| Rotavirus (rotavirus, RV) is the most important pathogen which causes acute diarrhea in infants and young children, and also contribute to the important cause of infant death in developing countries, accounting for more than 50% causes of intestinal infection. According to different epitopes in the inner capsid protein VP-6 group-specific antigen, RV can be divided into A～G 7 groups. Group A RV is the main reason of severe diarrhea in infants and young children. There is no specific drug treatment for RV diarrhea, although oral rehydration is effect, this therapeutic measure can not significantly reduce the mortality rate, particularly for children with severe diarrhea. Therefore, RV vaccine is the only effective mean for preventing and controlling RV infection. For any of the vaccine, its safety and efficacy are the most basic elements. It is very important to consider the security in the development of vaccines. There are many factors impacting safety of vaccines, one of which is matrix cell for the preparation of vaccines. Generally, the human cells are safer than other animal cells. Because of various reasons, many vaccines have been not yet prepared with human cells, but it is the development direction that human cells replace non-human derived cell in preparation of vaccine. Rotavirus vaccine play an important role in the prevention of RV infections, but the RV vaccines mainly use animal origin cells; whether a human-derived cells can be used for RV vaccine preparation is worthy of study.This study carried out on this thought, rotavirus P [2] G3 strains (SA11 strain) and rotavirus P [8] G1 strains (Wa strain) cultured in Vero cells were adapted to KMB17 cells by 10 continuous passages. The results showed that the time of producing cytopathogenic effect was progressively faster, viral infectious titer gradually increased and stabilized at a high level during continuous passage with rotavirus SA11 strain and Wa strain adapted in the KMB17 cells. SA11 strain reached peak titers 7.751gCCID50/ml, Wa strain reached peak titers 7.331gCCID50/ml with a fluorescent focus method. In the course of serial passage, the multiplicity of infection (MOI) was determined to be 0.05. Immunofluorescence confirmed the virus proliferation in KMB17 cell. Electron microscope showed typical rotavirus particles. Two strains of virus can form plaque in MA104 cells. RT-PCR amplification two viruses the 1st passage and 10th passage of VP7 gene sequence revealed that SA11 VP7 gene sequence did not change during subculture and maintained good genetic stability; Wa VP7 gene only occurred one nonsense mutation during subculture; By subcutaneous injection, the antibody titers of SA11 strain achieved 20480±8192, significantly higher than the antibody titers by oral immunization (10240±4096), by t test, P<0.05; The antibody titers of Wa strain was 7186±2048 by subcutaneous injection as well as the antibody titers 6144±2364 by oral immunization, by t test, P>0.05, no significant difference.This study succeeded in adapting two serotypes of rotavirus SA11 strain and Wa strain to KMB17 cells, and obtained progeny virus with high infectious titers. The virus showed good immunogenicity in mice immunized with progeny virus. This, study laid an important foundation for future large-scale preparation of rotavirus and the development of a safer human rotavirus vaccine. |
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