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Detection of La Crosse and Snowshoe Hare Viral Nucleic Acids by in Situ Hybridization
Laura J. Chandler
Laura J. Chandler
Colorado State University, Fort Collins, Colorado; NERC Institute for Virology, Oxford, United Kingdom; and Yale University, New Haven, Connecticut
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Barry J. Beaty
Barry J. Beaty
Colorado State University, Fort Collins, Colorado; NERC Institute for Virology, Oxford, United Kingdom; and Yale University, New Haven, Connecticut
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David H. L. Bishop
David H. L. Bishop
Colorado State University, Fort Collins, Colorado; NERC Institute for Virology, Oxford, United Kingdom; and Yale University, New Haven, Connecticut
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David C. Ward
David C. Ward
Colorado State University, Fort Collins, Colorado; NERC Institute for Virology, Oxford, United Kingdom; and Yale University, New Haven, Connecticut
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A molecular hybridization technique was developed to detect bunyavirus RNA in cells. Complementary DNAs (cDNAs) to the small (S) RNA segment of La Crosse (LAC) virus and to a portion of the middle (M) RNA segment of snowshoe hare (SSH) virus were used as probes to detect LAC or SSH viral RNA by in situ hybridization. Protocols were developed and standardized using radiolabeled DNA probes, and adapted for use with biotin labeled probes. The in situ hybridization procedure detected an estimated 3,600 copies of viral S RNA/cell at 24 hr postinfection. In growth curve studies, LAC nucleocapsid antigen was detectable slightly before S RNA. LAC S RNA synthesis was first seen about the nucleus. By 12 hr postinfection, hybridization signal was detected throughout the cytoplasm of the cell. The LAC S RNA probe was group-specific and cross-hybridized to 5 other California group viruses. The SSH M RNA probe was type-specific.
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