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

目的　了解我国不同疟疾流行区间日疟原虫裂殖子表面蛋白 - 1基因多态性及其分布。方法　用套式PCR扩增间日疟原虫现场分离株MSP - 1第五多态区 (ICB5 -ICB6 )基因片段 ,部分样本进行序列测定、分析和比对。结果　 82份间日疟原虫现场分离株扩增出 4 70bp片段 5 0份 ,4 0 0bp片段 39份 ,其中 7份为两种片段的混合型。海南分离株混合型为 2 0 %(6 / 30 ) ,平均克隆数为 1 2 0 (36 / 30 ) ,安徽分离株混合型仅为 2 3% ,平均克隆数 1 0 2。对 33份样本进行序列测定 ,Sal- 1型17份 ,Belem型 2份 ,12份重组型 (Ⅲ型 )和朝鲜型 2份为我国新发现基因型。结论　我国间日疟原虫PvMSP - 1存在 4种不同的等位基因型 ,以Sal- 1型和重组型 (Ⅲ型 )占优势 ,南部疟区基因型比北部复杂。     

辽宁丹东间日疟原虫裂殖子表面蛋白1等位基因型分析

用套式PCR方法扩增辽宁丹东地区间日疟原虫裂殖子表面蛋白1(MSP-1)基因ICB5-ICB6片段,经PvuⅡ酶切和琼脂糖凝胶电泳,对产物进行序列分析。结果显示,11份镜检确诊的间日疟患者血液标本经套式PCR扩增均出现大小约为470 bp(Sal-1型)或400 bp(Belem型)的特异性片段。经PvuⅡ内切酶消化后,其中5份血样(470 bp),出现120和350 bp酶切片段,为Sal-1型;其余6份血样(400 bp)中,1份出现400 bp片段,为Belem型,1份出现120和280 bp两种酶切片段,为重组Ⅲ型,4份出现120和240 bp两种酶切片段,为朝鲜型。辽宁省丹东地区的间日疟原虫PvMSP-1基因存在3种不同的等位基因型,以Sal-1型和朝鲜型为主,但无不同等位基因型的混合感染。     

间日疟原虫裂殖子表面蛋白的等位基因型检测

目的　建立一种间日疟原虫裂殖子表面蛋白 1(PvMSP-1)基因型检测技术。　方法　设计针对PvMSP 1ICB5～ICB6多态区的引物进行PCR ,其产物用PvuⅡ内切酶消化、琼脂糖凝胶电泳检测 ,鉴别我国间日疟原虫现场分离株基因型。　结果　 98份间日疟血样经套式PCR扩增均出现大小约为 40 0bp(Belem型 )或 470bp(Sal-1型 )的特异性片段。酶切消化后 ,45份 470bp样本出现 12 0bp和 3 5 0bp酶切片段 ,为Sal-1型 ;40份 40 0bp的样本中 3份仅出现 1条 40 0bp片段 ,为Belem型 ;3 5份出现 12 0bp和 2 80bp两种酶切片段 ,为Ⅲ重组型 ;2份 12 0bp和 2 40bp片段 ,为朝鲜型。结论　套式聚合酶链反应-限制性片段长度多态性 (PCR-RFLP)技术可用于检测我国间日疟原虫的 3种PvMSP-1等位基因型。     

我国华南间日疟原虫裂殖子表面蛋白1等位基因分型和序列分析

目的　了解我国间日疟原虫MSP1(PvMSP1)的等位基因类型和序列的多态性。方法　用特异的引物通过套式PCR方法体外扩增包含PvMSP1的ICB5与ICB6之间的基因片段 ,并对部分扩增产物进行序列测定和分析。结果　 2 7份间日疟原虫患者血样中 ,有 16份 (5 9 9% )扩增得到Belem型基因产物 ,2 5 (92 6 % )份扩增出Sal- 1型基因产物 ;两种不同等位基因型虫株的混合感染率为 5 1 9%。序列分析结果发现一个以前未见报告的基因内重组类型。结论　我国间日疟原虫虫株存在两种PvMSP1等位基因型 ,Sal- 1型可能是主导型 ;不同等位基因型的混合感染率较高     

