The coronavirus strain HECV-4408 was isolated from diarrhea fluid of a 6-year-old child with acute diarrhea and propagated in human rectal tumor (HRT-18) cells. Electron microscopy revealed coronavirus particles in the diarrhea fluid sample and the infected HRT-18 cell cultures. This virus possessed hemagglutinating and acetylesterase activities and caused cytopathic effects in HRT-18 cells but not in MDBK, GBK and FE cells. One of four S-specific monoclonal antibodies reacted in Western blots with HECV-4408, BCV-L9 and BCV-LY138 but not with HCV-OC43, and two reacted with BCV-L9 but not with HECV-4408, BCV-LY138 and HCV-OC43. One S-specific and two N-specific monoclonal antibodies reacted with all of these strains. cDNA encompassing the 3' 8.5 kb of the viral RNA genome was isolated by reverse transcription followed by polymerase chain reaction amplification had size and restriction endonuclease patterns similar to those of BCV-L9 and BCV-LY138. In contrast, the M gene of HCV-OC43 differed in restriction patterns from HECV-4408 and BCV. A genomic deletion located between the S and M within the non-structural genes of HCV-OC43 was not detected in HECV-4408. DNA sequence analyses of the S and HE genes revealed more than 99% nucleotide and deduced amino acid homologies between HECV-4408 and the virulent wild-type BCV. Forty-nine nucleotide and 22 amino acid differences were found between the HE genes of HECV-4408 and HCV-OC43, while only 16 nucleotide and 3 amino acid differences occurred between the HE genes of HECV-4408 and BCV-LY138. We thus conclude that the strain HECV-4408 is a hemagglutinating enteric coronavirus that is biologically, antigenically and genomically more closely related to the virulent BCV-LY138 than to HCV-OC43.

Hemagglutinating and acetylesterase functions as well as the 124 kDa glycoprotein were present in the highly cell-culture adapted, avirulent bovine coronavirus strain BCV-L9, in the Norden vaccine strain derived from it, and in 5 wild-type, virulent strains that multiplied in HRT-18 cells but were restricted in several types of cultured bovine cells. The BCV-L9 and the wild-type strain BCV-LY-138 agglutinated chicken and mouse erythrocytes. The acetylesterase facilitated break-down of the BCV-erythrocyte complex with chicken but only to a minimal extent with mouse erythrocytes in the receptor-destroying enzyme test. Purified preparations of the vaccine and the wild-type strains agglutinated chicken erythrocytes at low titers and mouse erythrocytes at 128 to 256 times higher titers whereas receptor destroying enzyme activity was detectable only with chicken erythrocytes. When wild-type strains were propagated in HRT cells at low passage levels, they produced 5 x 10(5) to 4.5 x 10(6) plaque forming units per 50 microliters which agglutinated erythrocytes from mice but not from chickens. Diisopropylfluoro-phosphate moderately increased the hemagglutination titers, but completely inhibited the receptor destroying enzyme of purified virus of all strains. It had virtually no influence on the plaque-forming infectivity of the different BCV strains. The acetylesterase of strain BCV-L9 reacting in the receptor-destroying enzyme test was stable for 3 h at 37 and 42 degrees C. It was inactivated within 30 min at 56 degrees C while the hemagglutinin function of this strain was stable for 3 h at 37, 42, and 56 degrees C, but it was inactivated at 65 degrees C within 1 h.

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...