Intracellular detection of sense and antisense enteroviral RNA by in situ hybridization.
We prepared sense (S) and anti-sense (AS) 3H-labelled single-stranded RNA probes by polymerase-directed in vitro transcription of a coxsackievirus B3-derived cDNA fragment cloned in the PGEM2 plasmid vector. The probes detected, by in situ hybridization, both S and AS forms of viral RNA in the cytoplasm of coxsackievirus-infected tissue culture cells. More S (genomic and messenger) RNA was present than AS (negative and replicative intermediate) RNA. We confirmed and quantitated this observation with slot-blot hybridization of lysates of infected cells in which the ratio of detectable S to AS RNA was 40:1. The selective detection and localization of both forms of RNA in infected cells with sensitive and specific bi-directional probes advances the applicability of in situ hybridization to the study of viral pathogenesis.
Rotbart
H A
HA
Department of Pediatrics, University of Colorado School of Medicine, Denver 80262.
Abzug
M J
MJ
Murray
R S
RS
Murphy
N L
NL
Levin
M J
MJ
D002460:Cell Line; D029822:Enterovirus B, Human / Q000302:isolation & purification* / Q000502:physiology; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000302:isolation & purification*; D015347:RNA Probes; D016372:RNA, Antisense; D012367:RNA, Viral / Q000302:isolation & purification*; D014779:Virus Replication
D015347:RNA Probes; D016372:RNA, Antisense; D012367:RNA, Viral; D012313:RNA