| Plasmodium vivax(P.vivax)is the most widely distributed malaria parasite species in the world,and also the main species in malaria endemic areas of China.Due to the unique biological characteristics of P.vivax,the control and elimination of P.vivax is more challenged than that of P.falciparum.In most epidemic areas with mixed species,the proportion of vivax malaria cases is found to be increasing while malaria is effectively control with decreasing incidence.Vivax malaria is still endemic years or even decades after the effective control or elimination of falciparum malaria in some countries.At present,China has entered the malaria elimination phase and most of malaria endemic areas have no indigenous malaria cases reported except for the border areas in Yunnan province,however,imported malaria from other countries has reached more than 3000 cases each year,which vivax cases accounted for about 20%-25%.One of the World Health Organization(WHO)standard for malaria elimination assessment is "No local mosquito-borne malaria cases for three consecutive three years".As mosquito vectors of the main original malaria endemic areas in China are still able to transmit P.vivax,when a new malaria cases is reported it is necessary to determine whether the case is local or imported and to assess the transmission risk locally.In China's Malaria Elimination Action Plan,the identification of imported malaria cases is currently dependent on the case epidemiological investigation,and all foci with imported vivax cases and mosquito vectors are considered to have a risk of transmission and need to take foci treatment including vector control measures.Therefore,it is of great importance for malaria elimination and preventing malaria re-establishment to explore the molecular tools for tracking the geographic origin of P.vivax as well as the adaptability(susceptibility)between imported P.vivax isolates and the local mosquito vectors,and further to assess the transmission risk.In this study,we used microsatellite markers to analyze the genetic diversity and population genetics of P.vivax isolates collected from different regions of China,and compare the genetic data of Plasmodium vivax isolates between China and other countries with the aim of exploring molecular tools for determining the origin of P.vivax infection.On the other hand,we analyzed the Pvs47 genetic diversity and population genetics of P.vivax isolates from different regions of China and compared them with the isolates from East Asia,Southeast Asia and South America with the aim of providing scientific evidence for the risk assessment of the local transmission by imported vivax cases.Part I Genetic Diversity and Population Genetics of Plasmodium vivax Using Microsatellite Markersi Genetic Diversity and Population Structure of Plasmodium vivax in the Central and Southern ChinaObjective:To analyze the genetic diversity and population structure of P.vivax isolates in the Central and Southern China,and to provide evidence for the establishment of molecular tool for determining the origin of P.vivax infection.Methods:The samples were collected from the P.vivax endemic areas in Central and Southern China from 2007 to 2012(n=236),and were genotyped by 9 microsatellite markers and detected by capillary electrophoresis.The genotype data was processed using Asia Pacific Malaria Elimination Network platform for Plasmodium vivax genotyping(VivaxGEN).The multiplicity of infection,population genetic diversity,level of linkage disequilibrium and population structure were analyzed.Results:1.The polyclonal infection rate of Southern China P.vivax isolates(70.4%,38/54)was higher than that in Central China(51.4%,93/181)and the difference was statistically significant(P<0.01).The expected heterozygosity(HE)of Southern China P.vivax isolates(HE=0.85 ± 0.07)was higher than that in Central China(HE=0.73:±0.13),and the difference was statistically significant(P<0.01).The allele richness(RS)of Southern China P.vivax isolates(Rs=11.89)was higher than that in Central China(Rs=8.17)and the difference was statistically significant(P<0.01).The average number of alleles and unique alleles of all loci in the Southern China were higher than those in Central China.2.A significant linkage disequilibrium were found both in Central China(IAS =0.147,P<0.01)and Southern China(IAS=0.136,P<0.01).A significant linkage disequilibrium was also found between different years in Central China and there was an increasing trend from 2008(IAS=0.137,P<0.01)to 2009(IAS=0.190,P<0.01)to 2010(IAS=0.264,P<0.01).3.STRUCTURE analysis demonstrated the clear sub-structure in the P.vivax populations of Central and Southern China.The delta K method demonstrated ^ K 4 as having the greatest likelihood.The genetic structure of the P.vz_vax population in Central China was complex while it was relatively simple in Southern China.At K = 2,92.59%(50/54)isolates of Southern China demonstrated predominant ancestry to pop2,1.85%(1/54)to popl and 5.56%(3/54)were mixed sources.43.09%(78/181)isolates of Central China demonstrated predominant ancestry to pop1,34.81%(63/181)to pop2,and 22.1%(40/181 were mixed sources.4.Population differentiation was observed between Central and Southern China(FsT=0.0696,P=0.00007).In Central China,the largest population differentiation was found between 2008 and 