Kempf Brian J, Blair Carol D, Beaty Barry J
Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, USA.
Am J Trop Med Hyg. 2006 Feb;74(2):224-32.
La Crosse (LAC) virus (family Bunyaviridae, genus Orthobunyavirus) small (S) segment negative-sense RNA genome (vRNA), positive-sense full-length RNA complement (vcRNA), and subgenomic mRNA were assayed in infected cell cultures and female Aedes (Ochlerotatus) triseriatus mosquito tissues using quantitative PCR (Q-PCR). During persistent infection of C6/36 (Aedes albopictus) and MAT (Aedes triseriatus) cultured cells and cytolytic infection of BHK-21 cultured cells, LAC vRNA was the most abundant RNA species, followed by mRNA and vcRNA. RNA copy numbers per cell were quantified and vRNA correlated to virus titer in cell culture medium. The Q-PCR assay proved more sensitive than reverse transcription (RT)-PCR and immunofluorescence assays (IFA) for detecting LAC virus infection of mosquitoes. After infection of female mosquitoes orally, quantities of LAC RNA increased in ovaries for 6 days, and as ovarian biosynthetic activity quiesced, LAC RNA quantities decreased then remained detectable at a low level. After a second, noninfectious blood meal, quantities of LAC RNA in ovaries increased significantly, quantitatively confirming correlation of LAC virus RNA synthesis with vector metabolic activity. Coregulation of viral replication and mosquito ovary metabolic activity may condition efficient transovarial transmission.
使用定量PCR（Q-PCR）对拉克罗斯（LAC）病毒（布尼亚病毒科正布尼亚病毒属）的小（S）节段负链RNA基因组（vRNA）、正链全长RNA互补物（vcRNA）和亚基因组mRNA在受感染的细胞培养物以及雌性三带喙库蚊（尖音库蚊）蚊虫组织中进行了检测。在C6/36（白纹伊蚊）和MAT（三带喙库蚊）培养细胞的持续感染以及BHK-21培养细胞的溶细胞感染过程中，LAC vRNA是最丰富的RNA种类，其次是mRNA和vcRNA。对每个细胞的RNA拷贝数进行了定量，并且vRNA与细胞培养基中的病毒滴度相关。Q-PCR检测法在检测蚊虫的LAC病毒感染方面比逆转录（RT）-PCR和免疫荧光检测法（IFA）更灵敏。雌性蚊虫经口感染后，卵巢中的LAC RNA量在6天内增加，随着卵巢生物合成活性静止，LAC RNA量随后减少，但仍可在低水平检测到。在第二次非感染性血餐后，卵巢中的LAC RNA量显著增加，从定量上证实了LAC病毒RNA合成与媒介代谢活性之间的相关性。病毒复制与蚊虫卵巢代谢活性的共同调节可能决定有效的经卵传递。
