Monoclonal antibodies differentiate between the haemagglutinating and the receptor-destroying activities of bovine coronavirus

Abstract

A relatively simple and sensitive method is described which enables the effect of monoclonal antibodies (MAbs) on the receptor-destroying enzyme (RDE) and the haemagglutination (HA) activity of bovine coronavirus (BCV) to be analysed in one assay. A lysate of HRT-18 cells infected with the L9 strain of BCV was found to have a higher RDE:HA ratio than purified virus. At 4 °C the lysate induced an HA pattern which completely disappeared upon raising of the temperature to 37 °C. This L9-infected cell lysate was used to determine the HA inhibition (HAI) titres of MAbs directed against the surface glycoproteins S and HE of BCV. Thereafter, the test plates were incubated at 37 °C to enable the ability of the MAbs to prevent elution of virus from BCV-erythrocyte complexes to be assessed. No inhibition of RDE was detectable with MAbs against glycoprotein S, which had HA1 titres ranging from 1:16 to 1:128. On the other hand, MAbs directed against glycoprotein HE had similar HA1 titres, but they inhibited elution of 8 HA units of BCV at titres of up to 1: 65 000.Bovine coronavirus (BCV) is an enteropathogen which causes severe diarrhoea in neonatal calves (Mebus et al., 1973) and which is implicated aetiologically in winter dysentery of adult cattle (Saif et al., 1988). BCV represents one of the better characterized haemagglutinating coronaviruses. Four major structural proteins are associated with infectious BCV (King & Brian, 1982;St Cyr-Coats et al., 1988), two of which are a phosphorylated nucleocapsid protein of Mr 50K to 54K and the integral membrane protein M, consisting of a family of glycoproteins of 23K to 26K.King et al. (1985) identified a haemagglutinin with an approximate Mr of 62K in the reduced, and of 124K in the non-reduced form. This structural protein forms the short spikes of the viral envelope (Doughri et al., 1976). Acetylesterase (AE) activity is associated with this glycoprotein, which is referred to as haemagglutininesterase (HE) (Vlasak et al., 1988a; Cavanagh et al., 1990). The enzyme is able to inactivate cellular receptors for BCV by hydrolysing an ester bond to release acetate from C-9 of sialic acid. The gene encoding HE is located upstream of the S gene, and encodes a protein of 424 amino acids Kienzle et al., 1990).The S glycoprotein is the third envelope-associated protein and forms the longer surface projections characteristic of BCV (Doughri et al., 1976). The nucleotide sequence of the S gene of BCV encodes 1363 amino acids, with an N-terminal signal sequence and a transmembrane sequence near the C-terminal end. Cleavage of the S glycoprotein into proteins S1 and $2 is predicted to occur at an RRSRR or RRSVR sequence at positions 764 to 768 (Zhang et al., 1991;Parker et al., 1990;Boireau et al., 1990;Abraham et al., 1990). The N-terminal moiety is the S1 glycoprotein and that at the C terminus represents the $2 subunit (Spaan et al., 1988). Cleavage of the S protein precursor is required for cell fusion activity (Storz et al., 1981 ;Sturman et al., 1985). The functions of the S prot...

Temperature-sensitive acetylesterase activity of haemagglutinin-esterase specified by respiratory bovine coronaviruses

“…This property indicates that HE interaction with other viral structural components may stabilise its structure and enzymic functions. Multiprotein interaction between S and HE glycoproteins was ®rst suggested by experiments with MAbs against HE of EBCV-L9 which inhibited both haemagglutination and AE activities [38,39].…”

In vitro studies on the use of clay, clay minerals and charcoal to adsorb bovine rotavirus and bovine coronavirus

“…Culturing and assaying for infectivity were performed in the BSC-1 cell line for rotavirus and the HRT-18 cell line for coronavirus. These methods have been described previously (Woode et al, 1987;Storz et al, 1991).…”

Bovine coronavirus

“…The HE gp also contains a receptor destroying enzyme (RDE) which cleaves an ester linkage to release 9-O-acetyl residues from sialic acids and which therefore inactivates the erythrocyte receptor (Neu 5, 9Ac,.,) for BCV. Activation of this enzyme causes the virus to elute from erythrocytes when the temperature is raised to 37°C (Vlasak et al, 1988a, b;Parker et al, 1990a;Vautherot et al, 1990;Schultze et al, 1991b;Storz et al, 1991). The RDE is probably involved in the early stages of virus replication, as enzyme inhibition greatly reduces viral infectivity (Vlasak et al, 1988a).…”

“…An amino acid sequence homology of. 29.7% has been demonstrated between the HE protein of BCV and the HA1 subunit of the influenza C virus HE protein (Kienzle et aL, 1990), and the HE proteins of both viruses induce haemagglutination, bind to receptors containing Neu 5, 9Ac,_, and possess a receptor destroying acetylesterase enzyme (Vlasak et aL, 1988a, b;Schultze et aL, 1990Schultze et aL, , 1991aStorz et al, 1991). On the basis of these observations it has been proposed that coronaviruses may have acquired the HE gene from the influenza C viruses by a non-homologous recombination event (Luytjes et al, 1988).…”