In vitro detection of canine distemper virus nucleic acid with a virus-specific cDNA probe by dot-blot and in situ hybridization

M. Oglesbee, D. Jackwood, K. Perrine, Michael Axthelm, S. Krakowka, J. Rice

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

A cDNA library was prepared from canine distemper viral (CDV) messenger RNA (mRNA) derived from Vero cells lytically infected with the Onderstepoort strain (Ond) of CDV. A 300 base pair insert was identified which, by Northern blot analysis and Sanger sequence data, was shown to be specific to the nucleocapsid gene. The nucleocapsid (NC) clone was radiolabeled with 32P using nick translation and used to detect viral RNA in both dot-blot and in situ preparations of Vero cells lytically infected with Onderstepoort CDV (Ond-CDV) and immortalized mink lung cells persistently infected with racoon origin CDV (CCL64-RCDV). Dot-blot hybridization results paralleled immunofluorescent results in the lytically infected cells. In 18 persistently infected cell lines from the RCDV-CCL64 parental stock, 13 lines were positive and two were negative on both immunofluorescence and dot-blot hybridization analysis for CDV antigen and RNA, respectively. Viral nucleic acid was detected in these persistently infected cells, where as few as 1.9% of the members of a line were positive on immunofluorescence. A dot-blot autoradiographic signal was obtained in three lines which were negative for CDV antigen. CDV RNA was detected in both lytically and persistently infected cell lines by in situ hybridization, where decreasing probe length was important in increasing the sensitivity of this assay. Viral RNA was detected in over 90% of the lytically infected cells, where only 70% were positive for viral antigen by immunofluorescence.

Original languageEnglish (US)
Pages (from-to)195-211
Number of pages17
JournalJournal of Virological Methods
Volume14
Issue number3-4
DOIs
StatePublished - 1986
Externally publishedYes

Fingerprint

Distemper
Canine Distemper Virus
Nucleic Acids
In Situ Hybridization
Viral RNA
Complementary DNA
Viruses
Viral Antigens
Fluorescent Antibody Technique
Nucleocapsid
Vero Cells
Mink
Cell Line
In Vitro Techniques
Gene Library
Base Pairing
Northern Blotting
Sequence Analysis
Clone Cells
Lung

Keywords

  • canine distemper virus nucleic acid
  • cDNA
  • dot-blot and in situ hybridization

ASJC Scopus subject areas

  • Virology

Cite this

In vitro detection of canine distemper virus nucleic acid with a virus-specific cDNA probe by dot-blot and in situ hybridization. / Oglesbee, M.; Jackwood, D.; Perrine, K.; Axthelm, Michael; Krakowka, S.; Rice, J.

In: Journal of Virological Methods, Vol. 14, No. 3-4, 1986, p. 195-211.

Research output: Contribution to journalArticle

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