| Background and Objective:Since the beginning of the 70’s rotavirus was found in the early 20th century so far it have still been the most important cause of diarrhea in infants and young children. About 500 thousand infants and young children died of severe disease due to infection with rotavirus. Diarrhea caused by rotavirus infection has no effective drug treatment, so vaccination is the best method. The current vaccine is a live attenuated vaccine. Whether preparation of live attenuated vaccine or inactivated vaccine, screening of vaccine strains is one of key factors. The principle of screening of strains in vaccine development and preparation include good safety, immunogenicity, genetic stability and production applicability. According to a general rule, the pathogenic strain (wild strain or a virulent strain) tends to drift or gene mutation after multiple passages in vitro. On the one hand this drift especially variation will cause a strong virulent strain to occur attenuated effects of and sreen of vaccine strains for the preparation of live attenuated; On the other hand, replicationand immunogenicity of attenuated strains will decline. Preparations of inactivated vaccine need the strain maintain the highly toxic substance to obtain a high yield of virus and stable immunogenicity. Working seed of vaccine strain is virus seed of vaccines prepared, and maintain its stability and immunogenicity are essential. This study is about the genetic stability and immunogenicity evaluation of rotavirus strains named ZTR-68 after in vitro passage. The rotavirus selected from a stool with severe diarrhea in children is prepared for rotavirus vaccine after in vitro passage.Methods:Subculture rotavirus ZTR-68 strain:the fourteenth generation of the rotavirus ZTR-68 strain (working seed strain of vaccine prepared) continued to subculture in Vero cells to the 40 generation (P40 generation). Genome banding patterns were detected by polyacrylamide gel electrophoresis in each generation. We use the RT-PCR to determination of VP4 and VP7 gene sequences of all generations of P14-P40 and by plaque forming units (PFU) detected in each pass from generation to generation of the PFU. The P15, P20, P25, P30, P35, P40 viral harvest were immunized animals. ICR female mice were selected for immunized animal which was injected intraperitoneally and immunized twice,4 weeks apart. In the second immunization, each immunization dose is 0.5ml,106 PFU/ml. The blood was collected in one week after the second immunization; the plaque subtrahend and experimental serum were used to detect antibody titers. Experimental group included P15, P20, P25, P30, P35, P40 and PBS group as control group.Results:Genomic Bands prove consistent among generations genetic band pattern; Gene sequencing of virus VP4 and VP7 found no mutation; infectious virus titers did not change significantly before the P38; the average infective titer was about 1×1065PFU/ml; infectious titer of P39 and P40 declined. The result in immunized animal experiments of rotavirus showed that the mean neutralizing antibody titer was 1:210, The geometric mean of each group (GMT) have no big difference. By analysis of variance, the difference in immune effect between groups was not statistically significant (P> 0.05).Conclusions:Rotavirus ZTR-68 strain passages to P40 generations in Vero cells whose genome banding pattern has not changed, the major antigen gene of VP4, VP7 genes are not mutated, the virus maintained a good ability to replicate before P38. The immunogenicity of various passages of the virus was no significant difference. It proof that rotavirus ZTR-68 strain after passage still maintain good genetic stability and immunogenicity. Rotavirus ZTR-68 strain is suitable for preparation of inactivated rotavirus vaccine. |
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