaqManR PCR.The acquired DNA sequences were aligned with reference sequences from P.ovale curtisi subspecies (PlasmoDB: PocGH01_12019400) and the wallikeri subspecies (GenBank: LT594516.1) using BioEdit software.The mutations of the obtained DNA sequences were analyzed using DNAstar software.The polymorphism of K13 gene was analyzed by DnaSP software.The phylogenetic tree was constructed using MEGA software.Results A total of 168 P.ovale infected blood samples were collected from malaria patients during 2012-2016 in Jiangsu Province.It was confirmed that the sources of infection were from Central African (95 cases),South Africa (37 cases),West Africa (34 cases) and one case from East and North Africa.A 1 500 bp fragment of K13 gene was successfully amplified from all samples and sequenced.The sequence results identified that half of the samples were P.ovale curtisi subspecies (84) or wallikeri subspecies (84).The nucleotide diversity index π of P.ovale K13 gene was 0.000 02.The haplotype diversity index Hd was 0.024.The K13 genes of two subspecies contained two haplotypes and one single nucleotide polymorphism site.There is only one nucleotide polymorphism within each P.ovale subspecies.The P.ovale curtisi had a A/G polymorphism at nucleotide 717 (amino acid 239E) and the P.ovale wallikeri had a T/A polymorphism at the nucleotide 1998 (amino acid 666P),both of them were synonymous mutations without the change of encoded amino acids.The two mutations identified in this study are not related to P.ovale artemisinin resistance.Neutral test was performed on the DNA sequences of both subspecies of P.ovale without statistical difference.The mutation in P.ovale K13 gene in this study is in accordance with the neutral evolution model.The phylogenetic tree shows that the two haplotypes of P.ovale curtisi subspecies are clustered into one branch,and the two haplotypes of P.ovale wallikeri subspecies are clustered into another branch.Conclusion No nonsynonymous mutation is found in K13 gene of P.ovale curtisi and wallikeri subspecies in this study.K13 gene has a low level of genetic polymorphism in Africa isolations and no mutation is related to P.ovale artemisinin resistance.