云南边境地区流行性出血热病毒血清学监测及血清型鉴定

为了解近几年云南边境地区牛、羊流行性出血热病毒（EHDV）的感染和流行情况，本研究从2014年起连续3年在与老挝、越南接壤的江城县设置EHDV监测点，每年选择投放EHDV抗体阴性的10头牛和5只山羊作为哨兵动物进行跟踪监测。每年5~10月份对哨兵动物采血，每周1次，11、12月每月采集一次，进行EHDV抗体、抗原监测和病毒分离。针对致细胞病变的样品，采用EHDV群特异性S7基因片段引物进行RT-PCR方法检测，同时利用EHDV-1、-5、-6、-7、-10标准阳性血清对分离到的病毒进行中和试验鉴定。结果显示，2014－2016年江城县牛EHDV抗体阳性率分别为41.9%、58.6%和75.4%；3年期间共监测到15头EHDV抗体阳性黄牛，并从中分离到20个可致细胞病变样品，经RT-PCR确认为EHDV，遗传进化分析发现有11个毒株与1997和2003年日本分离的EHDV毒株亲缘关系较近，9个毒株与1977和1981年澳大利亚分离的EHDV毒株亲缘关系较近，5个毒株与2015年广西分离株的亲缘关系较近；3年期间在山羊体内未检测出抗体，未发现抗原阳性动物；经中和试验血清型鉴定，确定20株毒株包括EHDV-5、-6、-7、-10型4种血清型，感染时间均在5~9月之间。本研究发现，江城县长期存在多种血清型EHDV同时流行，2014－2016年EHDV抗体阳性率逐年增加，亟需加强对EHDV感染情况及活动规律的持续研究，提高流行性出血热的防控效率。

Serological Investigation and Serotype Identification of Epidemic Hemorrhagic Virus in Yunnan Border Area

To study the epidemic situation of epidemic hemorrhagic disease virus (EHDV) in cattle and goats from Jiangcheng county in Yunnan province in recent years,in this study,three monitoring points were set up for three consecutive years from 2014 to 2016.Ten cattle and five goats with negative EHDV antibodies were selected as sentinel animals every year.Blood was collected once a week from May to October,and once a month in November and December,which were used to monitor antibody and antigen,and were used to isolate virus.A group specific S7 gene fragment primer was used to detect the samples of cytopathic lesions by RT-PCR method.At the same time,EHDV-1,-5,-6,-7,-10 standard positive serums were used to neutralize and identify the isolated viruses.The results showed that the positive rates of EHDV antibody from 2014 to 2016 were 41.9%,58.6% and 75.4%,respectively;In three years,15 cattle of EHDV positive were found in Jiangcheng county,and twenty samples were isolated from them.The RT-PCR result confirmed the EHDV,and genetic evolution analysis found that 11 strains were closely related to the EHDV strains isolated in Japan in 1997 and 2003,9 strains were closely related to the separation of Australia in 1977 and 1981,and 5 strains were closely related to the separation of Guangxi in 2015;Neither antibody nor antigen-positive animal was detected in goats for 3 years;After the neutralization test serotype identification,20 strains were determined to include 4 serotypes of EHDV-5,-6,-7,and -10,and the infection time was between May and September.This study found that there were multiple serotypes of EHDV in Jiangcheng county.The positive rate of EHDV antibodies increased year by year from 2014 to 2016.There was an urgen need to strengthen the continuous study of EHDV infection and activity patterns,and improve the prevention and control efficiency of epidemic hemorrhagic disease.