Analysis of genetic polymorphisms of merozoite surface protein-1 and circumsporozoite protein of Plasmodium vivax

doi:10.12140/j.issn.1000-7423.2020.03.011

Abstract

Abstract:

Objective This study aimed to conduct descriptive analysis of the genetic polymorphisms of Plasmodium vivax gene merozoite surface protein-1 (PvMSP-1) and circumsporozoite protein (PvCSP) in vivax malaria cases in Hangzhou City. Methods Blood samples and epidemiological information of vivax malaria cases reported in Hangzhou during 2008-2019 were collected. P. vivax genomic DNA was extracted by QIAamp DNA blood mini kit. Pvmsp-1 and Pvcsp were amplified by nested PCR and the amplification products were sequenced. The amino-acid sequences were analyzed using DNAStar and MEGA 6.0 softwares and the phylogenetic tree was constructed using the neighbor-joining method. The descriptive and inductive analysis of genetic polymorphism and place of infection origin were performed using SPSS 17.0. Results Fifty-six vivax malaria cases were sampled, of whom 44 and 54 samples were successfully sequenced for Pvmsp-1 and Pvcsp, respectively. Sequence analysis indicated that 44 Pvmsp-1 sequences were clustered into four genotypes: SalⅠ(18 samples), Belem (8), R-Ⅲ (17) and R-Ⅳ (1); 54 Pvcsp sequences were devided into two genotypes: VK210 (52 samples) and VK247 (2). Analysis of the genotyping and place of infection origin of Pvmsp-1 gene showed that the SalⅠtype was widely distributed in East China, Southeast Asia, India, and Ethiopia. The main type of Pvmsp-1 in East China was R-Ⅲ. In addition, the R-Ⅲ type was also distributed in South Asia and Southeast Asia. The Belem type was mainly distributed in Southeast Asia, South Asia and Ethiopia. Analysis of Pvcsp gene polymorphism and place of infection origin revealed four types of polymorphism with some regional features. Conclusion The Pvmsp-1 and Pvcsp genes in vivax malaria cases reported in Hangzhou during 2008-2019 were characterized being highly polymorphic, with some genotypes having certain regional features.

Key words: Plasmodium vivax, Merozoite surface protein-1, Circumsporozoite protein, Genetic diversity

CLC Number:

R531.31

JIN Hang-yi, ZHANG Ling-ling, ZHU Su-juan, XU Wei-min, CHEN Jun-fang, RUAN Wei, YAO Li-nong, CHEN Hua-liang. Analysis of genetic polymorphisms of merozoite surface protein-1 and circumsporozoite protein of Plasmodium vivax[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020, 38(3): 323-331.

