Study On The Polymorphism Of Plasmodium Vivax Merozoite Surface Protein 1 Gene

Aim To study the polymorphism of Plctsmodium vivax merozoite surface protein 1 gene in different Chinese malaria endemic area , develop a method for detection the genotype of Plasmodium vivax merozoite surface protein 1 (PvMSP-1) alleles, explore the geographical distribution of PvMSP-1 alleles , determinate the relationship between different genotypes and epidemiological characteristics , evaluate the significance of PvMSP-1 gene as genetic marker in malaria molecular epidemiological investigation . Method Blood samples and individual epidemicological information of malaria cases were collected from malaria endemic area in Hainan, Yunnan and Anhui province. P. vivax genomic DNA was isolated from filter blood sample and nested PCR was carried out to amplify the polymorphic region encompassing the inter-species conserved blocks(lCB) five and six of PvMSP-1 gene, which contains Q repeats and PvuII restriction site (Sal-1 type). The PCR products from 40 isolates were sequenced and aligned with previous published sequences include Belem and Salvador-1 sequences. According to the PvMSP-1 sequence characteristic, PCR product was digested by PvuII restriction endonuclease and the digested fragments were observed after 2% agarose gel electrophoresis. The allelic type was determined according to the banding pattern. Another important genetic marker --Circumsporozoite protein(CSP) was used to compare with MSP-1 gene for analysis of genetic diversity. The morphology of parasite in different genotype was observed. Result In 82 field isolates, two kinds of DNA fragment of 470bp and 400bp were produced respectively in 50 and 39 isolates, with 7 mixture infection. The mix-infection rate was 20%(6/30) in Hainan and 2.3% in Anhui isolates. The mean clones of multi-infection was 1.20 (36/30) and 1.02 (43/42) respectively in Hainan and Anhui. 33 sequenced fragments revealed 17 for Sal-l-like and2 for Belem-like , 12 recombination III type . 2 Korean- type were newly identified in China. Two size bands of 400 bp (Belem-like ) or/and 470 bp (Sal-1-like ) appeared in all 98 P.vivax isolates, no band was found in negative control. After PvuII endonuclease digestion, 45 samples of 470 bp were detected two fragments ( 120 bp and 350 bp.) -Sal-1 type. Single-band of 400 bp appeared in 3 of 40 samples with 400 bp-Belem type, other 35 samples appeared two bands of 120 bp and 280 bp-recombination III type, 2 with 120 bp and 240 bp-South korea type. 7 Sal-1 type, 1 Belem type and 8 recombination type were identified by nested PCR-RFLP in all 16 cases. 7 of the PCR products were sequenced and no 100% nucleotide similaiity was found in any of two sequence. The genetic diversity existed in different isolates of Plasmodium vivax. The infectious source might be determined both by the information of sequence and epidemiology. The two important markers of PvMSP-1 and CSP were used to evaluate parasite genetic diversity in field isolate in Hainan hyperendemic malaria area. Our study showed tha the parasite disply a high level of genetic complexity. The genotype multiple infection was 18.7% in MSP-1 and 35.3% in CSP, the mean clones were 1.16 in MSP-1 and 1.47 in CSP. Combined the two markers, the multiple infection was 50% in those isolates. Comparisons of MSP-land CSP genotypes, the correspondence analysis suggested that the genotype of Sal-1 and tropical zone family existed close relation, PvII and recombination III have certain relation, no relation was found in other genotypes. We observed parasite morphology by microscopy in 72 blood films. The typical Plasmodium vivax were found in all 72 films, most were trophozoites. There are no significance of morphologica difference in two genotype of Belem and Sal-1 strain. We calculate the size of ring form and found that the mean size of ring form of Belem strain was bigger than that of Sal-1 strain. The RBC infected more than two parasite was observed in Hainan isolates , 22.2% in Belem strain and 11.1% in Sal-1 strain, no RBC infected with morethan two parasite was found in Anhui isolates. Conclu...