间日疟原虫MSP1 C端编码基因的克隆及序列分析

目的 克隆间日疟原虫MSP1C端编码基因,并进行序列测定和分析。方法 根据间日疟原虫MSP1C端编码基因设计1对引物,采用PCR技术从深圳间日疟患者血样(编号为PvSZ1)的核酸提取物中扩增出MSP1C端编码基因,回收纯化后,与T载体连接构建重组子pMD/MSP1,并转化大肠杆菌JM109。阳性克隆以限制性酶切分析与PCR法鉴定后,双脱氧链末端终止法双向测定序列并分析。结果 从间日疟血样提取的DNA中扩增出1119bp的基因片段,所构建的pMD/MSP1阳性克隆重组子经双酶切和PCR鉴定与预期结果一致。所测定的间日疟原虫PvSZ1C端基因序列与国外Sal-1株比较,碱基数相同,未发现碱基缺失,相同的核苷酸占96.7％,序列中第542位碱基变化为同义突变(GTC→GTT,均编码缬氨酸),第375～542区间的碱基变化数占整个序列变化碱基总数的92.9％(34/37)。所推测的氨基酸序列与Sal-1株比较,同源性为92.5％。结论 成功克隆了间日疟原虫PvSZ1株MSP1C端编码基因,该基因在不同间日疟原虫地理株间相对保守,所克隆基因序列的第375～542核苷酸区域为高频变化区。     

Genetic diversity of merozoite surface protein-1 gene of Plasmodium vivax isolates in mining villages of Venezuela (Bolivar State)

The merozoite surface protein-1 gene of Plasmodium vivax is highly polymorphic and so, currently used in epidemiological studies of P. vivax malaria. We sequenced the variable block 5 of the gene from 39 Venezuelan isolates, 18 of which were co-infected with Plasmodium falciparum. We observed a limited variability with 34 isolates belonging to the type Salvador I, none Belem type and only five recombinants. Among the recombinants, only two types of sequences were observed with, respectively, 18 and 21 poly-Q residues. Nucleotide substitutions explained the major differences of the 11 patterns observed. We could evidence neither specific MSP-1 genotype associated with co-infected samples, nor peculiar MSP-1 genotype distribution inside the investigated areas. In comparison with other low endemic regions in the world, our sampling has a lower genetic diversity, which could be mainly explained by the lack of Belem type. In fact, the variable repeats of poly-Q residues involved in the polymorphism of Belem type and recombinant isolates are responsible for a great part of variability observed in MSP-1 block 5.     

Analysis of Plasmodium vivax merozoite surface protein-1 gene sequences from resurgent Korean isolates

The merozoite surface protein-1 (MSP-1) of Plasmodium vivax exhibits great antigenic diversity among different isolates of this parasite. This antigen is a useful genetic marker for studying the polymorphism of natural P. vivax parasite populations. One or more of these populations has been responsible for resurgent malaria now occurring in Korea. This paper reports the analysis of a highly polymorphic region between interspecies conserved blocks 5 and 6 of the MSP-1 gene, using the polymerase chain reaction to amplify the DNA fragment encompassing these regions from 25 Korean isolates, followed by sequencing. Almost all amino acid sequences of Korean isolates were nearly identical to that of Thai isolates TD525A (96.6-99.7%) and TD424 (96.3-99.5%), and very similar to that of the France-Belem strain when compared with other isolates (Sal-1, Sri Lanka, and Colombia). Interallelic recombination was found in the poly-Q repeat and a Sal-1 type amino acid structure was observed in all isolates. This study shows that the MSP gene nucleotide sequence of resurgent P. vivax in Korea is most similar to that of Thai isolates; however, the Korean strains are phylogenetically unique.     