Figures/Tables 6

Table 1

Table 2

Fig. 1

Fig. 2

Table 3

Table 4

References 28

[1]	World Health Organization. World malaria report 2018[R]. Geneva: WHO, 2018.
[2]	Talha AA, Pirahmadi S, Mehrizi AA, et al. Molecular genetic analysis of Plasmodium vivax isolates from Eastern and Central Sudan using pvcsp and pvmsp-3α genes as molecular markers[J]. Infect Genet Evol, 2015,32:12-22.
[3]	Huang B, Huang SG, Su XZ, et al. Genetic diversity of Plasmodium vivax population in Anhui Province of China[J]. Malar J, 2014,13:13. pmid: 24401153
[4]	Ruan W, Zhang LL, Feng Y, et al. Genetic diversity of Plasmodium vivax revealed by the merozoite surface protein-1 icb5-6 fragment[J]. Infect Dis Poverty, 2017,6:92. pmid: 28578709
[5]	Patil A, Orjuela-Sánchez P, da Silva-Nunes M, et al. Evolutionary dynamics of the immunodominant repeats of the Plasmodium vivax malaria-vaccine candidate circumsporozoite protein (CSP)[J]. Infect Genet Evol, 2010,10(2):298-303.
[6]	Kibria MG, Elahi R, Mohon AN, et al. Genetic diversity of Plasmodium vivax in clinical isolates from Bangladesh[J]. Malar J, 2015,14:267.
[7]	Zhang LL, Yao LN, Chen HL, et al. Genetic diversity analysis of PvCSP and its application in tracking of Plasmodium vivax[J]. Exp Parasitol, 2018,188:26-35.
[8]	Putaporntip C, Jongwutiwes S, Sakihama N, et al. Mosaic organization and heterogeneity in frequency of allelic recombination of the Plasmodium vivax merozoite surface protein-1 locus[J]. Proc Natl Acad Sci USA, 2002,99(25):16348-16353. pmid: 12466500
[9]	Premawansa S, Snewin VA, Khouri E, et al. Plasmodium vivax: recombination between potential allelic types of the merozoite surface protein MSP1 in parasites isolated from patients[J]. Exp Parasitol, 1993,76(2):192-199. pmid: 8454028
[10]	Kolakovich KA, Ssengoba A, Wojcik K, et al. Plasmodium vivax: favored gene frequencies of the merozoite surface protein-1 and the multiplicity of infection in a malaria endemic region[J]. Exp Parasitol, 1996,83(1):11-19.
[11]	Arnot DE, Barnwell JW, Tam JP, et al. Circumsporozoite protein of Plasmodium vivax: gene cloning and characterization of the immunodominant epitope[J]. Science, 1985,230(4727):815-818. pmid: 2414847
[12]	Rosenberg R, Wirtz RA, Lanar DE, et al. Circumsporozoite protein heterogeneity in the human malaria parasite Plasmodium vivax[J]. Science, 1989,245(4921):973-976. doi: 10.1126/science.2672336 pmid: 2672336
[13]	Zhang SY, Lu HM, Xu LS, et al. Polymorphism analysis of Plasmodium vivax merozoite surface protein 1 (PvMsp-1) gene from different malaria-endemic area of China[J]. Chin J Zoonoses, 2004,20(1):26-30. (in Chinese)
	( 张山鹰, 陆惠民, 许龙善, 等. 我国不同疟区间日疟原虫裂殖子表面蛋白1 (PvMSP-1) 基因多态性研究[J]. 中国人兽共患病杂志, 2004,20(1):26-30.)
[14]	Imwong M, Pukrittayakamee S, Grüner AC, et al. Practical PCR genotyping protocols for Plasmodium vivax using Pvcsp and Pvmsp-1[J]. Malar J, 2005,4:20.
[15]	Zhang L, Feng J, Zhang SS, et al. The progress of national malaria elimination and epidemiological characteristics of malaria in China in 2017[J]. Chin J Parasitol Parasit Dis, 2018,36(3):201-209. (in Chinese)
	( 张丽, 丰俊, 张少森, 等. 2017年全国消除疟疾进展及疫情特征分析[J]. 中国寄生虫学与寄生虫病杂志, 2018,36(3):201-209.)