2010(FST=0.0389,P=0.012)with smaller differentiation between 2008 and 2009(FST=-0.0001,P=0.422)as well as between 2009 and 2010(FST=0.034,P=0.034).5.Both phylogenetic tree and principal component analysis(PCA)showed that there was a cross in the genetic status for the P.vivax isolates in Central and Southern China.Mantel,s test showed that the genetic distance was correlated both with geographical distance(r=0.08,P=0.036)and the time sample collection(r=0.05,P=0.01).Conclusions:1.The genetic diversity of P.vivax isolates in Southern China was significantly higher than that in Central China both at individual level(polyclonal infection)and population level(expected heterozygosity)which is more consistent with the higher prevalence of P.vivax malaria in the Southern China compared to Central China.2.The significant linkage disequilibrium and the lower LD in Southern China also indicated that the P.vivax transmission intensity in Southern China was higher than that in Central China.3.The genetic structure of the P.vivax population in Central China was complex while it was relatively simple in Southern China.There was population differentiation between Central and Southern China.There was a cross in the genetic status for the P.vivax isolates in Central and Southern China.The genetic distance has a certain temporal and spatial correlation in P.vivax isolates of Central China.4.A genotype database of P.vivax in Central and Southern China based on microsatellite markers was established and included into APMEN Plasmodium vivax genotyping platform(VivaxGEN),which laid a foundation for further research on molecular tools to determine the origin of P.vivax infection.ii A Comparative Analysis of Genetic Diversity and Population Structure of Plasmodium vivax Gene between China and Other CountriesObjective:To compare the genetic diversity and population structure of P.vivax isolates from China and other countries and to explore the molecular tool for determining the origin of P.vivax infection.Methods:The genetic diversity and population structure were compared based on P.vivax isolates originated from Ethiopia,Iran,Bhutan,Malaysia,Indonesia,South Korea,Southern China and Central China through international cooperation.Data analysis was conducted using APMEN platform(VivaxGEN)for P.vivax genotype data sharing and comparative analyses.The origin of imported vivax malaria cases was analyzed using STRUCTURE and Weka software.Results:1.Polyclonal infection rate of P.vivax isolates was in a large range(4.2%-97.2%)with the highest rate in Iran(97.2%)and the lowest rate in South Korea(4.2%).higher in the central and southern parts of China,and the infection rate of Plasmodium vivax was 51.4%and 70.4%,respectively.The polyclonal infection rate of P.vivax isolates in Central and Southern China was 51.4%and 70.4%,respectively.2.A total of 78 unique alleles were found,among which the largest was Indonesia(25)and the lowest was Korea(0).7 unique alleles were found in Central China and 10 were found in Southern China.The short size allele(150bp)of locus MS20 were only found in samples of China and South Korea,and the allele frequency in South Korea,Central China and southern China was 98.8%,60.9%And 22.4%respectively.3.STRUCTURE analysis demonstrated that when K=2 the two genetic sub-populations were consistent with the geographical distribution of tropical and temperate P.vivax isolates.At K=4,the isolates of Africa demonstrated predominant ancestry to popl,the isolates of Iran demonstrated predominant ancestry to pop2,the isolates of Bhutan,Malaysia,Indonesia and Southern China demonstrated predominant ancestry to pop3,the isolates of Central China and South Kore China demonstrated predominant ancestry to pop4.At KF=6,the genetic sub-structure was also observed in the P.vivax population of central China and South Korea.Phylogenetic tree and principal component analysis showed that the genetic distance between Central China and South Korean was close,and the genetic distance between Central China and African as well as Southeast Asia was long.The genetic distance between Southern China and Southeast Asian countries was close.4.Based on STRUCTURE analysis,the average accuracy for assignment of 30 imported vivax malaria cases to their geographic region origin were 85%.The P.vivax isolates from Central China and Ethiopia can be divided into two genetic sub-populations(popl and pop2),and 98.20%of P.vivax isolates from Ethiopia demonstrated predominant ancestry to pop1,whereas 96.3%of P.vivax isolates from Central China demonstrated predominant ancestry to pop2.All the five vivax malaria cases with Ethiopia travel history can be assigned to pop1.The P.vivax isolates from Central China and Southeast Asia(Malaysia and Indonesia)can also be divided into two genetic sub-populations(pop1 and pop2),and 97.50%of P.vivax isolates from Southeast Asia demonstrated predominant ancestry to pop1 whereas 94.20%of P.vivax isolates from Central China demonstrated predominant ancestry to pop2.Among the 10 vivax malaria cases with Southeast Asian travel history,9 cases can be assigned to pop2 with the accuracy of 90%.5.Based on Weka analysis,the average accuracy for assignment using four microsatellite markers(MS8,MS 10,MS 16 and MS20)for 493 vivax malaria cases from Africa,Middle East,South/Southeast Asia and East Asia was 