| Coxsackievirus A16(CVA16) is one of the main pathogens of hand, foot and mouth disease(HFMD), which is a common acute infectious disease affecting young children. CVA16 is a member of Human Enterovirus A(HEV-A) species of the Enterovirus genus of Picornaviridae family. CVA16 is a small, non-enveloped, icosahedral particle that contains a single-stranded, positive-sense and polyadenylated viral RNA genome of approximately 7.4 kb.CVA16 is generally thought to cause mild symptoms, but it has been reported that a small number of patients also developed central nervous system symptoms and myocarditis. Recent studies have showed that severe and fatal cases caused by CVA16 are increasing, and the co-circulation of CVA16 and EV71 has increased the possibility of co-infections and viral genetic recombination, which may lead to serious nervous system symptoms. CVA16 vaccine is still under research, while phase â…¢ clinical trials for the inactivated EV71 whole virus vaccines have been completed by three manufacturers in mainland China. So it is urgent to speed up the development of the CVA16 vaccine.During the related study of a vaccine candidate strain CVA16-P4-L731, it was found that the virulence of CVA16-P4-L731 had become attenuated in sucking mouse after continuous passaging in Vero cells. Sequencing analysis of CVA16-P4-L731-P6 revealed seven nucleotide mutations at positions 1434(C to T), 2744(A to G), 2747(A to G), 3161(G to A), 3182(A to G), 4968(C to T) and 6064(C to T). All seven mutations resulted in amino acid changes of VP2 T161M(VP2 T to M at position 161), VP1 N102 D, VP1 T103 A, VP1 E241 K, VP1 T248 A, 2C S297 F and 3D E42 A. The full-length c DNA was constructed based on the template of CVA16-P4-L731-P1. The c DNA was demonstrated infectious by characterizing rescued virus in producing CPE, viral nucleotide and protein synthesis. The availability of a CVA16 infectious clone will greatly facilitate the investigation of the genetic determinants of its virulence. An attenuated, plaque-purified strain of the CVA16-P4-L731-P6 and the virulent strain CVA16-P4-L731-P1, as well as other four rescued mutants, were used to infect 1-day-old Balb/C mice. The LD50 of each strain was determined. Comparison of pathogenecity between these strains indicates that mutations at amino acids at positions 2C 297, VP1 102 and VP1 241 might been associated with attenuation of CVA16-P4-L731-P6 in mouse. Meanwhile, A CVA16 strain was also rescued from c DNA-derived viral RNA in 2BS cells and reached high titers. This demonstrated that a Vero-isolated CVA16 could be adapted to 2BS cells depriving of the original genetic backgound of Vero cell line. The data showed that an infectious c DNA could be used as virus bank of a vaccine strain and prepare vaccine lots through a few passages avoiding high posibility of mutations not needed for vaccines. |
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