Outbreak of epizootic haemorrhagic disease on the island of Reunion
Outbreak of epizootic haemorrhagic disease on the island of Reunion
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DOI：
10.1136/vr.155.14.422
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发表时间：
2004-10-02
影响因子：
2.2
通讯作者：
Zientara, S
中科院分区：
文献类型：
--
作者：
Bréard, E;Sailleau, C;Zientara, S
从2003年1月至2003年4月，该岛大部分地区出现了以前从未在岛上观察到的几起牛病病例。临床症状与感染蓝舌病病毒的症状相似，但岛上的绵羊没有临床疾病的证据。牛的疾病特征为发热（40 ℃）、充血、口腔溃疡、流涎以及鼻和眼分泌物过多。受累动物表现为麻木和跛行。在某些情况下，死亡发生在8至10天内，但死亡率尚未确定。感染牛的发病率约为7%，在涉及3607头牛的流行病期间，报告了235例EHD临床病例。留尼汪的绵羊数量非常有限（18个羊群共2，000头）;牛和山羊的数量估计分别为30，000头和40，000头，从24头有急性疾病迹象的牛身上采集的EDTA血样被送往提交人的实验室。将每份EDTA血样的等分试样用于病毒分离或RNA提取。使用选择的引物进行逆转录酶PCR（RT-PCR），以扩增蓝舌病病毒的S7和S10区域（Zientara等人，2002年; Bréard等人，2003年）。用RT-PCR方法扩增EHDV的S7和S10区，引物选自GenBank上EHDV的S7或S10区序列。用蓝舌特异性引物未获得扩增产物。与此相反，EHDV S7和S10特异性引物分别获得1162碱基对（bp）和809 bp的扩增产物。所有的S7核苷酸序列均为1162 bp，有一个开放阅读框，编码349个氨基酸残基的预测蛋白。将S7序列与GenBank上的序列进行比较（表2）;在核苷酸水平上分别发现与EHDV血清型1和2的同源性为77%和77.1%，当比较氨基酸序列时，分别增加到94.3%和94.9%。EHDV的最小基因（S10）在病毒感染的细胞中表达为两种蛋白质。EHDV的S10的核苷酸序列包含两个框内起始密码子，其允许类似于蓝舌病毒的NS 3和NS 3A的蛋白质的翻译（詹森等1994）。留尼汪岛EHDV毒株S10的核苷酸序列与血清1型和血清2型EHDV毒株S10的同源性分别为87.3%和87.8%（表2）。与EHDV血清型1和2相比，留尼汪EHDV毒株NS 3的预测氨基酸序列分别显示96.9%和96.5%的相似性。对于病毒分离，用已储存的血液样品的等分试样接种含胚鸡蛋。含胚鸡蛋在35 ℃下孵育5 - 7天.将它们取出并在补充有抗生素的Eagle最低必需培养基中均质化。通过离心澄清组织匀浆，并用上清液接种BHK-21细胞（Bréard等，2003年）。在样品接种后5天，含胚卵未被血液接种物杀死，并且未观察到任何病变。然而，在通过BHK-21细胞两次传代后，观察到病毒感染的特征性细胞病变效应。根据EHDV特异性引物S7和S10的PCR结果，将该病毒鉴定为EHDV。病毒株和…
From January 2003 to April 2003, several cases of disease in cattle which had never been observed on the island before occurred over a large part of the island. The clinical signs were similar to those of infection with bluetongue virus but there was no evidence of clinical disease in sheep on the island. The disease in cattle was characterised by pyrexia (40 C), hyperaemia, oral ulcerations, ptyalism and excessive nasal and ocular secretion. Affected animals presented with torpor and lameness. In some cases, death occurred in eight to 10 days, but the mortality rate was not determined. The morbidity rate in the infected cattle was approximately 7 per cent. During the epizootic, which involved 3607 cattle, 235 clinical cases of EHD were reported. The sheep population of Réunion is very limited (a total of 2000 animals in 18 flocks); the cattle and goat populations are estimated at 30,000 and 40,000 animals, respectively.EDTA-blood samples collected from 24 cattle displaying signs of acute disease were sent to the authors’ laboratory. An aliquot of each EDTA-blood sample was used for virus isolation or RNA extraction. Reverse-transcriptase PCRs (RT-PCRs) were carried out, using primers selected to amplify the S7 and S10 regions of bluetongue virus (Zientara and others 2002, Bréard and others 2003). RT-PCRs were also carried out to amplify the S7 and S10 regions of EHDV, using primers that were selected from sequence data on S7 or S10 of EHDV available on GenBank. No amplification products were obtained with the bluetongue-specific primers. In contrast, 1162 base pair (bp) and 809 bp amplification products were obtained with the EHDV S7-and S10-specific primers, respectively. All the complete S7 nucleotide sequences were found to be 1162 bp and to have a single open reading frame encoding a predicted protein of 349 residues. The S7 sequences were compared with those available on GenBank (Table 2); 77 per cent and 77· 1 per cent homology, respectively, were found at the nucleotide level with EHDV serotypes 1 and 2, which increased to 94· 3 per cent and 94· 9 per cent, respectively, when the amino acid sequences were compared. The smallest gene (S10) of EHDV is expressed as two proteins in virus-infected cells. The nucleotide sequence of S10 of EHDV contains two in-frame initiation codons, which allow the translation of proteins analogous to NS3 and NS3A of bluetongue viruses (Jensen and others 1994). The nucleotide sequence of S10 of the EHDV strain from Réunion showed 87· 3 per cent and 87· 8 per cent homology, respectively, when compared with S10 of EHDV serotypes 1 and 2 (Table 2). The predicted amino acid sequence of the NS3 from the Réunion EHDV strain showed 96· 9 per cent and 96· 5 per cent similarity when compared with EHDV serotypes 1 and 2, respectively. For virus isolation, embryonated chicken eggs were inoculated with aliquots of blood samples which had been stored. The embryonated chicken eggs were incubated for five to seven days at 35 C. They were removed and homogenised in Eagle’s minimum essential medium supplemented with antibiotics. The tissue homogenates were clarified by centrifugation and BHK-21 cells were inoculated with supernatants (Bréard and others 2003). The embryonated eggs were not killed by the blood inoculum five days after the samples had been inoculated, and no lesions were observed in them. However, after two passages through BHK-21 cells, a characteristic cytopathic effect of a viral infection was observed. On the basis of the results of PCR using the EHDV-specific S7 and S10 primers, the virus was identified as EHDV. The virus strain and …