Limited polymorphism of the Plasmodium vivax merozoite surface protein 1 gene in isolates from Turkey

The 200-kD merozoite surface protein of Plasmodium vivax (PvMSP-1) is one of the leading vaccine candidates against P. vivax malaria. However, the gene encoding PvMSP-1 (pvmsp1) is highly polymorphic and is a major obstacle to effective vaccine development. To further understand polymorphism in pvmsp1, we obtained 30 full-length pvmsp1 sequences from southeastern Turkey. Comparative analysis of sequences from Turkey and other areas showed substantially limited polymorphism. Substitutions were found at 280 and 162 amino acid sites in samples from other regions and those from Turkey, respectively. Eight substitutions were unique to Turkey. In one of them, D/E at position 1706 in the C-terminal 19-kD region, the K/E change at 1709 was the only polymorphism previously known. Limited diversity was also observed in microsatellites. Data suggest a recent population bottleneck in Turkey that may have obscured a signature for balancing selection in the C-terminal 42-kD region, which was otherwise detectable in other areas.     

Genetic diversity in the C-terminal 42 kDa region of merozoite surface protein-1 of Plasmodium vivax (PvMSP-1(42)) among Indian isolates

Plasmodium vivax merozoite surface protein 1 (PvMSP-1) is a leading malaria vaccine candidate. This protein is processed to give rise to various sized fragments during merozoite maturation. Here, we describe the analysis of genetic diversity in the 42 kDa C-terminal part of this protein among 33 Indian P. vivax isolates. A total of 27 haplotypes with 72 mutations and 0.0212 ± 0.0005S.D. over all π nucleotide diversity were observed among the isolates. Twenty-six of 27 haplotypes reported here were new as they have not been reported so far from any other country. The difference between non-synonymous (dN) and synonymous (dS) mutations was found to be positive (0.0081 ± 0.0051) for the entire 42 kDa region. Further analysis revealed that 33 kDa (MSP-1₃₃) fragment of the MSP-1₄₂ was highly polymorphic with π nucleotide diversity 0.0290 ± 0.0007S.D. The dN-dS for this region of MSP-1 was also positive (0.0114 ± 0.0071S.E.). On the other hand, there was no non-synonymous mutation in the 19 kDa (MSP-1₁₉) fragment of the MSP-1₄₂ and thus it was highly conserved. In conclusion, MSP-1₃₃ fragment was highly polymorphic and appeared to be under diversifying selection whereas there was no selection at MSP-1₁₉ region among the isolates. Present study will be helpful for the development of PvMSP-1 based vaccine against P. vivax malaria.     

间日疟原虫不同MSP-1等位基因型形态学观察

目的　比较两种不同等位基因型间日疟原虫生物学形态特征。方法　现场采集间日疟病人血样并进行基因分型 ,镜下观察形态并测量大小。结果与结论　 72份现场分离株基因分型为Sal- 1型 4 0份 ,Belem型 2 5份 ,混合感染 7份 ,均查到典型的间日疟原虫 ,6例多重感染病例均发现在海南分离株 ,多重感染与基因型混合感染无关联。测量正常红细胞、寄生红细胞、环状体以及核大小差别有显著意义 ,Belem型比Sal- 1型大 ,两种不同基因型差异的分子机制需进一步探讨     

间日疟原虫裂殖子表面蛋白3α基因多态性研究

目的分析我国部分地区间日疟原虫裂殖子表面蛋白3α(PvMSP-3α)基因多态性。方法套式PCR分别对PvMSP-3α和间日疟原虫裂殖子表面蛋白1(PvMSP-1)基因进行扩增,并应用限制性片段多态性(RFLP)对PvMSP-3α的PCR扩增产物酶切进行分析。结果31个PvMSP-3α基因型中Ⅰ型、Ⅱ型和Ⅲ型比例分别占77.42%、6.45%和16.13%。其中云南勐腊24份样本中Ⅰ型、Ⅱ型和Ⅲ型分别占79.17%、4.17%和16.17%。PvMSP-3α的Ⅱ型和Ⅲ型均为PvMSP-1的Belem型,而PvMSP-1的Sal-1型均为PvMSP-3α中的Ⅰ型。结论云南分离株的PvMSP-3α存在广泛多态性,PvMSP-3α和PvM-SP-1之间的联系有待于进一步探讨。     