90.47%and for 172 vivax malaria cases from Central China and Ethiopia was 99.42%.The average accuracy for assignment using two microsatellite loci(MS8 and MS20)for 235 vivax malaria cases from Central China and Southeast Asia was 98.70%.Conclusions:1.The short size allele(150bp)of MS20 was found for the first time in P.vivax isolates of Central China,which can be used as a molecular marker for the identification of local and imported vivax malaria in Central China.2.A molecular tool based on microsatellite markers for determining the origin of P.vivax infection has been preliminarily established in this study.The accuracy reached 99.42%when four microsatellite markers(MS8,MS 10,MS 16,MS20)were used to assign P.vivax isolates from Central China and Africa.The accuracy reached 98.70%when two microsatellite markers(MS 8 and MS20)were used to assign P.vivax isolates from Central China and Southeast Asia.Part ? Genetic Diversity and Population Genetics of Plasmodium vivax Pvs47Objective:To study the genetic diversity and population genetics of Plasmodium vivax based on Pvs47 using isolates from different endemic regions,and to provide scientific evidence for the risk assessment of the local transmission caused by imported vivax malaria cases.Methods:Plasmodium vivax parasite isolates from Central and Southern China as well as those imported from Southeast Asian countries to China(n=212)were used to sequence the Pvs47 gene,and compared to the Pvs47 sequence previously reported to GenBank(n=76)and PlasmoDB(n=68).Results:1.Based on analysis of Pvs47 in 356 P.vivax isolates,34 single nucleotide polymorphisms(SNPs)were identified.The overall nucleotide diversity(?)was 0.0039.The highest nucleotide diversity was observed in Southeast Asia(?=0.00424)and the lowest nucleotide diversity was observed in East Asia(?=0.00029).The nucleotide diversity of P.vivax isolates from Central China(?=0.00091)was slightly higher than that of East Asia(?=0.00029),but significantly lower than that of Southern China(?=0.00253).2.The largest genetic differentiation was found between East Asia and South America(FST=0.92,GST=0.60)and the smallest genetic differentiation was found between Central China and East Asia(FST=0.06,GsT=0.04).The genetic differentiation between Southern China and Southeast Asia was small(FST=0.08,GST=0.03),but the genetic differentiation between the Central China and Southeast Asia was big(FST=0.37,GST=0.20).3.A total of 52 Pvs47 DNA haplotypes and 47 Pvs47 amino acid haplotypes were identified.Among the 47 haplotypes,the largest number was found in Southeast Asia(n=28)and the least number was found in East Asia(n=3).The numbers of haplotype in Central and southern China was 12 and 11,respectively.Unique amino acid haplotype(n=1-20)was found in each P.vivax population.There was 1 in Southeast Asia(Hap_aa40),1 in Southern China(Hap_aa8)and 2 in South America(Hap_aal and Hap_aa45)among the unique amino acid haplotype which frequency was greater than 1%.Hap_aa2 was the dominant amino acid haplotype both in Central China and East Asia which accounted for 80%(112/140)and 95.1%(39/41),respectively.In contrast,Hap_aa2 accounted for only 3.0%(2/67)in Southeast Asia,28.9%(13/45)in Southern China and was not found in South America.Hap_aal was the dominant haplotype in South America which accounted for 74.6%(47/63)and was not found in other regions.4.Phylogenetic analysis of the 47 Pvs47 amino acid haplotypes identified two main clades.One clade(cladel)included 10 haplotypes that were more frequent in Central China;whereas the second clade(clade2)included 23 haplotypes that were more frequent in Southeast Asia.Seven popular haplotypes in Southern China were found in both clades.One unique haplotype to East Asia was clustered with haplotypes of Central China.Four unique haplotypes to South America were located in the clade 2 with close genetic status.5.Network analysis demonstrated that the clustered distribution of Pvs47 DNA haplotypes relevant to the geographic origin.Haplotypes of Central China clustered with ones of East Asia,haplotypes of Southern China clustered with ones of Southeast Asia and haplotypes of South America clustered individually.Conclusions:1.The obvious difference in genetic diversity and population structre for P.vivax isolates from different regions were found for the first time.The distribution of Pvs47 haplotypes clustered relevant to the geographic origin.2.The Pvs47 genetic distance of P.vivax isolates between Southern China and Southeast Asia was close with haplotypes clustered into one clade,which suggested that imported P.vivax parasites have strong adaptability to local mosquito vectors and there was a higher local transmission risk caused by imported vivax cases from close distance.3.The Pvs47 genetic distance of P.vivax isolates between Central China and Southeast Asia was long with haplotypes specific to geographical origin,which suggested that imported P.vivax isolates have poor adaptability to local mosquito vectors and there was a lower local transmission risk caused by imported vivax cases from long distance importation.This might be one of the important molecular mechanisms that the imported vivax malaria cases from Southeast Asia and Africa have not yet caused local transmission in Central China. |
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