Genetic Diversity And Population Genetics Of Pvs48/45 And Pvs47 Along The China-myanmar Border

Objective: Plasmodium vivax has become one of the important obstacles to eliminate malaria.On the one hand,a latent hypnozoite stage which can cause malaria relapses contributes to continuous P.vivax transmission.On the other hand,P.vivax produces infective gametocytes before the onset of the clinical symptoms,allowing the transmission of the parasites to mosquitoes prior to the anti-malaria treatments implementation.Globally,almost more than 50% of the burden of P.vivax infection occurs in Southeast Asia,especially in the Greater Mekong Subregion(GMS).The China-Myanmar border is one of the areas with the highest burden of P.vivax malaria in the GMS and is also an important source of imported malaria in China.Since 2014,decline rates of malaria in the GMS have stalled even reversed.In this case,goals against P.vivax began to focus on developing transmission blocking vaccines(TBVs).The polymorphism of TBVs candidate antigens is very limited when comparing with pre-erythrocyte and erythrocytic malaria vaccines candidates.Gametocyte is the only stage for Plasmodium to infect Anopheles from a human host.Therefore,the surface proteins of gametocyte are important candidates for TBVs.P48/45 and P47 are the gametocyte surface proteins that have attracted much attention in recent years.P48/45 plays an indispensable role in fertilization of male gametes.P47 not only plays an important role in optimizing fertilization of female gametes but also help P.falciparum to escape the complement immune system of the local mosquito vector.Based on the determination of the antigen function,studying the characteristics of genetic diversity is one of the important contents to explore whether the antigen can be applied for vaccine.However,in recent years,there have few studies on Pvs48/45 polymorphism in the China-Myanmar border,and Pvs47 have never been studied in this region.Therefore,this article studied the genetic diversity and population genetics of Pvs48/45 and Pvs47 in clinical samples along the China-Myanmar border to explore whether they can be valuable candidates for TBVs in this region.Methods: Using TIANamp Blood Spots DNA Kit to extract genomic DNA(gDNA)of Plasmodium vivax from the endemic area along the China-Myanmar border.Nest-PCR was used to amplify the open reading frame(ORF)of Pvs48/45 and Pvs47 and the final sequences were obtained by sequencing.MEGA 7.0 and DnaSP v5.10 were used to analyze the genetic diversity and natural selection characteristics of Pvs48/45 and Pvs47.The linkage disequilibrium(LD)and recombination of Pvs48/45 and Pvs47 were detected by DnaSP v5.10 and the geographic differences between the China-Myanmar border and other populations previously reported were assessed through DnaSP v5.10 and STRUCTURE v2.3.4.In the last,using PHYLOVIZ 2.0 explore the global haplotype distributions of Pvs48/45 and Pvs47.Results: 1.Compared with the reference sequence,9 mutations were detected in the Pvs48/45 sequences,of which 7 were non-synonymous,namely E35 K,H211N,K250 N,T273S,D335 Y,A376T,K418R;2 are synonymous,respectively K89 K and F352 F.A total of 16 mutations were found in the Pvs47 sequences,including 14 non-synonymous(F6V,F22 L,F24L,K27 E,S57T,S62 N,L82V,V230 I,M233I,F237 I,I262K,I262 T,I273V,A373V)and 2 synonymous(T26T and F356F).2.9 mutations in Pvs48/45 resulted in 9 haplotypes with a haplotype diversity of 0.796.The level of nucleotide polymorphism in Pvs48/45 was extremely low,only 0.00094,of which the highest in the CRD? domain was 0.00265.A total of 24 haplotypes were found in the Pvs47 along the China-Myanmar border,with a haplotype diversity of 0.875.The nucleotide polymorphism of Pvs47 was 0.00183,and the CRDIII domain also showed higher than the other two(? =0.00179).3.Multiple neutral tests were not statistically significant in the Pvs48/45.Except for the CRD? domain,the results of intra-specific neutrality tests were almost greater than zero,especially the CRD? domain reached a higher Tajima' D value.The MK test showed that the neutrality index(NI)was greater than zero,indicating the accumulation of synonymous substitutions between species.The Tajima' D of Pvs47 ORF was 0.071,and the Fu and Li's D * and F * values were-0.413 and-0.286,respectively.The neutral results were all negative in the CRD?domain,but almost positive in the CRD?and CRD?.The MK test showed significantly positive NI values in the ORF and CRD?of Pvs47(P<0.01 and P<0.05,respectively).Z-test showed ORF and CRD?of Pvs47 experienced significant positive selection.4.Analysis of the ORF of Pvs48/45 and Pvs47 from the China-Myanmar border provided estimates of the minimum number of recombination events of two and three,while values of the recombination parameter C between adjacent sites and per gene were 1.718,2275,and 0.046,60.1,respectively.The linkage disequilibrium indexes Zns and Za were not statistically significant.5.Population differentiation showed that the global pair genetic differentiation(Fst)of Pvs48/45 was 0.00-0.83,and Pvs47 ranged from 0.07 to 0.98.The population structure shows that Pvs48/45 formed four clusters,while Pvs47 formed three clusters.6.The global haplotype network diagram showed that Pvs48/45 had 23 haplotypes in the world,among which H5 was the dominant haplotype.Pvs47 had a total of 42 haplotypes and H21 with the highest frequency.Both Pvs48/45 and Pvs47 haplotypes showed significant continental aggregation.Conclusion: 1.Pvs48/45 and Pvs47 in the China-Myanmar border had limited genetic diversity,and the CRD? of Pvs48/45 and CRD? and CRD? of Pvs47 have undergone positive selection,indicating that they can be candidates for TBVs.2.The recombination level of Pvs48/45 and Pvs47 gene was very low,and there was no significant linkage disequilibrium,which may be the main reason for the low level of gene diversity.3.Pvs48/45 and Pvs47 had strong population differentiation,and the distribution of haplotypes also had geographical clustering and diversity,suggesting that TBVs should be developed based on the characteristics of major haplotypes on each continent.