Screening Of Coxsackie Virus A6 Vaccine Candidate Strains And Research On Related Sites Of Cellularity

CV-A6(Coxsackievirus A6)can cause hand,foot and mouth disease,and has become a major serotype in recent years,gradually becoming a global public health threat.CV-A6 can infect human malignant embryonic rhabdomyoma cells(RD)to cause cytopathic effect(CPE)and can proliferate therein.However,it is extremely difficult to proliferate on the human vaccine cell substrates(such as Vero cells).Obtaining CV-A6 strain adapted to grow in cells allowed for human vaccine is the difficulty and focus of current work.The purpose of this project is to obtain an adapted strain of CV-A6 on Vero cells,and to study sites related to the tropism and growth ability of CV-A6 Vero cells.This subject is based on 8 CV-A6 RD cell-adapted strains obtained from more than 2,000 CV-A6 positive samples in Xiangyang in the laboratory's previous research.Through continuous passage on RD cells,a CV-A6-adapted strain CV-A6-HEV3391/XY/2017(hereinafter referred to as 3391)which can grow on Vero cells was obtained in the 16th generation of RD pasages.Three consecutive rounds of plaque purification were performed on 3391-R16V6(passage of RD cells to passage 16 and Vero cells to passage 6).Through the comparison of growth characteristics,antigen content,virus titer,etc.,the clone P49-21-21 was selected for construction of infectious cDNA and virus rescue.The CV-A6 vaccine candidate strain rescue-3391(hereinafter referred to as res3391)without RD cell background was obtained.At the same time avoid the risk of virus mutation during the passage.In order to explore the key sites related to CV-A6 and Vero cell tropism the adaptability to Vero cells from the early generations of RD passages was traced by IFA.It was found that Vero cell tropism has appeared since the R2 generation.The3391-R1 was subjected to three consecutive terminal dilution purifications to obtain a pair of cloned HEV3391-712-clone(hereinafter referred to as c712)strains and HEV3391-1023-clone(hereinafter referred to as c1023)strains with different adaptability of Vero cells.712 strains can grow on Vero cells and RD cells at the same time,while 1023 strains can only grow on RD cells(hereinafter phenotypes of the two clones referred to as RD~+/Vero~+,RD~+/Vero~-).Infectious cDNAs were constructed to rescue these two strains to obtain rescue strain rescue-712(hereinafter referred to as res712)and rescue-1023(hereinafter referred to as res1023).Through genome-wide sequencing and comparison,there were four differences in nucleotides resulting in two differences in amino acids.Through point mutations,the effects of these two amino acid sites on the tropism of viral cells were preliminarily explored.The results suggest that changes in the tropism of viral cells may be affected by both mutations at these sites.Nest,in order to explore the sites that affect the growth ability of CV-A6 on Vero cells,the res3391 strain of the the 16th generation(derived from RD16-Vero4)and the res712 strain of the early generation were compared on Vero cells.The titer of res3391 was about 400 times higher than that of the res712 strain.Through genome-wide sequence comparison,there are 14 nucleotide differences between the two genomes,which are relatively large.Two rounds recombinants were generated.The first round takes res712 as the genetic backbone and replaces the P1 and P2/P3segments with those from res3391,respectively,to obtain the recombinants recombine-712P1(hereinafter referred to as r712-P1)and recombine-712P2P3(hereinafter referred to as r712-P2P3).And the titer of r712-P1 strain on Vero cells has been significantly improved,while r712-P2P3 strain has no significant change.In the second round of generation of recombinants,res712 was used as the backbone,and the VP1 and VP2-VP4 segments of res3391 were replaced with those of res3391,respectively,to obtain the recombinants recombine-712VP1(hereinafter referred to as r712-VP1)and recombine-712VP2-VP4(hereinafter referred to as r712-VP24).However,the titers of both of them on Vero cells did not change significantly.This result suggests that the growth ability of CV-A6 on Vero cells may be determined by multiple sites located in the P1 segment,and the involvement of specific residues need further study.