IgG subclasses pattern and high-avidity antibody to the C-terminal region of merozoite surface protein 1 of Plasmodium vivax in an unstable hypoendemic region in Iran

The C-terminal region of Plasmodium vivax merozoite surface protein 1 (PvMSP-1₁₉) is a leading vaccine candidate for inclusion in a polyvalent malaria vaccine. In the present study, the IgG subclasses profile and the avidity of IgG to PvMSP-1₁₉ were evaluated in individuals (n = 94) naturally exposed to P. vivax parasite in malaria endemic areas in Chabahar districts, Iran. In individuals with patent P. vivax malaria, 86.1% was sero-positive to PvMSP-1₁₉ and IgG1 (81.9%) was the predominant subclass. In addition, to determine the persistence of specific IgG, IgG1 and IgG3 antibodies to PvMSP-1₁₉, the frequency of antibodies was determined in the infected subjects (n = 74) after treatment with standard chloroquine and it was detected that the frequency of responders was significantly reduced to 51.3%, 51% and 16.2%, respectively. The antigen-binding avidity of IgG antibodies to PvMSP-1₁₉ was measured in sero-positive sera and the high-avidity of IgG, IgG1 and IgG3 was found in 66.6%, 61% and 47% of the infected subjects with P. vivax, respectively. The present result shows that individuals who exposed to vivax malaria in the endemic region in Iran develop antibodies with high-avidity to PvMSP-1₁₉. These results could help to understand the interactions between the host and P. vivax parasite in development of MSP-1₁₉-based vaccine.     

Population structure analysis of Plasmodium vivax in areas of iran with different malaria endemicity

To obtain the genetic structure of Plasmodium vivax populations in the northern and southern malaria-endemic areas in Iran, which differ in endemicity, sequence diversity in the variable block 5 and the C-terminal part of P. vivax merozoite surface protein 1 (Pvmsp 1) was analyzed. The variable block 5 fragment from 52 northern and 94 southern isolates was amplified and sequenced. Type 1, type 2, and recombinant type 3 allelic variants were found in both northern and southern isolates, with type 1 predominant in parasites from the north and type 2 in those from the south. A total of 7 and 27 distinct variants were detected among northern and southern isolates, respectively. A single variant predominated (71%) in the northern isolates, whereas variants were evenly distributed among southern isolates, with only two exceeding 10%. Thus, parasites from the southern malaria-endemic area were more polymorphic than those circulating in the northern area, where malaria is a re-emerging disease. Sequence alignments showed that although some variants were found only in northern or southern isolates, some were common to both and had also been observed in parasites from Azerbaijan, Turkey, Thailand, Bangladesh, and China. The Pvmsp 1 fragment corresponding to the C-terminal region was also amplified and the sequences derived from 20 northern and 50 southern isolates were identical. This high degree of conservation reinforces the potential of this polypeptide fragment for inclusion in synthetic vaccines being developed against P. vivax.     

