Dengue Virus Ban18HK20 Strain Virulence Site Study And Dengue Chimeric Virus Construction And Identification

[Objective]To construct infectious clones of the dengue virus type 4 Ban18HK20strain and establish a stable dengue virus?DENV?reverse genetics platform,which will be more conducive to explore the important virulence genes of dengue virus and construct its attenuated strains.To construct the dengue chimeric virus rDENV4/2 with Ban18HK20 as the backbone,and lay the foundation for the development of dengue vaccine.[Methods]First,with the specific primers of the viral genome for dengue type 4 virus Ban18HK20 strain,the six cDNA fragments were subcloned respectively,and then linked into the high-copy plasmid pSPTM.To overcome the bacterial toxicity of the viral genome by screening many competent cells and culture conditions,a stable viral full-length cDNA infectious clone pSPTM-DENV was obtained.RNA was transcribed in vitro from the full-length cDNA as a template and electrotransfected into Vero cells to rescue the virus.Secondly,the amino acid sites in the dengue virus Ban18HK20 were reverse-mutated by single-point or multi-point to the corresponding genes of its parental virus Ban18 strain by homologous recombination cloning technology,and the reverse mutation dengue virus were rescued.Finally,the prM-E gene of the infectious clone pSPTM-DENV was replaced with corresponding genes of DENV type 2 16681strain to construct the chimeric plasmid pSPTM-DENV4/2 by fusion PCR and homologous recombination.The chimeric plasmid pSPTM-DENV4/2 was then linearized with XhoI,and transcribed into RNA,and then transfected Vero cells to rescue the dengue chimeric virus rDENV4/2.Biological characteristics of the recovered dengue virus Ban18HK20,reverse mutant dengue virus and chimeric virus rDENV4/2 were studied by virus plaque assay,indirect immunofluorescence assay,growth kinetics assay,and mouse neurovirulence assay.The genetic stability of the recovered virus Ban18HK20 and chimeric virus rDENV4/2 was studied by gene sequencing.The immunogenicity and immunoprotective effect of dengue chimeric virus rDENV4/2 in mice were studied.At the same time,the safety of dengue chimeric virus rDENV4/2 and dengue virus type 4Ban18HK20 strain was studied by monkey model.[Results]The Ban18HK20 infectious clone pSPTM-DENV was constructed successfully.The first-generation recovery virus titer obtained by electroporation of Vero cells can reach 6.3 lg PFU/mL.The recovered virus was consistent with its parental Ban18HK20 strain in terms of plaque morphology.Viral E protein expression,growth characteristics,mouse neurovirulence,full-length sequence of the genome were same as that of Ban18 strain,and the genetic characteristics were stable.The genetic analysis revealed that there were differences in three amino acids in structural protein and one in non-coding region between the dengue virus Ban18HK20strain and its parental virus Ban18 strain.By reverse genetics,the four differential genes in the dengue virus BanHK20 strain were reverse mutated into the corresponding genes of Ban18,and the reverse mutant viruses were rescued.The plaque of the E155 reverse mutant virus is clear,and the plaques of the other reverse mutant viruses are similar to Ban18HK20 strain,which are plaque-blurred.The replication efficiency of multi-point mutant viruses was relatively low,while single-point mutant viruses exhibit similar growth characteristics to Ban18HK20.The reverse mutant viruses rDENV1?E155?,rDENV3?E155&E369?and rDENV6?E155&E369&C111&3'UTR219?showed strong neurovirulence to mice with lg PFU/LD₅₀ of 0.1,0.4,1.1,and AST of 7.8d,8.1d,8.1d respectively,while the other reverse mutant viruses showed lower neurovirulence in mice.The chimeric virus rDENV4/2 based on the dengue virus type 4 Ban18HK20 with the prM-E genes of dengue virus type 2 16681 strain was rescued successfully.The chimeric virus can form a small plaque similar to the dengue virus type 2 16681 strain,which is significantly different from the large plaque of the Ban18HK20 strain.The optimal MOI of the chimeric virus rDENV4/2 cultured on Vero cells was 0.01,and the virus reached a titer peak on the 4th day after infection.The growth curve is similar to the parental viruses,but the virus titer on C_6/36 is slightly lower than that of the parental viruses.The chimeric virus rDENV4/2 showed its genetic stability on the Vero cells for ten consecutive passages.The chimeric virus has no neurovirulence to mice and strong neurovirulence to suckling mice.After immunizing the mice intraperitoneally with the7.0 lgPFU chimeric virus,a relatively high anti-DENV-2 neutralizing antibody was induced at 6 weeks after the initial immunization,which can protect mice from intracerebral inoculation by DENV-2 NGC.The rDENV4/2 and Ban18HK20 have no neurovirulence to monkeys.[Conclusion]A stable Ban18HK20 infectious clone and the reverse genetics technology platform for DENV research were constructed successfully.E155 is an important amino acid site that might allow Ban18HK20 to be non-neurovirulent to mice.The chimeric virus rDENV4/2 was non-neurovirulent to both mice and monkeys,and can protect mice from intracerebral inoculation of DENV-2 NGC virus.This study provides a theoretical value and practical significance for the research of the pathogenesis of dengue virus and development of dengue fever vaccine.