Use of in Situ Hybridization for the Detection of Foot-and-Mouth Disease Virus in Cell Culture

Abstract

Biotinylated complementary DNA (cDNA) and RNA probes were prepared from a specific and highly conserved section of the foot-and-mouth disease virus (FMDV) genome coding for the RNA-dependent RNA polymerase. Hybridization was conducted on FMDV-infected, bovine enterovirus (BEV)-infected, and noninfected swine kidney cell cultures. The detection system utilized the enzyme system streptavidin-alkaline phosphatase, the substrate phosphate, and the chromogen nitroblue tetrazolium. Intense cytoplasmic granular staining was present at 2 and 4 hr postinfection (hpi), with less staining observed at 24 hpi. The staining was specific for FMDV, as indicated by a lack of staining of noninfected cells and BEV-infected cells. With the RNA probe, positive cells were detected up to the highest viral dilution assayed, which was approximately 96 TCID50. The cDNA probe was slightly less sensitive, detecting positive cells at 10-fold lower dilutions. This technique could prove useful in the diagnosis of foot-and-mouth disease in animals or in the detection of FMDV in biologics submitted for importation.

Detection of Porcine Parvovirus using Nonradioactive Nucleic Acid Hybridization

“…1,7,10,14,17,22 The purpose of the study reure in swine, 11,15 characterized by fetal death and mum-ported here was to evaluate 2 nonradioactive hybridmification. Identification of viral antigens by immu-ization assays for detection of PPV: a digoxigenin-lanofluorescent microscopy and detection of viral beled DNA probe and a biotin-labeled RNA probe.…”

“…Nucleic acid hybridization has been developed and has far ranging applications for routine analysis of virus, including herpesvirus, 2,8,14,22 picornavirus, 4,5,17,23 adenovirus, 10,26 papilloma virus, l African swine fever virus, 25 and parvovirus infection, 3,7,21 in clinical samples. Hybridization techniques have been primarily based on systems employing radiolabeled probes.…”