Genetic Polymorphism Of Circumsporozoite Protein Of Imported Plasmodium Falciparum In Henan Province

ObjectiveBy analyzing the genetic sequence characteristics of the imported plasmodium falciparum circumparzoite protein(PfCSP)in Henan,and comparing with the global PfCSP,the polymorphism pattern and distribution of PfCSP gene can be further understood,the causes and mechanisms of PfCSP gene polymorphism were preliminarily explored,and the differences of PfCSP gene in different geographical populations were clarified.To provide scientific basis for the prevention and control of imported falciparum malaria in the process of preventing malaria re-establishment after malaria elimination.To provide molecular basis for the design and improvement of PfCSP-based vaccine,so as to propose more accurate malaria cases responses.MethodsCases data and whole blood samples of imported falciparum malaria patients in Henan from 2016 to 2018 were collected.DNA of Pf was extracted by QIAamp DNA mini kit,and the complete fragment of CSP was amplified by PCR and DNA sequencing was performed.Molecular biology software such as Dna SP and MEGA were used to analyze polymorphism,neutral test and population differentiation of the sequencing results and the global PfCSP sequences downloaded from the NCBI Gen Bank.Web Logo online program was used to construct amino acid logo map.Popart software was used to construct haplotype network diagrams.The CSP sequence of 3D7 strain was compared as the standard sequence.Results1.There were 287 DNA samples of imported P.falciparum,with a positive rate of PCR amplification of 91.29%(262/287).After sequencing,201 samples were available,including 10 from East Africa,83 from West Africa,57 from South Africa,3from North Africa,and 48 from Central Africa.A total of 1156 full-length PFCSP sequences were obtained from the NCBI Gen Bank.2.At the amino acid level: there were 2 mutations at the N-terminal of imported PfCSP in Henan,the insertion of 19 amino acids(58.71%,118/201)and A98G(38.81%,78/201).The number of repeats of the tetrapeptide motif in the central repeating region was mainly 39 to 42(97.51%,196/201).A total of 14 non-synonymous amino acid changes were found at the C-terminal.There are 5 mutations at the N-terminal of the global PfCSP.The insertion frequency of 19 amino acids ranged from 98.95% to 100%in Asia,Oceania and America,and 48.11%(127/264)in Africa.The frequency of A98 G was higher in Asia and Oceania(90.91% to 100%),but lower in America(52.38%)and Africa(29.55%).The number of repeats of the tetrapeptide motif in Asia were mainly 43 to 48(96.61%,524/561),and in Oceania was mainly 41 to 44(93.66%,251/268),and in America was mainly 40 to 41(93.65%,59/63),and in Africa was mainly 39 to 43(94.32%,149/264).A total of 15 dimorphic or polymorphic amino acid changes were found at the C-terminal.L320 I and N325 Y were found only in Asia,S326 A was found only in America,and wild-type A361 was mainly found in Africa and Henan imported PfCSP.3.According to the analysis of C-terminal nucleotide polymorphism,neutral test and gene recombination,the average number of nucleotide differences(K=5.566),haplotype diversity(Hd > 0.9)and nucleotide diversity(π > 0.2)of PfCSP from Africa were higher than those from other regions,and the corresponding values of PfCSP from Henan were close to them.The values of global PfCSP(d N-d S)are all positive,indicating that they are experiencing positive selection,but this trend is not statistically significant(P > 0.05).Except for the Philippines,other neutral test results were not statistically significant(P > 0.05).The Tajima’s D value of global PfCSP deviates from0 to varying degrees,with the exception of Asia(D =-0.65174)which may experience negative selection,Oceania(D = 0.65860),America(D = 0.00900)and Africa(D =0.19441)may experience positive selection.The minimum number of recombination events(Rm)of imported PfCSP in Henan is close to that in Africa,higher than that in other regions.4.Population differentiation analysis showed that there was significant differentiation between the four continents(Fst > 0.15,P < 0.05),but there was a genetic relationship between Africa and America(Fst = 0.15929,P < 0.05)and between Oceania and Asia(Fst = 0.06705,P < 0.05).The imported PfCSP in Henan had no differentiation with Africa(Fst = 0.00101).Conclusions1.The N-terminal of PfCSP is relatively conserved,and the central repeating region and the Th2 R and Th3 R regions of C-terminal are highly polymorphic.Natural selection and gene recombination may be the main reasons for maintaining and producing the genetic polymorphism of global PfCSP population.2.The genetic polymorphisms of PfCSP populations in the world have obvious geographical differences.The genetic polymorphism pattern of imported PfCSP in Henan is consistent with that in Africa.3.The geographical pattern of population differentiation and the evidence of natural selection and gene recombination suggest that the effect of polymorphism on efficacy of PfCSP-based vaccines should be considered.