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QUANTITATIVE ANALYSIS OF LA CROSSE VIRUS TRANSCRIPTION AND REPLICATION IN CELL CULTURES AND MOSQUITOES

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.

Received February 17, 2004. Accepted for publication February 5, 2005.

Acknowledgments: We acknowledge the technical assistance of Cynthia Meredith and Elizabeth Gabitzsch.

Financial support: This work was supported by Public Health Services grant AI32543 from the National Institutes of Health.

^* Address correspondence to Barry J. Beaty, Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523. E-mail: barry.beaty{at}colostate.edu

Authors’ addresses: Brian J. Kempf, Department of Microbiology, School of Medicine, UCHSC at Fitzsimmons, Mailstop 8333, P.O. Box 6511, Aurora, Colorado 80045, Telephone: (303) 724-4263, Fax: (303) 724-4226, E-mail: brian.kempf{at}uchsc.edu. Carol D. Blair, Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado 80523, Telephone: (970) 491-8243, Fax: (970) 491-8323, E-mail: cblair{at}colostate.edu. Barry J. Beaty, Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado 80523, Telephone: (970) 491-2988, Fax: (970) 491-8323, E-mail: barry.beaty{at}colostate.edu.

Reprint requests: Barry J. Beaty, Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado 80523, Telephone: (970) 491-2988, Fax: (970) 491-8323, E-mail: barry.beaty{at}colostate.edu.

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