| Coxsackievirus group A type 6(CVA6),a pathogen of hand,foot and mouth disease(HFMD).In recent years,CVA6 has even replaced EV71 and CA16 as the main pathogen of HFMD in some places of mainland China.CVA6-associated HFMD has several clinical features that differ from classic HFMD caused by EV71 or CA16.CVA6 infection can lead to severe herpetic pharyngitis,meningitis and myocarditis,and even cause neonatal death.Adults with normal immune function can also get sick after being infected with CVA6.At the same time,CVA6 virus is easy to be co-infected with other types of enterovirus or form new variant strains after gene recombination during the epidemic season,which brings many difficulties to the diagnosis and treatment of HFMD.In order to prevent the occurrence of HFMD in an all-round way,the research and development of CVA6 vaccine are urgent tasks.In this study,we isolated and identified the virus from feces and swabs of HFMD children in Yunnan Province.Then we analyzed the biological characteristics of the virus identified as CVA6,and the CVA6 vaccine candidate strains were screened according to the key screening indicators.This study is mainly divided into three parts.The first part is the isolation and identification of CVA6 virus.According to the results of RT-PCR identification of viral nucleic acid VP1 and standard serum neutralization,5 of the 20 samples were infected with CVA6 alone,4 by CVA6 and CVA10 mixed infection,and the others were infected with CVA10.five CVA6 strains(57,94,113,129,162),the CVA6 viruses in four mixed samples and the CVA6 reference strains(GS2015-399,GS2015-495),all belong to the D3 subtype of CVA6 virus.GS2015-399,57,94,113,129,162 and the CVA6 viruses in 4 mixed samples were all located in Yunnan's unique Yunnan cluster 2 branch.The analysis of VP1 amino acid sequence shows that there are several amino acid diversity sites.The isolation rate of 7 strains of CVA6 virus(57,94,113,129,162,GS2015-399,GS2015-495)and 4 mixed samples on RD cells was 100%,and only 129 and 94 appeared to proliferate in Vero cells.The second part is the analysis of the biological characteristics of CVA6 virus strain.In this part,we analyzed the growth characteristics,plaque morphology,infectious titer and pathological changes in suckling mice.Among the five CVA6 strains,129 has the highest infective titer and the fastest proliferation rate on Vero and RD cells.In RD cells,the plaque morphology of five strains of CVA6 virus was not significantly different.The plaque morphology formed by 129 on Vero and RD cells was different,and 94 could not form obvious plaques on Vero cells.The five CVA6 strains were pathogenic to suckling mice.After intraperitoneal injection of CVA6 virus,the neonatal mice began to develop the disease one after another in 4 to 6 days,and the neonatal mice with severe symptoms showed paralysis of the forelimbs hind limbsand and death.Under the same infection route and immune dose,129 suckling mice were more pathogenic.The third part is the screening of CVA6 vaccine candidate strains.Whether the strain can proliferate and grow on KMB17 cells and has high infectious titer as the primary screening index.In five CVA6 viruses,only 129 could proliferate and grow on KMB17 cells,and its infectious titer can reach 7.541gCCID50/ml.The other 4 strains did not meet the primary screening conditions,so 129 was selected as the strain for subsequent plaque cloning and purification.One strain(8.05lg CCID50/ml)with the highest infectious titer after the third plaque purification was selected as the vaccine candidate for the subsequent study,named as the 129-1 clone strain.The 129-1 clone strain could induce limb paralysis and death in suckling mice by intraperitoneal and intramuscular.The viral load in tissues and pathological changes showed that the viral load in the hind limb muscle was the highest,the heart and skeletal muscle showed obvious pathological damage,and the muscle should be the main replication site of the virus.Before or after plaque purification,129 shows that it can induce a good humoral immune response in mice,and its immunogenicity is good.Formaldehyde-inactivated 129-1 clone strain can induce high-titer neutralizing antibodies in mice after two doses of intraperitoneal immunization,and the serum with 128U of the experimental inactivated vaccine can provided complete protection against 57 strain of 105CCID50.The 129-1 clone strain was uploaded to 17 generations on KMB17 cells,and its viral infectivity titer remained stable.In this study,we explored the biological characteristics of CVA6 clinical isolates and finally screened a 129-1 clone strain which could proliferate and grow on KMB17 cells with high infective titer and good immunogenicity as a candidate vaccine strain.The experimental inactivated vaccine of 129-1 clone strain could produce high-titer neutralizing antibody,and its immunogenicity and protective effect were ideal.In the follow-up,it is still necessary to subculture the strain,and complete the systematic study of infectious titer,genetic stability,antigen content and so on,so as to establish a strain bank that meets the requirements. |
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