Summary
Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned inE. coli. Three clones (174, 160, PG 78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes, were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabelling with32P and enzymatic labelling through covalent linkage to peroxidase and chemiluminescence detection. The radioactive probe 174 detected as little as 1 to 3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the familyCoronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in faecal samples using enzymatic probe was compared with conventional diagnostic methods.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Boireau P, Cruciere C, Laporte J (1990) Nucleotide sequence of the glycoprotein S gene of bovine enteric coronavirus and comparison with the S proteins of two mouse hepatitis virus strains. J Gen Virol 71: 487–492
Brian DA, Dennis DE, Gut JS (1980) Genome of porcine transmissible gastroenteritis virus. J Virol 34: 410–415
Bridger JC, Woode GN, Meyling A (1978) Isolation of coronaviruses from neonatal calf diarrhoea in Great Britain and Denmark. Vet Microbiol 3: 101–113
Chasey D, Lucas M (1977) A bovine coronavirus identified by thin-section electron microscopy. Vet Rec 100: 530–531
Crouch CF, Raybould TJG, Acres SD (1984) Monoclonal antibody capture enzyme-linked immunosorbent assay for detection of bovine enteric coronavirus. J Clin Microbiol 19: 388–393
Crucière C, Laporte J (1988) Sequence and analysis of bovine enteritic coronavirus (F15) genome. I Sequence of the gene coding for the nucleocapsid protein; analysis of the predicted protein. Ann Inst Pasteur Virol 139: 123–138
Czerny CP, Eichhorn W (1989) Characterization of monoclonal and polyclonal antibodies of bovine enteric coronavirus: establishment of an effecient ELISA for antigen detection in feces. Vet Microbiol 20: 111–122
Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13
Gouet P, Contrepois M, Dubourguier HC, Riou Y, Scherrer R, Laporte J, Vautherot JF, Cohen J, L'Haridon R (1978) The experimental production of diarrhoea in colostrum deprived axenic and gnotoxenic calves with enteropathogenicEscherichia coli, rotavirus, coronavirus and in a combined infection of rotavirus andE. coli. Ann Rech Vet 9: 433–440
Kienzle TE, Abraham S, Hogue BG, Brian DA (1990) Structure and orientation of expressed bovine coronavirus hemagglutinin-esterase protein. J Virol 64: 1834–1838
Kapke PA, Brian DA (1986) Sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene. Virology 151: 41–49
Macnaughton MR (1978) The genomes of three coronaviruses. FEBS Lett 94: 191–194
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, New York
Mebus CA, Newman LE, Stair EL (1975) Scanning electron, light, and immunofluorescent microscopy of intestine of gnotobiotic calf infected with calf diarrheal coronavirus. Am J Vet Res 36: 1719–1725
Mebus CA, Stair EL, Rhodes MB, Twiehaus MJ (1973) Pathology of neonatal calf diarrhea induced by a coronavirus-like agent. Vet Pathol 10: 45–64
Laporte J, Bobulesco P (1981) Polypeptide structure of bovine enteritic coronavirus: comparison between a wild strain purified from feces and a HRT-18 cell adapted strain. In: V ter Meulen, S Siddell, H Wege (eds) Biochemistry and biology of coronavirus, Plenum, New York, pp 181–184
Laporte J, Bobulesco P, Rossi F (1980) Une lignée cellulaire particulièrement sensible à la réplication du coronavirus entérique bovin: les cellules HRT-18. CR Acad Sci Paris D 290: 623–626
Renz M, Kurz C, (1984) A colorimetric method for DNA hybridization. Nucleic Acids Res 12: 3435–3444
Reynolds DJ (1983) Coronavirus replication in the intestinal and respiratory tracts during infection of calves. Ann Rech Vet 14: 445–446
Reynolds DJ, Chasey D, Scott AC, Bridger JC (1984) Evaluation of ELISA and electron microscopy for the detection of coronavirus and rotavirus in bovine faeces. Vet Rec 114: 397–401
Reynolds DJ, Debney TG, Hall GA, Thomas LH, Parsons KR (1985) Studies on the relationship between coronaviruses from the intestinal and respiratory tracts of calves. Arch Virol 85: 71–83
Sato K, Inaba Y, Kurogi H, Takahashi E, Satoda K, Omori T, Matumoto M (1977) Hemagglutination of calf diarrhea coronavirus. Vet Microbiol 2: 83–87
Sato K, Inaba Y, Tokuhisa S, Miura Y, Kaneko N, Asagi M, Matumoto M (1984) Detection of bovine coronavirus in feces by reversed passive hemagglutination. Arch Virol 80: 23–31
Savoysky E, Boireau P, Finance C, Laporte J (1990) Sequence and analysis of BECV F 15 matrix protein. Res Virol 141: 411–425
Shockley LJ, Kapke PA, Lapps W, Brian DA, Potgieter LND, Woods R (1987) Diagnosis of porcine and bovine enteric coronavirus infections using cloned cDNA probes. J Clin Microbiol 25: 1591–1596
Spaan W, Cavanagh D, Horzinek MC (1988) Coronaviruses: structure and genome expression. J Gen Virol 69: 2939–2952
Van Balken JAM, De Leeuw PW, Ellens DJ, Straver PJ (1978) Detection of coronavirus in calf faeces with haemadsorption-elution-haemagglutination assay (HEHA). Vet Microbiol 3: 205–211
Vautherot JF (1981) Plaque assay for titration of bovine enteric coronavirus. J Gen Virol 56: 451–455
Verbeek A, Dea S, Tijssen P (1990) Detection of bovine enteric coronavirus in clinical specimens by hybridization with cDNA probes. Mol Cell Probes 4: 107–120
Verbeek A, Tijssen P (1988) Biotinylated and radioactive cDNA probes in the detection by hybridization of bovine enteric coronavirus. Mol Cell Probes 2: 209–223
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Collomb, J., Finance, C., Alabouch, S. et al. Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization. Archives of Virology 125, 25–37 (1992). https://doi.org/10.1007/BF01309626
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01309626