Mosaic organization and heterogeneity in frequency of allelic recombination of the Plasmodium vivax merozoite surface protein-1 locus

The organization and allelic recombination of the merozoite surface protein-1 gene of Plasmodium vivax (PvMsp-1), the most widely prevalent human malaria parasite, were evaluated in complete nucleotide sequences of 40 isolates from various geographic areas. Alignment of 31 distinct alleles revealed the mosaic organization of PvMsp-1, consisting of seven interallele conserved blocks flanked by six variable blocks. The variable blocks showed extensive variation in repeats and nonrepeat unique sequences. Numerous recombination sites were distributed throughout PvMsp-1, in both conserved blocks and variable block unique sequences, and the distribution was not uniform. Heterozygosity of PvMsp-1 alleles was higher in Asia (0.953 +/- 0.009) than in Brazil (0.813 +/- 0.047). No identical alleles were shared between Asia and Brazil, whereas all but one variable block nonrepeat sequence found in Brazil occurred in Asia. These observations suggest that P. vivax populations in Asia are ancestral to Brazilian populations, and that PvMsp-1 has heterogeneity in frequency of allelic recombination events. Recurrent origins of new PvMsp-1 alleles by repeated recombination events were supported by a rapid decline in linkage disequilibrium between pairs of synonymous sites with increasing nucleotide distance, with little linkage disequilibrium at a distance of over 3 kb in a P. vivax population from Thailand, evidence for an effectively high recombination rate of the parasite. Meanwhile, highly reduced nucleotide diversity was noted in a region encoding the 19-kDa C-terminal epidermal growth factor-like domain of merozoite surface protein-1, a vaccine candidate.     

不同疟区恶性疟原虫地理株谷氨酸富有蛋白基因R2区序列分析及分型

[目的 ]测定云南与海南两省不同疟区恶性疟原虫分离株谷氨酸富有蛋白 (GLURP)基因的部分序列及了解其分型。[方法 ]采用套式PCR方法特异性扩增GLURP基因R2区片段 ,并将该基因片段克隆于T载体 ,用双脱氧末端终止法测定不同长度的阳性克隆核苷酸序列 ,并应用DNAStar软件对云南与海南两省分离株GLURP基因及蛋白序列进行比较和分析。[结果 ]首次发现云南与海南两省恶性疟原虫至少存有 7个大小不同的GLURP等位基因型虫株 ,其基因片段变化范围为 6 0 0～ 15 0 0bp。不同分离株的GLURP基因R2区具高度保守性 ,其由编码 19～ 2 0个氨基酸的碱基的基本重复单位构成 ,该基因具有长度的多态性 ,表现在碱基基本重复单位的数目不同。序列分析结果表明 ,我国不同分离株之间或同一地区不同株之间GLURP基因及氨基酸序列具有高度的同源性 ,无明显的地理差异。 [结论 ]不同分离株GLURP基因结构的高度保守性及碱基重复片段数目的多态性 ,对研究疫苗候选抗原和建立疟原虫基因分型方法具有一定理论价值。     

Parasites bearing a single copy of the multi-drug resistance gene (pfmdr-1) with wild-type SNPs predominate amongst Plasmodium falciparum isolates from Malawi

We genotyped 160 P. falciparum infections from Malawi for pfmdr-1 copy number changes and SNPs associated with in vivo tolerance and poor in vitro sensitivity to the component drugs of Coartem. We also measured in vitro susceptibility of 49 of these isolates to a variety of drugs in clinical use or with a potential for use in Africa. All 160 infections carried a single copy of pfmdr-1 but 34% exhibited sequence variation at 4 of the 5 polymorphic sites in pfmdr-1. Isolates carrying 86-Asn and 184-Tyr pfmdr-1 alleles were significantly less sensitive (p < 0.001) to mefloquine, lumefantrine, artemether and dihydroartemisinin compared with those bearing 86-Tyr and 184-Phe polymorphisms. This study provides baseline measures prior to policy change: continued surveillance for changes in baseline drug susceptibility, pfmdr-1 copy number and SNPs, and other putative Coartem resistance loci will be necessary to provide an early warning of emerging Coartem resistance in this setting.     

