Adaptation Of Coxsackievirus A10 And Analysis Of The Biological Characteristics In The Representative Strains

Objective Coxsackievirus A10(CVA10)is one of the major pathogens causing hand,foot and mouth disease(HFMD).Until now,there were still no specific antiviral drugs or vaccine for CVA10.Vaccine is an effective measure for the prevention and control of infectious disease.However,the research of CVA10 vaccine was seriously limited,because CVA10 was hard to propagate in the vaccine-produced cell lines,such as African green monkey kidney(Vero)cell.In this study,we selected the Vero cell-adapted strains or the Human embryonic lung diploid cell(KMB17)cell-adapted strains of CVA10,and preliminarily researched the one-step growth curve,pathogenicity,and immunogenicity of the representative CVA10 strains.This study provided the foundation for the research of CVA10 vaccine and related mechanism.Method1.The stool samples and anal swab samples(the total number of samples were 327)from patients with hand,foot and mouth disease(HFMD)were collected from Kunming Yunnan 2009-2015 and Xiangyang Hubei 2017,respectively.The clinical samples were inoculated into human rhabdomyosarcoma(RD)cell,Vero cell and KMB17 cell,respectively.The CVA10 isolates were identified and the phylogenetic analysis was based on the entire VP1 sequences of CVAIO.2.The isolates propagated in RD cell were continuously passaged in Vero cell and KMB17 cell,respectively.3.The representative strains of four genotypes with high virus titer were cloned by plaque cloning technique.Then,micro tissue culture technique(MCT)were used to detect the virus titer at different inoculated time.We drew the one-step growth curves of four representative strains growing in Vero cell and KMBI7 cell,respectively.4.After the intracranial injection of 105TCID50 of virus suspension in the 3-day BalB/C neonatal mouse model,we recorded the weight of neonatal mouse,clinical score,and survival curve.The tissues of neonatal mice with a clinical score of 3 were used to detect the virus load(The Absolute quantitation was performed using real-time PCR taqman probe method).The hematoxylin-eosin(HE)staining and immunohistochemical staining of parafin section were also analysed.5.The strain V6-19/XY/CHN/2017 was inactive by p-propiolactone,and then concentrated by ultra-filtration.We immunized 6?8 week-old BalB/C mice with the antigens,and detected the neutralizing antibody titers in sera of mice.6.After the continuously passages of V6-19/XY/CHN/2017 strain in Vero cell,the 1st,5th,10th,and 15th-generation strains were compared in complete genome sequences.7.The complete genomes of four representative strains were identified by RT-PCR.Result1.14 CVA10 isolates were identified in RD cell,after the inoculation of three cell lines,respectively,and the isolates were all belonged to C genotype.The detection rate ot CVA10 in this study was 5.2%.2.2 Vero cell-adapted strains and 11 KMB 17 cell-adapted strains were identified after the adaptive propagation.3.The strain V6-19/XY/CHN/2017 was strongly sensitive to both Vero cell and KMB17 cell The Virus titer could reach 107 63TCID50/mL and 107 32TCID50/mL in Vero cell and KMB17 cell,respectively.4.The four representative strains all exhibited strong tropism to muscle tissue,but showed different virulence(K1554/YN/CHN/2015>V6-19/XY/CHN/2017>K975/YN/CHN/2010>K136/YN/CHN/2013).5.The inactive vaccine was made of the strain V6-19/XY/CHN/2017 propagated in Vero cell.It could induce the neutralizing antibody in mice(GMT=462),and had good cross-neutralizing ability to the other three KMB17 cell-adapted strains in vitro(GMT was 266.4-401.7).6.Strain V6-19/XY/CHN/2017 had good genetic stability in Vero cell within 15 generations.7.The analysis of comparison of nucleotide(amino acid)sequences indicated that the similarities of nucleotide(amino acid)sequences between four strains were 91.5%?96.0%(98.4%?98.9%).However,the similarities of 5'UTR and 3'UTR between four strains were 90.5%?97.4%and 88.4%?97.6%,respectively.Many different amino acid sites were found in the amino acid sequences.Conclusion1.The CVA10 isolates in this study all belonged to C genotype,which was the epidemic genotype in the Chinese mainland recent years.2.We identified 2 Vero cell-adapted strains and 11 KMB17 cell-adapted strains in this study.3.CVA10 exhibited strong tropism to muscle tissue.4.The Vero cell strain V6-19/XY/CHN/2017 showed good immunogenicity and genetic stability.This study could provide important reference for the research of inactive vaccine,and could help us to reveal the interaction mechanism between CVA10 and the host cell.