Strand-specific detection of enteroviral RNA in myocardial tissue by in situ hybridization.
In this report we describe the development and application of single-stranded RNA probes for strand-specific detection of enterovirus RNA in infected heart tissue by in situ hybridization. For synthesis of RNA probes a full-length reverse-transcribed, recombinant CVB3 cDNA was inserted into the transcription vector pSPT18. Run-off transcripts of plus-strand and minus-strand orientation were produced using either T7 or SP6 RNA polymerase. Binding specificity and sensitivity of the radioactively labelled RNA probes were determined by slot-blot hybridization. Due to the high degree of genetic identity among enteroviruses, the in vitro transcribed CVB3 RNA probes hybridized with various enterovirus serotypes, including group A and B coxsackieviruses and echoviruses, which are commonly implicated in human viral heart disease. Strand-specific in situ hybridization led to detection of viral plus-strand or minus-strand RNA in infected cell cultures and in myocardial tissue sections of infected mice. In consecutive sections either viral genomic plus-strand RNA or complementary minus-strand RNA were localized in the same infected myocardial cells. In situ hybridization with enterovirus-specific and highly sensitive single-stranded RNA probes is of particular interest for the diagnosis of myocardial infections and for studies concerning viral RNA replication.
Hohenadl
C
C
Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
Klingel
K
K
Mertsching
J
J
Hofschneider
P H
PH
Kandolf
R
R
D000818:Animals; D003384:Coxsackievirus Infections / Q000175:diagnosis* / Q000382:microbiology; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D004770:Enterovirus / Q000235:genetics; D029822:Enterovirus B, Human / Q000235:genetics / Q000302:isolation & purification*; D005814:Genes, Viral; D006321:Heart / Q000382:microbiology*; D008297:Male; D051379:Mice; D009205:Myocarditis / Q000175:diagnosis* / Q000382:microbiology; D009693:Nucleic Acid Hybridization; D010849:Picornaviridae / Q000235:genetics; D015347:RNA Probes; D016372:RNA, Antisense; D012367:RNA, Viral / Q000032:analysis*; D014709:Vero Cells
D015347:RNA Probes; D016372:RNA, Antisense; D012367:RNA, Viral; D004247:DNA; D012321:DNA-Directed RNA Polymerases