Chandler L J, Wasieloski L P, Blair C D, Beaty B J
Department of Microbiology, Colorado State University, Fort Collins 80523, USA.
J Virol. 1996 Dec;70(12):8972-6. doi: 10.1128/JVI.70.12.8972-8976.1996.
La Crosse (LAC) virus is an important cause of pediatric arboviral encephalitis in the United States. LAC virus is biologically transmitted by the mosquito Aedes triseriatus, and, like other arthropod-borne viruses, it establishes a persistent, nonpathogenic infection in its vector following oral infection. To investigate LAC virus persistent infection of mosquitoes, a reverse transcription-PCR assay was developed for the amplification of LAC virus negative-sense small (S) genome RNA segment, its full-length complement, and its mRNA transcript for qualitative analysis of transcription and replication in persistently infected mosquito tissues. RNAs were assayed from midguts removed at predetermined times after infection with a LAC virus-containing blood meal. LAC virus genome was detected almost uniformly in midguts at days 3 to 28 postinfection (p.i.) and, as the time p.i. progressed, in more of the samples than either mRNA or viral cRNA (vcRNA). Thus, persistent LAC virus infection of A. triseriatus midguts was correlated with a reduction in detectable viral mRNA and vcRNA. The assay was also used for analysis of virus-specified RNA in both quiescent and biosynthetically active mosquito ovaries. Viral replication decreased, as indicated by the absence of viral mRNA and vcRNA, in the ovaries of mosquitoes that did not receive further blood meals after their original oral infection. Viral replication increased in ovaries of mosquitoes that took an additional blood meal 30 days p.i. and was continuous in mosquitoes that took multiple meals to stimulate oogenesis. Thus, virus replication in persistently infected mosquito ovaries was dependent on host cell biosynthetic status.
拉克罗斯（LAC）病毒是美国儿童虫媒病毒性脑炎的一个重要病因。LAC病毒通过三带喙库蚊进行生物传播，并且与其他节肢动物传播的病毒一样，在经口感染后会在其传播媒介中建立持续的、非致病性感染。为了研究LAC病毒在蚊子中的持续感染，开发了一种逆转录聚合酶链反应（RT-PCR）检测方法，用于扩增LAC病毒负义小（S）基因组RNA片段、其全长互补序列及其mRNA转录本，以对持续感染蚊子组织中的转录和复制进行定性分析。在用含LAC病毒的血餐感染后，在预定时间取出中肠进行RNA检测。在感染后第3至28天（p.i.），几乎在所有中肠中都检测到了LAC病毒基因组，并且随着感染后天数的增加，检测到病毒基因组的样本比检测到mRNA或病毒互补RNA（vcRNA）的样本更多。因此，三带喙库蚊中肠的LAC病毒持续感染与可检测到的病毒mRNA和vcRNA的减少相关。该检测方法还用于分析静止状态和具有生物合成活性的蚊子卵巢中的病毒特异性RNA。在初次经口感染后未接受进一步血餐的蚊子卵巢中，病毒复制减少，表现为不存在病毒mRNA和vcRNA。在感染后30天额外进食一次血餐的蚊子卵巢中，病毒复制增加，而在多次进食以刺激卵子发生的蚊子中，病毒复制持续进行。因此，在持续感染的蚊子卵巢中，病毒复制取决于宿主细胞的生物合成状态。