[16]	Zhang L, Feng J, Zhang SS, et al. Epidemiological characteristics of malaria and the progress towards its elimination in China in 2018[J]. Chin J Parasitol Parasit Dis, 2019,37(3):241-247. (in Chinese)
	( 张丽, 丰俊, 张少森, 等. 2018年全国疟疾疫情特征及消除工作进展[J]. 中国寄生虫学与寄生虫病杂志, 2019,37(3):241-247.)
[17]	Zhang L, Feng J, Xia ZG, et al. Epidemiological characteris-tics of malaria and progress on its elimination in China in 2019[J]. Chin J Parasitol Parasit Dis, 2020,38(2):133-138. (in Chinese)
	( 张丽, 丰俊, 夏志贵, 等. 2019年全国疟疾疫情特征分析及消除工作进展[J]. 中国寄生虫学与寄生虫病杂志, 2020,38(2):133-138.
[18]	Cao J, Liu YB, Cao YY, et al. Sustained challenge to malaria elimination in China: imported malaria[J]. Chin J Parasitol Parasit Dis, 2018,36(2):93-96. (in Chinese)
	( 曹俊, 刘耀宝, 曹园园, 等. 中国消除疟疾的持续挑战: 输入性疟疾[J]. 中国寄生虫学与寄生虫病杂志, 2018,36(2):93-96.)
[19]	Feng J, Zhou SS. From control to elimination: the historical retrospect of malaria control and prevention in China[J]. Chin J Parasitol Parasit Dis, 2019,37(5):505-513.(in Chinese)
	( 丰俊, 周水森. 从控制走向消除:我国疟疾防控的历史回顾[J]. 中国寄生虫学与寄生虫病杂志, 2019,37(5):505-513.)
[20]	Zhang L, Feng J, Tu H, et al. Challenges in malaria eliminations: the epidemiological characteristics of Plasmodium vivax in China from 2011 to 2018[J]. Chin J Parasitol Parasit Dis, 2019,37(5):532-538. (in Chinese)
	( 张丽, 丰俊, 涂宏, 等. 消除疟疾的挑战: 2011-2018年全国间日疟疫情分析[J]. 中国寄生虫学与寄生虫病杂志, 2019,37(5):532-538.)
[21]	Dong Y, Sun AM, Chen MN, et al. Polymorphism analysis of the block 5 region in merozoite surface protein-1 gene of imported and local Plasmodium vivax in Yunnan Province[J]. Chin J Parasitol Parasit Dis, 2017,35(1):1-7. (in Chinese)
	( 董莹, 孙艾明, 陈梦妮, 等. 云南省输入性及本地感染间日疟原虫裂殖子表面蛋白1基因5区序列的多态性分析[J]. 中国寄生虫学与寄生虫病杂志, 2017,35(1):1-7.)
[22]	Putaporntip C, Jongwutiwes S, Sakihama N, et al. Mosaic organization and heterogeneity in frequency of allelic recombination of the Plasmodium vivax merozoite surface protein-1 locus[J]. Proc Natl Acad Sci USA, 2002,99(25):16348-16353. doi: 10.1073/pnas.252348999 pmid: 12466500
[23]	Gutierrez A, Vicini J, Patarroyo ME, et al. Plasmodium vivax: polymorphism in the merozoite surface protein 1 gene from wild Colombian isolates[J]. Exp Parasitol, 2000,95(3):215-219.
[24]	Liu Y, Zhou RM, Chen WQ, et al. Polymorphism analysis on the genotypes of circumsporozoite protein of Plasmodium vivax with PCR-RFLP[J]. Chin J Parasitol Parasit Dis, 2013,31(6):483-485. (in Chinese)
	( 刘颖, 周瑞敏, 陈伟奇, 等. 间日疟原虫环子孢子蛋白PCR-RFLP多态性分析[J]. 中国寄生虫学与寄生虫病杂志, 2013,31(6):483-485.)
[25]	Liu Y, Qian D, Chen WQ, et al. Tracing investigation of one vivax malaria case by detecting the gene encoding circumsporozoite protein in Henan[J]. Chin J Parasitol Parasit Dis, 2015,33(2):156-158. (in Chinese)
	( 刘颖, 钱丹, 陈伟奇, 等. 环子孢子蛋白检测法对1例间日疟病例的溯源[J]. 中国寄生虫学与寄生虫病杂志, 2015,33(2):156-158.)
[26]	Raza A, Ghanchi NK, Thaver AM, et al. Genetic diversity of Plasmodium vivax clinical isolates from southern Pakistan using pvcsp and pvmsp1 genetic markers[J]. Malar J, 2013,12:16.
[27]	Zakeri S, Raeisi A, Afsharpad M, et al. Molecular characterization of Plasmodium vivax clinical isolates in Pakistan and Iran using Pvmsp-1, Pvmsp-3alpha and Pvcsp genes as molecular markers[J]. Parasitol Int, 2010,59(1):15-21. pmid: 19545647
[28]	Kim JR, Imwong M, Nandy A, et al. Genetic diversity of Plasmodium vivax in Kolkata, India[J]. Malar J, 2006,5:71. doi: 10.1186/1475-2875-5-71 pmid: 16907979

