Antigenic and Polypeptide Structure of Turkey Enteric Coronaviruses as Defined by Monoclonal Antibodies

Abstract

SUMMARYTwenty-nine hybridoma cell lines, producing monoclonal antibodies (MAbs) to the Minnesota strain of turkey enteric coronavirus (TCV), have been established by fusion of Sp2/0 myeloma cells with spleen cells from BALB/c mice immunized with purified preparations of the egg-adapted or tissue culture-adapted virus. The hybridomas produced mainly IgG2a or IgG1 antibodies. Western immunoblotting experiments with purified virus, and immunoprecipitation tests with [35S]methionine-labelled infected cell extracts, allowed assessment of the polypeptide specificity of the MAbs. Sixteen hybridomas secreted antibodies directed to the peplomeric protein (E2, gp200/gpl00) and putative intracellular precursors of apparent Mr 170K to 180K and 90K. Four hybridomas produced antibodies that selectively reacted with a glycoprotein with an Mr of 140K (E3). This polypeptide species corresponded to the major structural component of small granular projections, located near the base of the larger bulbous peplomers, and was found to be responsible for haemagglutination. The major neutralization-mediating determinants were found to be carried by both E2 and E3 glycoproteins. Eight hybridomas produced MAbs directed to the major nucleocapsid protein (N, 52K), and only one MAb reacted with a low Mr structural glycoprotein (24K), corresponding to the matrix (El) protein. By indirect immunofluorescence, MAbs of different specificity also revealed distinct patterns of distribution of the viral antigens within the cells. The location on the virion of the antigenic determinants recognized by MAbs of different specificity was determined by the use of an immunogold electron microscopy technique. Comparison of nine TCV Quebec strains, using MAbs directed to peplomer and haemagglutinin proteins of the prototype Minnesota strain, confirmed their close antigenic relationship, but also revealed the occurrence of at least two distinct antigenic groups.

Identification of Coronaviruses by the Use of Indirect Protein A-Gold Immunoelectron Microscopy

“…5,7 Both viruses possess an hemagglutinating activity that has been associated to a 140-kD envelope glycoprotein (a disulfide-linked dimer) and additional small granular surface projections located near the base of the typical large bulbous peplomers of the coronavirions. 4,6 Both viruses are also antigenically related to the porcine HEV and human HCV-OC43 coronaviruses, 2 other viruses that belong to the subgroup of mammalian hemagglutinating coronaviruses. 26 Previous studies have demonstrated that these viruses all share antigenic determinants located on their homologous structural proteins.…”

Sequence of the 3′-terminal end (8·1 kb) of the genome of porcine haemagglutinating encephalomyelitis virus: comparison with other haemagglutinating coronaviruses

“…The significance of specific amino acid residues for HEV strains, in view of their virulence for pigs and their respiratory and neurological tropism, is difficult to interpret, considering that they are apparently distributed randomly among the previously mentioned proteins, except for the S protein. The S1 portion of the envelope-associated S glycoprotein is known to carry major antigenic determinants of coronaviruses that trigger the host immune response to induce the production of virus-neutralizing (VN) antibodies (Dea & Tijssen, 1989 ;Deregt & Babiuk, 1987 ;Vautherot et al, 1992). Consequently, sequence differences in S1 are thought to be responsible for many of the antigenic differences between coronaviruses .…”

“…The HE is associated with granular projections located near the base of the typical large bulbous peplomers and displays haemagglutinating (HA), acetylesterase (AE) or receptor-destroying enzyme (RDE) activities (Parker et al, 1990a, b ;Vlasak et al, 1988). The S and HE glycoproteins both possess antigenic determinants that trigger the immune response for the production of antibodies that neutralize virus infectivity and inhibit HA activity (Dea & Tijssen, 1989 ;Deregt & Babiuk, 1987 ;Michaud & Dea, 1993 ;Vautherot et al, 1992). Except for HCoV-OC43, BCoV and TCoV, these viruses do not seem to cross-react by seroneutralization or haemagglutination inhibition, but they all share common antigenic determinants on their structural proteins, as demonstrated by indirect immunofluorescence, Western blotting and immunoprecipitation assays (Dea & Tijssen, 1989).…”

“…Recent data from the USA and the UK indicate that coronaviruses associated with acute enteritis in turkey poults are genetically related to infectious bronchitis virus (IBV) of chicken and not to BCoV (Breslin et al, 1999;Cavanagh et al, 2001;Ismail et al, 2002;Loa et al, 2000), contrary to Canadian and US isolates characterized in the early 1990s (Dea et al, 1990 ;Verbeek & Tijssen, 1991) and even until very recently (Lin et al, 2002). In addition to the four structural proteins of all coronaviruses -N (nucleophosphoprotein, 50-60 kDa), M (matrix glycoprotein, 20-26 kDa), E (small membrane protein, 9n5 kDa) and S (peplomeric glycoprotein, 180-200 kDa) -coronaviruses of the second subgroup (haemagglutinating coronaviruses) also possess a 140 kDa envelope-associated glycoprotein, the haemagglutinin-esterase (HE), which is made of two 65 kDa subunits linked together by disulfide bonds (Dea & Tijssen, 1989 ;Hogue & Brian, 1986 ;King et al, 1985). The HE is associated with granular projections located near the base of the typical large bulbous peplomers and displays haemagglutinating (HA), acetylesterase (AE) or receptor-destroying enzyme (RDE) activities (Parker et al, 1990a, b ;Vlasak et al, 1988).…”

“…The S and HE glycoproteins both possess antigenic determinants that trigger the immune response for the production of antibodies that neutralize virus infectivity and inhibit HA activity (Dea & Tijssen, 1989 ;Deregt & Babiuk, 1987 ;Michaud & Dea, 1993 ;Vautherot et al, 1992). Except for HCoV-OC43, BCoV and TCoV, these viruses do not seem to cross-react by seroneutralization or haemagglutination inhibition, but they all share common antigenic determinants on their structural proteins, as demonstrated by indirect immunofluorescence, Western blotting and immunoprecipitation assays (Dea & Tijssen, 1989). The S glycoprotein possesses an N-terminal signal sequence, which is cleaved during intracellular processing, and thus is absent from the mature S protein (Abraham et al, 1991 ;Boireau et al, 1990 ;Parker et al, 1990a, b).…”