Genetic Diversity And Population Structure Of Plasmodium Vivax In The Greater Mekong Subregion With Duffy-binding Protein Gene And SNP Barcode

Objective:Malaria is a major public health problem in the Greater Mekong subregion of Asia.In recent years,malaria control has been strengthened,resulting in substantial improvements in malaria in most countries.But there are still some areas in the region such as Myanmar,the epidemic cannot be ignored,and China's Yunnan Province belongs to the Greater Mekong Subregion,imported malaria is inevitable.A notable change in the malaria epidemic is that most endemic areas in the Greater Mekong are dominated by Plasmodium vivax.because P.vivax is of relatively little concern,although its global distribution is much larger than that of Plasmodium falciparum,and because of the long-term incidence of recurrent infections,it has a striking impact on the economy and public health.Plasmodium krypton proteins involved in erythrocyte invasion are selected by host immunity and their diversity is strongly influenced by malaria epidemiological changes.In the Greater Mekong Subregion,malaria transmission is concentrated on international borders,and malaria epidemiology has changed significantly,with Plasmodium vivax becoming a dominant species in many regions.herein,we investigated the genetic diversity of the interhemiparietal plasmodium duffy-binding protein domain II?pvdbp-II?along the eastern and western border isolates of myanmar and compared it with the global interhemitral plasmodium population.the pvdbp-II sequences were obtained from the clinical isolates of 85 and 82 P.vivax isolates isolated from eastern and western myanmar,respectively.in addition,504 pvdbp-II sequences of the world's nine inter-rimalarial populations were retrieved from genbank and their genetic diversity,recombination,and population structure were compared.Furthermore,we used 42 SNP bar codes to conduct genetic polymorphisms and population structure analyses among the Greater Mekong Subregion,including in central and western China?Anhui and Yingjiang?,in the north-eastern and western regions of Myanmar?Laiza,Meomauk,Paletwa,Kyauktaw,Thanbyuzayat and Kawthaung?,and in the western part of Thailand?Tak?,to determine the genetic characteristics of insect strains in the endemic areas.Methods:1.A total of 167 PvDBP-II sequences were obtained by PCR,cloning and sequencing on the border between China and Myanmar and Western Myanmar.The genebank database was used to obtain a total of 504 pvdbp-II sequences from nine other regions of the world.software such as MEGA7.0?DnaSP 5.10.01?NETWORK v4.6.1.3?STURCTURE v2.3.4 were used for genetic diversity,natural selection,recombination,linkage imbalance analysis and genetic differentiation,haplotype network and population genetic structure analysis of pvdbp-II genes,and the essentially unstructured/disordered regions?IURS?were evaluated through the RONN server for polymorphism analysis associated with B and T cell epitopes.2.Collection of blood samples from fingertips of patients with P.vivax malaria infection from 2011 to 2018,multiple genotyping of 42SNP barcodes using MassARRAY technique,analysis of genetic characteristics of population response such as GeneAlEx,Haploview 4.1,STRUCTURE v2.3.4,AREQUINv3.5,MEGA 7.0 and online program ClustVis in the GMS region of complex infection,genetic diversity,linkage imbalance,molecular analysis of variance,and Mantal test,as well as analysis of main components,genetic differentiation,population structure and so on.Result:1.The PvDBP-II R391C mutation is unique to isolated strains in Western Myanmar.2.The highest peak of PvDBP-II nucleotide diversity was identified between the China-Myanmar border and the P.vivax nucleotide position in western Myanmar;the degree of polymorphism at the amino acid level was enhanced;the results showed that the central region of PvDBP-II was more polymorphic than other regions.3.The positive selection of PvDBP-II gene for the Myanmar and Myanmar border isolates?P<0.05?was analyzed by Fu and Li's F*tests and Tajima'D analysis,indicating that the P.vivax population in the region was under pressure positive selection.4.LD analysis showed that R² values decreased more rapidly in the P.vivax population in western Myanmar,indicating a larger number of parasites and higher levels of intra-genomic recombination in western Myanmar.5.The P.vivax population along the border with western Myanmar is poorly differentiated,with a higher degree of genetic differentiation in Colombia(F_ST=0.232753 and 0.38761,respectively),and with the P.vivax population in the rest of the world(F_ST:0.02760 to 0.21909).6.Population structure and cluster analysis revealed that the P.vivax population in the GMS region was divided into three sub-categories and showed a high degree of gene mixing;a total of 60 unique haplotypes were identified in the PvDBP-II sequence of the 210 GMS-derived Plasmodium vivax,65%of which