Characterization of monoclonal antibodies to the hemagglutinin-esterase glycoprotein of a bovine coronavirus associated with winter dysentery and cross-reactivity to field isolates.

Seven hybridoma cell lines producing monoclonal antibodies (MAbs) to the hemagglutinin-esterase (HE) glycoprotein of bovine coronavirus (BCV) were obtained from BALB/c mice that were immunized with an enriched peplomeric fraction of the winter dysentery (WD)-associated strain BCQ.2590. The specificities of these MAbs to either the dimeric (140-kDa) or the monomeric (65-kDa) form of the HE glycoprotein were determined by Western immunoblotting experiments with purified virus and immunoprecipitation tests with [35S]methionine-labelled infected cell extracts. Four of these anti-HE MAbs inhibited the hemagglutinating activity of the virus and three weakly neutralized its infectivity in vitro. In addition, competition binding assays allowed for the definition of two independent antigenic domains (domains A and D) and two overlapping antigenic domains (domains B and C) for the HE glycoprotein of the WD-associated strain; epitopes located within antigenic domain A were not associated with hemagglutination inhibition (HAI) and virus neutralization activities. In HAI tests, the four anti-HA MAbs defined two distinct antigenic subgroups among 24 BCV field isolates that have been associated with either typical outbreaks of WD or neonatal calf diarrhea (NCD) in Quebec dairy herds from 1986 to 1996. The Quebec WD-associated strains of BCV, as well as some of the NCD-associated strains isolated since 1991, fell within a subgroup distinct from that of the prototype Mebus strain.

Antigenic variability among North American and European strains of porcine reproductive and respiratory syndrome virus as defined by monoclonal antibodies to the matrix protein.

Two hybridoma cell lines producing monoclonal antibodies (Mabs) to the 19-kDa matrix (M) protein of porcine reproductive and respiratory syndrome virus (PRRSV) were obtained from BALB/c mice that were immunized with a reference Quebec tissue culture-adapted strain (strain IAF-Klop). The polypeptide specificities of the MAbs were determined by immunoblotting and immunoprecipitation tests with concentrated and purified preparations of the virus and by determining their reactivities with the Escherichia coli-expressed gene products of open reading frames 5 to 7. The two anti-M protein MAbs (MAbs IAFK3 and IAFK6) and another MAb (MAb IAFK8) directed to the 15-kDa nucleocapsid (N) protein were devoid of virus-neutralizing activity. A library of four anti-N protein MAbs (MAbs IAFK8, SDOW17, VO17, and EP147) and two anti-M protein MAbs (MAbs IAFK6 and IAFK3) was used to investigate, by an indirect fluorescent-antibody assay, the antigenic diversity of 15 Canadian PRRSV isolates, in comparison with those of the U.S. ATCC VR2332 attenuated vaccine strain and two reference European (Lelystad and Weybridge) PRRSV strains. The North American and European PRRSV isolates tested shared the epitopes recognized by anti-N protein MAbs IAFK8 and SDOW17, but three distinct patterns could be identified on the basis of their reactivities with the other anti-PRRSV MAbs. No reactivity to the anti-M protein MAbs was observed by either European PRRSV isolate or the attenuated U.S. vaccine strain.

Comparison of bovine coronavirus isolates associated with neonatal calf diarrhoea and winter dysentery in adult dairy cattle in Québec.

Cytopathic coronaviruses were isolated in HRT-18 cells from bloody faecal samples collected from cows in Québec dairy herds having experienced typical outbreaks of winter dysentery (WD). The formation of polykaryons in the infected cell cultures was found to be dependent on the presence of trypsin in the medium. The WD isolates differed from the prototype Mebus strain of bovine enteropathogenic coronavirus (BCV.Meb) in respect to haemagglutination inhibition (HI), haemagglutination patterns at 4 degrees C and 37 degrees C, and receptor destroying enzyme activity with rat erythrocytes. Other field strains of BCV associated with outbreaks of neonatal calf diarrhoea (NCD) also differed from the BCV.Meb strain by demonstrating differences in HI. In all cases, no differences were detected by virus neutralization and Western immunoblotting. Analysis and comparison of the nucleotide and deduced amino acid sequences of the PCR-amplified haemagglutinin esterase (HE) genes of one representative WD strain (BCQ.2590) and two highly cytopathic NCD strains (BCQ.3 and BCQ.571) revealed high degrees of similarities (nt and aa sequence homologies > 98%) with the BCV.Meb strain. The putative esterase active site FGDS was conserved among these four BCV strains, indicating that this domain is probably not a determinant for BCV virulence. Six amino acid substitutions occurred between the HE glycoproteins of BCV.Meb and BCQ.2590 strains; two proline substitutions occurred respectively in the signal peptide (at aa 5) and near the sequences of the putative esterase domain (at aa 53).