Genetic Diversity Of A Novel Plasmodium Vivax Erythrocyte Binding Protein(PvEBP)

Objective: Clinical isolates of Plasmodium vivax from eight endemic areas were analyzed to clarify the genetic diversity,natural selection characteristics and population genetic differences of the novel Plasmodium vivax erythrocyte binding protein(PvEBP),and to understand the epidemic characteristics of PvEBP gene,providing basic information for PvEBP as a vaccine candidate antigen.Methods: Asking for the informed consent of patients with Plasmodium vivax in endemic areas,blood samples of the patient’s filter paper were obtained.The genomic DNA of Plasmodium vivax patients from China(Anhui)(n=31),China-Myanmar border(n=44),Thailand(n=38),India(n=28)and Ethiopia(n=13)were extracted,amplified by PCR and sequenced.The sequences of 50 samples in Myanmar,Vietnam and Papua New Guinea were obtained from GenBank.Using MEGA 7.0,DnaSP 6.12.03 and ARLEQUIN v3.5.2.2 to analyze PvEBP gene polymorphism,selection pressure and genetic differentiation.PopART 1.7 was used to construct haplotype network maps in different regions;STRUCTURE v2.3.4 software and principal component discriminant analysis were aimed to characterize population genetic structure differences;online website IEDB was ordered to predict linear B cell epitopes.Results: 1.The degree of nucleotide polymorphism and haplotype diversity of PvEBP were lower in China(Anhui),and relatively higher in Ethiopia.The mutations were concentrated in PvEBP-RII,which region corresponded to higher polymorphism,and D311N site mutations existed in isolates from all regions.2.PvEBP was affected by natural selection,in which PvEBP-RII had experienced positive selection,and PvEBPRIII-V might have undergoing purification selection.3.The Rm of PvEBP gene of all isolates in the epidemic areas was only five,the degree of linkage disequilibrium was little,and the correlation between loci was weak.4.The degree of genetic differentiation of PvEBP genes among different populations was relatively higher.China(Anhui)and other regions were quite different,and gene exchanges were not frequent.5.The phylogenetic tree showed that there was no obvious geographic clustering of PvEBP gene,Hap＿9 was the haplotype with the highest frequency,Hap＿4 was a haplotype shared by other regions except Myanmar and had regional adaptability.6.The eight populations were all composed of four genetic components,but there were obvious differences in genetic structure.There was some genetic crossover in geographically close populations.7.A total of eight B cell epitopes were predicted,with distinct polymorphism and dN > dS.Conclusions: 1.The genetic polymorphism characteristics,degree of genetic differentiation and population genetic structure of PvEBP isolates in various epidemic areas were quite different due to geographical isolation.2.PvEBP-RII presented diversified positive selection,which could be used as an promising target for the candidate vaccine of Plasmodium vivax in the red stage.