were isolated;and there were shared haplotypes in the GMS epidemic regions,indicating the existence of gene exchange.7.All predicted B-cell and T-cell epitopes and MHC binding regions were polymorphic and Tajima'D values were positive,indicating a decrease in population size and balance selection,possibly related to the diversity of PvDBP-II.8.SNP barcode analysis showed that 158 strains?53.7%?of Plasmodium vivax in GMS region contained multiple infections and there were significant differences among epidemic ranges,indicating that the load of P.vivax in GMS regions varied.9.Of the 42 SNPs on the 14 chromosomes of the Plasmodium vivax genome,the AMAF values of 37 SNPs were higher than 0.1?Table 2.4?,indicating a higher diversity of the Plasmodium vivax population in the GMS region.10.The results of both SHDI and HE observations showed that there was no significant difference in genetic diversity among the different regions of GMS.11.Whether based on the IAM or SMM model,the highest number of Ne was found in Southern Myanmar,followed by Western Myanmar,North-Eastern Myanmar and Western Thailand?table 2.5?,with little difference in the overall effective population.12.LD analysis showed that most of the SNP combinations in the P.vivax population in the town of Meomauk in North-Eastern Myanmar were less than 0.3 except for R²;regardless of the size of the population,the LD coefficient R²decreased slowly with increasing genetic distance.After several comparative corrections,there was no significant difference between the R² value and Beijing LD level.13.STRUCTURE showed that the eight population groups were mainly clustered into two subpopulations;among them,the population of P.vivax in the town of Laiza in North-Eastern Myanmar was dominated by green subpopulations,while the two subpopulations in the town of Meomauk were more evenly distributed,while the other epidemic areas were dominated by red subpopulations.The results indicated that the main haplotypes in the epidemic area of North-Eastern Myanmar were different from the other epidemic areas,while the other epidemic areas had similar P.vivax haplotype genetic patterns.14.Molecular variation?AMOVA?analysis showed that 13 percent of the genetic variation of P.vivax in the GMS range came from between populations,whereas 87 percent of the genetic variation was attributed to within populations;in the GMS range,some regions showed moderate genetic differentiation?Fst:0.06-0.07?,while some regions showed very low genetic differentiation?Fst 0.02-0.03?;in combination with the Molecular Variation Model AVA analysis and Fst results,it was shown that the genetic differentiation of the eight geographical populations of P.vivax in the GMS region mainly came from within populations.the results of Fst analysis of the genetic differentiation of the global plasmodium P.vivax showed that the differentiation between GMS and inter-malarial populations in South America,Africa and South Asia was higher?Fst:0.40–0.52?,indicating that the SNPs barcode-labeled plasmodium had varying degrees of genetic differentiation among populations worldwide.15.PCA analysis showed that the screening 38 SNPs barcode-labeled P.vivax had distinct clusters among GMS groups and could not distinguish between P.vivax whereas in the global snps marker data,the GMS clustered in a cluster with its geographical location as the center,only a few of them crossed among the other regions of the inter-rimalarial population16,phylogenetic tree analysis found consistent with the PCA results,the P.vivax population in the GMS region was clearly clustered and closely related;the mriplasma parasites in South America were earlier in a single cluster,showing a distant kinship to the other populations;although Africa and South America had one branch at the end of each other branches,the two showed a closer relationship with the GMS in South America.Conclusion:1.The PvDBP-II gene shows genetic diversity in the P.vivax population on the border between China and Myanmar and in Western Myanmar,comparable to the global population.2.The presence of positive natural selection and recombination of the PvDBP-II gene,as well as polymorphisms in T,B cell epitopes and MHC binding regions,suggested that P.vivax in the China-Myanmar Border and Western Myanmar might be in the choice of host immune stress.3.The PvDBP-II gene showed slight genetic differentiation on the China-Myanmar B order and in western Myanmar,indicating that P.vivax had gene flow in both regions.4.The SNPs barcode-labeled GMS regions exhibited high intra-population diversity in the P.vivax population.5.The SNPs barcode could not distinguish the P.vivax population in the GMS region,but it could distinguish the plasmodium population in the subtropical and tropical regions.6.A GMS regional P.vivax genotype database based on SNPs barcode was established to provide a theoretical basis for further molecular epidemiological studies of Plasmodium vivax.