Chandler L J, Hogge G, Endres M, Jacoby D R, Nathanson N, Beaty B J
Department of Microbiology, Colorado State University, Fort Collins 80523.
Virus Res. 1991 Jul;20(2):181-91. doi: 10.1016/0168-1702(91)90108-8.
Experiments were conducted to determine if La Crosse (LAC) and Tahyna (TAH) viruses reassort in Aedes triseriatus mosquitoes and to determine the genotypic frequencies of viruses selected by in vivo vector interactions. A molecular hybridization technique was used to analyze progeny viruses. Probes specific for the La Crosse L, M and S segments (pLAC4.16: LAC L RNA; pLAC4.27: LAC M RNA; pLAC4C-26: LAC S RNA) were used to determine the parental origin of the progeny RNA segments. Following infection with a mixture of LAC and TAH viruses, mosquitoes were held for 23 days extrinsic incubation, then assayed for reassortment. Individual progeny viruses were isolated by plaque assay and propagated in BHK-21 cells. Cytoplasmic RNA was extracted from the cells, blotted in triplicate to Nytran, and each blot was hybridized with 32P-labelled pLAC4.16, pLAC4.27 or pLAC4C-26 to determine the parental origin of each RNA segment. High frequency reassortment occurred in these mosquitoes. All of the expected genotypes resulting from a cross of LAC and TAH were obtained from these mosquitoes. Genotypic frequencies of 708 virus isolates from 39 mosquitoes were: LLL, 150 (21%); LLT, 71 (10%); LTL, 39 (5.5%); LTT, 109 (15%); TTT, 259 (36%); TTL, 16 (2.2%); TLT, 55 (7.8%); TLL, 9 (1.2%).
开展实验以确定拉克罗斯（LAC）病毒和塔希纳（TAH）病毒是否在三带喙库蚊中发生重配，并确定通过体内媒介相互作用选择的病毒的基因型频率。使用分子杂交技术分析子代病毒。使用针对拉克罗斯病毒L、M和S片段的特异性探针（pLAC4.16：LAC L RNA；pLAC4.27：LAC M RNA；pLAC4C - 26：LAC S RNA）来确定子代RNA片段的亲本来源。用LAC和TAH病毒混合物感染蚊子后，将蚊子进行23天的外在潜伏期培养，然后检测重配情况。通过蚀斑测定法分离单个子代病毒，并在BHK - 21细胞中进行繁殖。从细胞中提取细胞质RNA，一式三份印迹到Nytran上，每个印迹与32P标记的pLAC4.16、pLAC4.27或pLAC4C - 26杂交，以确定每个RNA片段的亲本来源。这些蚊子中发生了高频重配。从这些蚊子中获得了LAC和TAH杂交产生的所有预期基因型。来自39只蚊子的708个病毒分离株的基因型频率为：LLL，150个（21%）；LLT，71个（10%）；LTL，39个（5.5%）；LTT，109个（15%）；TTT，259个（36%）；TTL，16个（2.2%）；TLT，55个（7.8%）；TLL，9个（1.2%）。