基因 Genes	引物名称 Primer names	引物序列（5′→3′） Primer sequences (5′→3′)
Pvmsp-1	Pvmsp-1-F1	CCCTACTACTTGATGGTCCTC
	Pvmsp-1-R1	CCTTCTGGTACAGCTCAATG
	Pvmsp-1-F2	AGCATGATCGCCACTGAGAAG
	Pvmsp-1-R2	GTGCTTGTGACATGCGTAAGC
Pvcsp	VCSF1	ATGTAGATCTGTCCAAGGCCATAAA
	VCSR1	TAATTGAATAATGCTAGGACTAACAATATG
	VCSF2	GCAGAACCAAAAAATCCACGTGAAAATAAG
	VCSR2	CCAACGGTAGCTCTAACTTTATCTAGGTAT

来源地 Source area	Pvmsp-1/Pvcsp												合计Total
来源地 Source area	2008	2009	2010	2011	2012	2013	2014	2015	2016	2017	2018	2019	合计Total
中国安徽Anhui, China	1, 2	0, 0	4, 4	4, 4	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	9, 10
中国浙江Zhejiang, China	0, 0	0, 0	0, 0	1, 1	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	1, 1
中国四川Sichuan, China	0, 1	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 1
柬埔寨Cambodia	0, 0	0, 0	0, 0	2, 2	2, 3	2, 2	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	6, 7
缅甸Myanmar	0, 0	0, 0	0, 0	0, 1	0, 1	0, 0	0, 0	2, 2	0, 0	0, 0	0, 0	0, 0	2, 4
印度尼西亚Indonesia	0, 0	0, 0	0, 0	0, 0	1, 1	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	1, 1
印度India	0, 0	0, 0	0, 0	2, 2	0, 0	0, 0	1, 1	0, 0	0, 0	1, 1	0, 0	0, 0	4, 4
巴基斯坦Pakistan	0, 0	0, 0	0, 0	2, 3	0, 0	1, 2	0, 0	1, 1	1, 3	1, 1	0, 0	1, 1	7, 11
阿富汗Afghanistan	0, 0	0, 0	0, 0	0, 0	0, 0	2, 2	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	2, 2
埃塞俄比亚Ethiopia	0, 0	0, 0	0, 0	0, 1	0, 0	0, 0	0, 0	2, 2	0, 0	3, 3	0, 0	0, 0	5, 6
尼日利亚Nigeria	0, 0	1, 1	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	1, 1	2, 2
苏丹Sudan	0, 0	0, 0	0, 0	0, 0	0, 0	1, 1	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	1, 1
科特迪瓦Cote d′ivoire	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 1	0, 0	0, 1
非洲（不详）Africa	0, 0	1, 1	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	1, 1
未知来源地Unknown	0, 0	0, 0	2, 1	1, 1	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	0, 0	3, 2
合计Total	1, 3	2, 2	6, 5	12, 15	3, 5	6, 7	1, 1	5, 5	1, 3	5, 5	0, 1	2, 2	44, 54

来源地Area	V1				V2		V3				V4						V5
来源地Area	a	b	c	d	e	f	g	h	i	j	k	l	m	n	o	p	q	r	s	t	u	v	w
中国安徽Anhui, China	1	0	8	0	10	0	0	10	0	0	0	1	0	3	4	2	0	2	0	1	3	4	0
中国浙江Zhejiang, China	0	0	1	0	1	0	0	1	0	0	0	1	0	0	0	0	0	0	0	0	0	0	1
中国四川Sichuan, China	0	0	0	0	1	0	1	0	0	0	0	1	0	0	0	0	0	1	0	0	0	0	0
柬埔寨Cambodia	3	1	2	0	5	2	4	1	1	1	0	2	4	1	0	0	1	5	1	0	0	0	0
缅甸Myanmar	2	0	0	0	1	1	0	1	1	0	0	1	0	1	0	0	0	1	1	0	0	0	0
印度尼西亚Indonesia	0	1	0	0	0	1	0	0	1	0	0	0	0	1	0	0	0	1	0	0	0	0	0
印度India	4	0	0	0	4	0	4	0	0	0	1	3	0	0	0	0	0	4	0	0	0	0	0
巴基斯坦Pakistan	3	1	3	0	11	0	11	0	0	0	0	11	0	0	0	0	0	11	0	0	0	0	0
阿富汗Afghanistan	0	0	2	0	2	0	2	0	0	0	0	2	0	0	0	0	0	2	0	0	0	0	0
埃塞俄比亚Ethiopia	1	3	1	0	6	0	6	0	0	0	0	6	0	0	0	0	0	6	0	0	0	0	0
尼日利亚Nigeria	1	1	0	0	2	0	2	0	0	0	0	2	0	0	0	0	0	2	0	0	0	0	0
苏丹Sudan	1	0	0	0	1	0	1	0	0	0	0	1	0	0	0	0	0	1	0	0	0	0	0
科特迪瓦Cote d’ivoire	0	0	0	0	1	0	1	0	0	0	0	1	0	0	0	0	0	1	0	0	0	0	0
非洲（不详）Africa	0	0	0	1	1	0	1	0	0	0	0	1	0	0	0	0	0	1	0	0	0	0	0
未知Unknown	2	1	0	0	2	0	1	1	0	0	0	1	0	0	0	1	0	1	0	0	1	0	0

基因 Gene	多态性特征 Polymorphism characteristics	地域分布 Regional distribution
基因 Gene	多态性特征 Polymorphism characteristics	中国华东地区 East China	东南亚地区 Southeast Asia	南亚地区 South Asia	非洲地区 Africa
Pvmsp-1	SalⅠ	√	√	√	√
	Belem		√	√	√
	R-Ⅲ	√	√	√	√
	R-Ⅳ				√
Pvcsp	中央重复区后不具有12肽插入序列 Without a 12-peptide insertion sequence after the central repeat region		√
	12肽插入序列后GGNA重复次数 Number of repeats of the GGNA unit after the 12-peptide insertion sequence	2	1~4	1	1
	中央重复区9肽单位重复次数 Number of repeats of peptide repeat motif in central repeat region	18~22	18~20	18	18
	9肽单位（GNGAGGQAA）重复次数 Number of repeats of peptide repeat motif (GNGAGGQAA)	1, 13~15	1~2	1	1