| The Chuzan virus(CHUV),a member of the Palyam serogroup within the genus Orbivirus of the family Reoviridae,was first demonstrated to be the causative agent of bovine disease among the Palyam serogroup.CHUV is responsible for many reproductive disorders,including abortion,stillbirth,and congenital malformation,which result in considerable economic loss in the cattle industry.The Chuzan disease caused by CHUV originated in Japan,related research data is less.In order to understand the prevalence of Chuzan disease in China and to further control the disease occurrence and development,this study performed an epidemiological investigation of Chuzan disease in China and established an indirect ELISA assay.A total of 543 sera of cattle and goat derived from Heilongjiang,Inner Mongolia,Xinjiang,Shandong,Jiangsu,Hubei,Guangxi,Yunnan and Hainan provinces were detected for the neutralizing antibodies against CHUV by micro-neutralization test.The results indicated that Chuzan disease was epidemic severely in 8 Provinces except Heilongjiang.The highest positive rate of CHUV was up to 57.35%(39/68)in Hainan province,and the samples from Heilongjiang province were all negative.The positive rates in other provinces were between 6% and 48.65 %.The Chuzan disease was epidemic severely in 8 of 9 provinces,and the infection rate in the south part of China was higher.The positive rate of bovine sera(55.56%-58.6%)was higher than goat sera(32.73%-33.33%)in seven provinces except of Xinjiang province.In addition,the infection rate in autumn(September to November)was the highest(16.67%-43.33%),which was significantly higher than that in spring and summer.A Chuzan virus(CHUV)was initially isolated from the sentinel cattle in Guangxi province of China in 2014.In order to establish a simple and rapid method to detect Chuzan disease,in this study an indirect ELISA was successfully developed to detect antibody to Chuzan Virus using the recombinant VP7 protein as antigen expressed in Escherichia coli BL21.CHUV VP7 gene was amplified by RT-PCR,then was cloned to p ET-28a(+)expression vector,and recombinant VP7 protein was identified by SDS-PAGE and Western blot.The indirect ELISA(VP7-ELISA)to detect CHUV antibody was established,using phalanx titration method to determine the optimal concentration of VP7 antigen,through optimized and improved the ELISA procedure.The cut-off value was determined as 0.384-0.347,and OD450 nm values more than 0.384 were positive,the values less than 0.347 were as negative,and the values between 0.383 and 0.347 were as ambiguous.The VP7-ELISA revealed a higher sensitivity and specificity,and had no cross reaction with Akabane virus(AKAV),Rotaviruses(RV),Bluetongue virus(BTV),Foot and mouth virus(FMDV),Bovine viral diarrhoea virus(BVDV),Infectious bovine rhinotracheitis virus(IBRV)and Parainfluenza virus type3(PIV3).The variation coefficient of the inter-batch and inter-batch repeated trials was less than 7.1% and 13.2% respectively.The concordance of VP7-ELISA relative to neutralization test was 83.05%.121 The bovine sera from heilongjiang,xinjiang,guangxi,and yunnan provinces of China were detected by the indirect VP7-ELISA and the results was consistent with the results of epidemiological investigation by micro-neutralization test.Therefore,the present study revealed that the indirect VP7-ELISA would be useful tool for epidemiological surveys of the CHUV infection.In conclusion,the Chuzan disease was popular in most parts of China.The infection rate of tropical and subtropical regions was higher.The positive rate in autumn was higher than summer and spring.Cattle were more susceptible than sheep.In this study the indirect ELISA method was established using recombinant VP7 protein as antigen,and revealed an good specificity and sensitivity,and can be used for the serological high-throughput detection to Chuzan disease. |
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