Studdert M J, Azuolas J K, Vasey J R, Hall R A, Ficorilli N, Huang J-A
Centre for Equine Virology, School of Veterinary Science, The University of Melbourne, Parkville, Victoria 3010.
Aust Vet J. 2003 Jan-Feb;81(1-2):76-80. doi: 10.1111/j.1751-0813.2003.tb11438.x.
To develop and validate specific, sensitive and rapid diagnostic tests using RT-PCR for the detection of Ross River virus (RRV), Kunjin virus (KV) and Murray Valley encephalitis virus (MVEV) infections in horses.
Primer sets based on nucleotide sequence encoding the envelope glycoprotein E2 of RRV and on the nonstructural protein 5 (NS5) of KV and MVEV were designed and used in single round PCRs to test for the respective viruses in infected cell cultures and, in the case of RRV, in samples of horse blood and synovial fluid.
The primer pairs designed for each of the three viruses amplified a product of expected size from prototype viruses that were grown in cell culture. The identity of each of the products was confirmed by nucleotide sequencing indicating that in the context used the RT-PCRs were specific. RRV was detected in serums from 8 horses for which there were clinical signs consistent with RRV infection such that an acute-phase serum sample was taken and submitted for RRV serology testing. The RRV RT-PCR was analytically sensitive in that it was estimated to detect as little as 50 TCID50 of RRV per mL of serum and was specific in that the primer pairs did not amplify other products from the 8 serum samples. The RRV primers also detected virus in three independent mosquito pools known to contain RRV by virus isolation in cell culture. Samples from horses suspected to be infected with KV and MVEV were not available.
Despite much anecdotal and serological evidence for infection of horses with RRV actual infection and associated clinical disease are infrequently confirmed. The availability of a specific and analytically sensitive RT-PCR for the detection of RRV provides additional opportunities to confirm the presence of this virus in clinical samples. The RT-PCR primers for the diagnosis of KV and MVEV infections were shown to be specific for cell culture grown viruses but the further validation of these tests requires the availability of appropriate clinical samples from infected horses.
开发并验证使用逆转录聚合酶链反应（RT-PCR）检测马感染罗斯河病毒（RRV）、库京病毒（KV）和墨累谷脑炎病毒（MVEV）的特异性、敏感性和快速诊断试验。
基于编码RRV包膜糖蛋白E2的核苷酸序列以及KV和MVEV的非结构蛋白5（NS5）设计引物组，并用于单轮PCR，以检测感染细胞培养物中的相应病毒，对于RRV，还用于检测马血样和滑液样本。
为三种病毒各自设计的引物对从在细胞培养物中生长的原型病毒中扩增出预期大小的产物。通过核苷酸测序确认了每个产物的身份，表明在所使用的情况下RT-PCR具有特异性。在8匹马的血清中检测到RRV，这些马具有与RRV感染一致的临床症状，因此采集了急性期血清样本并提交进行RRV血清学检测。RRV RT-PCR在分析上具有敏感性，估计每毫升血清中可检测到低至50个半数组织培养感染剂量（TCID50）的RRV，并且具有特异性，因为引物对未从8个血清样本中扩增出其他产物。RRV引物还在通过细胞培养物中的病毒分离已知含有RRV的三个独立蚊虫样本中检测到病毒。没有疑似感染KV和MVEV的马的样本。
尽管有许多关于马感染RRV的传闻和血清学证据，但实际感染和相关临床疾病很少得到证实。用于检测RRV的特异性和分析敏感的RT-PCR的可用性为确认临床样本中该病毒的存在提供了更多机会。用于诊断KV和MVEV感染的RT-PCR引物对细胞培养生长的病毒具有特异性，但这些试验的进一步验证需要从感染马获得适当的临床样本。
