| Bovine group A rotavirus a（BRVA）is one of the major etiological agents of acute diarrhea in newborn calves,resulting in significant losses to the cattle industry.The current major strategy for preventing bovine rotavirus is to generate passive immunity by vaccinating pregnant cows with maternal antibodies delivered to newborn calves,but there is currently no commercially available vaccine in China.The aim of this study was to use the prevalent strain of BRVA in China to prepare inactivated vaccine and provide technical support for diarrhoea of calves in China.The main results achieved in this study are as follows:1.Optimization of culture conditions for SDA2 strainsThere are many genotypes of group a bovine rotavirus,but homotypic strains do not produce potent protection against heterotypic strains,and previous epidemiological data have indicated that G6P1 bovine rotavirus is the dominant genotype in China.The purpose of this experiment was to screen cultured cell lines isolated and purified from our laboratory for the G6P1 rotavirus SDA2 strain,to optimize the virus inoculum size and time of collection,and thus to improve virus yield.Results the CT value of SDA2 in Vero cell culture was 34.214±0.557,and that of SDA2 in MA104 was 15.121±1.219after other incubation under the same conditions for 19 h,indicating that the SDA2 strain proliferated on MA104 cells significantly more efficiently than on Vero cells.MA104cells were inoculated in 25 cm2 cell flasks with 400μL,600μL,800μL,1000μl of four virus inoculations,respectively,and after incubation for 1 h,the virus solution was discarded to add 5 ml of maintenance solution and incubated for 19H after collection,the virus loads in the four inoculated cell cultures were 2.39×100.59,2.39×101.62,2.39×103.75,2.39×102.76,which were quantified using fluorescence quantitative RT-PCR,indicating that 800μL（8×105.39 TCID50）of virus solution was optimal.Fluorescence quantitative RT-PCR was used to quantify the virus in cell cultures at 10 time points（4,8,12,16,20,24,28,32,36 and 40 h）after SDA2 inoculation on MA104 cells,and one-step growth curves of the virus were plotted.The results showed that the virus load was less at 4～28h after inoculation,followed by a slow increase in the virus load,the virus began to proliferate greatly at 28h,reached a peak at 36 h,the virus load was 2.39×104.39,and the virus load maintained a stable change from 36 to 40H.Based on the data from this study,the maximum viral load was achieved at 36 h after virus inoculation,providing a reliable basis for antigen production in subsequent vaccine development.2.Immunogenicity of a G6P1 bovine rotavirus inactivated vaccine in miceVirus titers were determined to be 10-5.39TCID50/0.1 ml of virus fluid was inactivated withβ-propiolactone and emulsified with 201 adjuvant to make inactivated vaccine,mice were vaccinated according to the dose of 0.2 ml/piece,0.5 ml/piece,1.0ml/piece.In this experiment,0.5 ml of mouse serum from group II immunized for 28days was diluted into six serum samples to be examined,and the neutralization test and indirect ELISA were performed,respectively,for correlation verification and for establishing a linear equation using SPSS software,and the correlation coefficient between the two results was 0.997,which indicated that the neutralization test results had a significant correlation with the ELISA test results,and the indirect ELISA can be used instead of the neutralization test on mouse blood The clear antibody was tested,and the neutralization test measured that the neutralizing antibody titer of 0.5 ml group mouse secondary immunization for 28 days was 1:3556,indicating that the vaccine of this experiment could produce a higher level of neutralizing antibody.Mouse anti BRVA antibodies were detected by indirect ELISA after the secondary immunization,and the results showed that the serum antibody titers of mice in 0.2 ml group were 1:348±49.05,1:540±45.03,1:1440±50.70,1:1872±72.72,1:1535±58.66,1:981±54.18,and that of mice in 0.5 ml group were 1:680±52.47,1:1672±58.90,1:2860±6 at 7 d,14 d,21 d,28 d,35 d,and 42 d after the secondary immunization,respectively3.75,1:3556±61.72,1:3926±63.35,1:2142±63.65,respectively,the serum antibody titers of mice in 1.0ml group were 1:524±47.59,1:1492±50.16,1:2750±61.16,1:3672±100.44,1:4018±66.99,1:2251±88.93.The results showed that the serum antibody level in the 0.2 ml group was significantly lower than that in the other two groups,and the serum antibody peaked at 1:1872±72.72 on the 28th D;the 0.5 ml group and the 1.0 ml group had an increase in the serum antibody level from 7 d to 35 d,and peaked at 1:3926±63.35 and1:4018±66.99 on the 35th D,respectively.The 0.5 ml group had a slightly higher antibody level than 1.0 on the 7th to 21 d after the second immunization Ml,the antibody levels of the two groups from 28d to 35d were flat step by step,the results showed that the antigen content had an effect on the antibody levels,in a range of antibody levels with the increase of antigen amount,the results also showed that the optimal dose was0.5 ml/piece on the mice. |
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