Detection of La Crosse and snowshoe hare viral nucleic acids by in situ hybridization.
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.
Chandler
L J
LJ
Colorado State University, Fort Collins.
Beaty
B J
BJ
Bishop
D H
DH
Ward
D C
DC
D000818:Animals; D000956:Antigens, Viral / Q000032:analysis; D002043:Bunyaviridae / Q000302:isolation & purification*; D002460:Cell Line; D015342:DNA Probes; D002141:Encephalitis Virus, California / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D005455:Fluorescent Antibody Technique; D008433:Mathematics; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011237:Predictive Value of Tests; D012367:RNA, Viral / Q000032:analysis*; D013045:Species Specificity
D000956:Antigens, Viral; D015342:DNA Probes; D012367:RNA, Viral