Allelic dimorphism in the merozoite surface protein-3alpha in Korean isolates of Plasmodium vivax

To study the genetic diversity of re-emerging Plasmodium vivax in the Republic of Korea, nucleotide sequence variations at the merozoite surface protein-3alpha (PvMSP-3alpha) locus were analyzed using 24 re-emerging isolates and 4 isolates from imported cases. Compared with the well known Belem strain (Brazil), a large number of amino acid substitutions, deletions, and insertions were found at the locus of the isolates examined. The Korean isolates were divided into two allelic types; type I (15 isolates), similar to the Belem strain, and type II (9), similar to the Chess strain (New Guinea). Isolates from imported cases were classified into three types; type III (1 from Malaysia), similar to type B from western Thailand, type IV (1 each from Indonesia and India), and type V (1 from Pakistan), both being new types. Our results have shown that the MSP-3alpha locus of re-emerging Korean P. vivax is dimorphic with two allelic types coexisting in the endemic area.     

Plasmodium vivax dihydrofolate reductase point mutations from the Indian subcontinent

Mutations in Dihydrofolate Reductase (dhfr) gene of Plasmodium vivax are known to be associated with resistance to antifolate drugs. To analyze the extent of these mutations in P. vivax population in India, dhfr gene was isolated and sequenced for 121 P. vivax isolates originating from different geographical regions of Indian subcontinent. These sequences were compared with the gene sequence that represent wild type sequence (accession no. X98123). P. vivax dhfr (Pvdhfr) sequences showed limited polymorphism and about 70% isolates showed wild type dhfr sequence. A total of 36 mutations were found at 11 positions in 121 isolates. A majority of mutant isolates showed double mutations at residues 58 (S-->R) and 117 (S-->N), known to be associated with pyrimethamine resistance, but only 19% showed double mutations at residues 57 (F-->L) and 58 (S-->R). Pvdhfr alleles showing quadruple mutation (F57L, S58R, T61M and S117T) were found in two isolates. Three other mutations reported earlier at residue 13, 33 and 173 were not found in any of the Isolates. Six novel mutations at residues 38 (R-->G), 93 (S-->C), 109 (S-->H), 131 (R-->G), 159 (V-->A) and 188 (I-->V) were observed in seven isolates. Whether these novel mutations are linked to pyrimethamine resistance remains to be established.     

Markers for population genetic analysis of human plasmodia species, P. falciparum and P. vivax

Present report deals with the genetic diversity existing among the field isolates of Plasmodium falciparum and P. vivax in India. Isoenzymes and molecular markers were used to analyse field isolates of P. falciparum and P. vivax. High level of length polymorphism was observed in repeat nucleotide sequences of MSP-1, MSP-2 and GLURP in P. falciparum isolates and CSP, GAM-1 and MSP-3 alpha in P. vivax isolates. In study populations a high proportion of isolates (up to 60%) were comprised of more than one genetically distinct parasite type--multiclonal. Presence of identical allelic forms of enzyme and DNA variations in different geographical areas and in different years suggest that isolates belong to a single random mating population of P. vivax and P. falciparum. Observed random combination of alleles in the field isolates suggest the unlinked nature of loci studied. Study supports the feasibility of using molecular markers for the identification of recrudescence in P. falciparum from fresh infection.     

从Giemsa染色的血膜中扩增间日疟原虫DNA方法探讨

目的 建立从Giemsa染色的血膜中提取疟原虫DNA的方法。 方法 分别采用Na₂HPO₄法和Chelex-100离子交换法，经过反复优化，提取血膜中的DNA，进行套式PCR扩增鉴定PvMSP-1等位基因型。 结果 用Na₂HPO₄法和Chelex-100离子交换法分别提取40张不同类型的间日疟原虫阳性血膜DNA，未染色的厚血膜全部扩增出目的基因条带，而染色的薄血膜未能扩增出目的基因条带。两种方法均可检测红细胞间日疟原虫感染率≥0.01％的血涂片。 结论 从多年保存的标准血膜中提取疟原虫DNA进行基因型鉴别是可行的。