Analysis Of Gene Sequence Variation Of Japanese Encephalitis Virus Under Host Selection Pressure

Japanese encephalitis virus(JEV)is a member of the family Flaviviridae.It is a single-stranded positive-strand RNA virus of 10976 nucleotides in length,consisting of a 5 ’and 3’ untranslated region and a single open reading frame(ORF)in the middle.The precursor of polyprotein was first encoded by the ORF and then digested by the enzyme to forming ten different proteins,including three structural proteins,such as protein C,protein PrM and protein E as well as seven non-structural proteins,such as protein NSl,protein NS2 A,protein NS2 B,protein NS3,protein NS4 A,protein NS4 B and protein NS5.Viral replication can only be carried out in host cells.Therefore,the selection pressure of the host certainly influences the gene mutation of JEV.Due to the error-prone nature of the RNA-dependent RNA polymerase,which lacks an exonuclease-proofreading activity,the viral genome is prone to mutations.Mutant strains have different survival adaptability under host selection pressure.The higher the relative fitness of the mutant strains,the stronger the ability of growth and replication in the host environment,and the easier to obtain the growth advantage and become the dominant strain.In-depth study of the JEV genetic variation and evolutionary rules under the host selection pressure can provide a theoretical basis for molecular epidemiological surveillance and epidemic prevention and control.Meanwhile,it is required for future vaccine research and development.In this study,JEV BJ-1 strain and SA14 strain were used as experimental objects.In order to simulate the natural process of JEV transmission between mosquito vectors and vertebrate hosts,we passaged the JEV strains alternately between mosquito C6/36 cells and mammalian BHK cells as in vitro dual-host experimental model.Meanwhile,we passaged the JEV strains alternately between vector mosquito(Cx.)and mammalian(Kunming neonatal rats)as in vivo dual-host experimental model.Besides that,in order to simulate the natural process of JEV sustained infection in a single storage host,the BJ-1 strain and SA14 strain were passaged continuously in BHK and C6/36 cells,respectively.We monitored the changes in the JEV genome sequence in order to identify the mutation hot spots and understand the genetic evolution of JEV under host selection pressure.Then,by comparing our results with the published GenBank sequences,we explored the molecular epidemiological characteristics and evolution rules of JEV in nature and evaluated the experimental models both in vivo and in vitro.The main results are as follows:1.Typical Cell pathological effects(CPE)appeared about 3 days after virus inoculation both in C6/36 cells and BHK cells.2.The neonatal rats showed symptoms of scattered nest,convulsions and arched back at days 3–5 post infection with both BJ-1 and SA14 passage strains,which lasted for about 6 hours before moribund status.The natural course show no significance different(P>0.05).3.The average value of viral titer of each series of BJ-1 passage strains was all higher than the parental strain.The series of SA14 passage strains were different from each other.The average value of viral titer of continuous BHK passage strains has no significant difference compared with the parental strain.The average value of viral titer of continuous C6/36 passage strains was lower than the parental strain.The average value of viral titer of alternate passage strains both in vivo and in vitro was higher than the parental strain.4.The average value of viral titer of BJ-1 passage strains has no significant difference among four series.The average value of viral titer of SA14 passage strains has significant difference among four series.The series from high to low was alternate passage strains in vitro,alternate passage strains in vivo,continuous BHK passage strains and continuous C6/36 passage strains.5.The number of the nucleotide variation sites of each series of BJ-1 passage strains was all more than 100.The number of the nucleotide variation sites of alternate passage strains in vivo and continuous BHK passage strains was also more than 100.However,the number of the nucleotide variation sites of alternate passage strains in vitro and continuous C6/36 passage strains was less than 40.6.The ratios of the reverse nucleotide mutation occurring in both BJ-1 and SA14 passage strains were high(94.7% and 91.8%,respectively).More specifically,the ratios of the reversible nucleotide mutation occurring in BJ-1 alternate passage strains in vivo and continuous C6/36 passage strains were higher than others.Meanwhile,the ratio of the reversible nucleotide mutation occurring in SA14 continuous C6/36 passage strains was higher than others.7.The most common types of nucleotide mutations were T→C in BJ-1 passage strains and C→T in SA14 passage strains.8.The total number of the mutation sites of amino acids occurring in both BJ-1 and SA14 passage strains was 32.The ratios of the reverse mutation were high(87.5% and 59.4%,respectively).Moreover,the number of the mutation sites occurring in alternate passage in vivo was more than others.The number of the mutation sites occurring in protein E was higher than the other proteins.9.The amino acid sites closely related to virulence and antigenicity of both BJ-1 and SA14 passage strains were fairly stable.10.The total number of the reverse mutation sites of amino acids in BJ-1 alternate passage strains in vivo was higher than others.Meanwhile,the total number of the reverse mutation sites of amino acids in SA14 continuous BHK passage strains was higher than others.11.The total synonymous mutation rate of BJ-1 passage strains was 85.6% and the dN/dS was 0.17.The total synonymous mutation rate of SA14 passage strains was 77.3% and the dN/dS was 0.29.The selection pressure acts on BJ-1 passage strains was stronger than that acts on SA14 passage strains.12.In the analysis of similarity and clustering,the similarities of eleven BJ-1 passage strains(BP28,CP24,BCP8,BCP12,BCP16,BCP20,BCP24,BCP28,M10R10,M12R12 and M14R14)and the SA14 parental strain were relatively high,while the similarities of seven SA14 passage strains(BP12,M4R4,M6R6,M8R8,M10R10,M12R12 and M14R14)and the BJ-1 parental strain were relatively high.Conclusions:1.The alternating passage in vivo experimental model and single cell continuous passage experimental model can comprehensively simulate JEV alternating propagation process and persistent infection state in nature,which are useful for laboratory studies on the evolution of JEV and other arboviruses,such as the Zika virus.2.There were significant differences in the nucleotide sequence variation,amino acid variation and survival fitness changes between in vivo experimental model and alternate passage in vitro experimental model.This suggests that alternate passage in vitro experimental model is not suitable for the pathogenic biology of JEV.3.The survival fitness of alternate passage strains both in vitro and in vivo were higher than that of continuous cell passage strains,suggesting that the "trade-off hypothesis" is not suitable for JEV.Considering that the virulence of JEV is stronger than that of most other arboviruses,this evolutionary feature is conducive to JEV persistent infection.4.There were significant changes in the survival fitness among BJ-1 and SA14 passage strains.It was speculated that the variations of some amino acid sites were related to the changes of survival fitness.For example,the mutations of E protein(E49K)and NS5 protein(R29K)may have the relationship with the survival fitness decreasing and the mutation of the NS1 protein(E127D)may have the relationship with the survival fitness increasing.5.There was a relatively high mutation rate in the nucleotides over the entire coding genome of BJ-1 and SA14 passage strains,and most of the mutations were single synonymous nucleotide substitutions without amino acid changes.Neither insertions nor deletions were observed.The JEV mutation site in the different experimental models had high degree of coincidence.6.The viral regions involved in viral infectivity and immunogenicity were well conserved,indicating the virulence and antigenicity having no significant changes and that live attenuated vaccine SA14-14-2 having protective effect against passage strains.7.The dN/dS strains of BJ-1 and SA14 passage strains were both lower than 0.6,suggesting that the JEV genome shows a slow evolutionary rate under a conservative selection pressure.8.The similarities between part of BJ-1 passage strains and SA14 parental strain were improved while the similarities between part of SA14 strains and BJ-1 parental strain were improved.It suggested that the nucleotide sites which are prone to variation in JEV nucleotide sequences are relatively fixed,and the nucleotide variation types are relatively fixed.