Johannes Storz

Biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child

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 nonstructural 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 BCVLY138. 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 BCVLY138 than to HCV‐OC43.

Comparison of the nucleotide and deduced amino acid sequences of the S genes specified by virulent and avirulent strains of bovine coronaviruses

Abstract The entire nucleotide sequences of the spike glycoprotein (S) genes of the highly virulent bovine coronavirus (BCV) strain BCV-LY138, the avirulent BCV-L9 and related Norden Vaccine (BCV-Vaccine) strains were determined using the polymerase chain reaction (PCR) to amplify cDNAs obtained by reverse transcription of viral RNA, and to produce single strand cDNAs for DNA sequencing. The S gene sequences of these viral strains were compared with those of recently published strains BCV-Mebus, BCV-Quebec, and BCV-F15. An open reading frame of 4092 nucleotides, encoding a protein of 1363 amino acid residues, was found in all six strains. Frameshifts and insertions or deletions were not observed except for the BCV-Fl 5. The S gene sequences were more than 98% conserved overall inspite of different origins of the six viruses. There were 45 to 56 nt differences between the virulent and avirulent groups while there were 6 to 14 nt differences among four avirulent strains. Comparison of the deduced amino acid sequences indicated that the S proteins had typical properties of membrane glycoproteins. Nineteen Winked glycosylation sites were predicted in five strains, and 18 of them were conserved in the avirulent strain BCV-L9. The sequence KRRSRR at the predicted proteolytic cleavage site was identified in five strains while the sequence KRRSVR was found in BCV-Fl 5. Substitutions of few amino acids in the putative fusogenic domains and two prolines at 507 and 567 in the antigenic domains may cause altered immunogenic and other functional properties of the S proteins specified by the virulent and avirulent BCV strains. Nine amino acid substitutions between the virulent and avirulent groups may correlate with BCV virulence.

Nucleotide and Predicted Amino Acid Sequences of All Genes Encoded by the 3′ Genomic Portion (9.5 kb) of Respiratory Bovine Coronaviruses and Comparisons Among Respiratory and Enteric Coronaviruses

The 3′-ends of the genomes (9538 bp) of two wild-type respiratory bovine coronavirus (RBCV) isolates LSU and OK were obtained by cDNA sequencing. In addition, the 3′-end of the genome (9545) of the wild-type enteric bovine coronavirus (EBCV) strain LY-138 was assembled from available sequences and by cDNA sequencing of unknown genomic regions. Comparative analyses of RBCV and EBCV nucleotide and deduced amino acid sequences revealed that RBCV-specific nucleotide and amino acid differences were disproportionally concentrated within the S gene and the genomic region between the S and E genes. Comparisons among virulent and avirulent BCV strains revealed that virulence-specific nucleotide and amino acid changes were located within the S and E genes, and the 32 kDa open reading frame.

The hemagglutinin/esterase gene of human coronavirus strain OC43: Phylogenetic relationships to bovine and murine coronaviruses and influenza C virus☆

Abstract The complete nucleotide sequences of the hemagglutinin/esterase (HE) genes of human coronavirus (HCV) strain OC43 and bovine respiratory coronavirus (BRCV) strain G95 were determined from single-stranded cDNA fragments generated by reverse transcription of virus-specific mRNAs and amplified by polymerase chain reaction. An open reading frame of 1272 nucleotides was identified as the putative HE gene by homology to the bovine coronavirus HE gene. This open reading frame encodes a protein of 424 amino acids with an estimated molecular weight of 47.7 kDa. Ten potential N-linked glycosylation sites were predicted in the HE protein of HCV-OC43 while nine of them were present in BRCV-G95. Fourteen cysteine residues were conserved in the HE proteins of both viruses. Two hydrophobic sequences at the N-terminus and the C-terminus may serve as signal peptide and transmembrane anchoring domain, respectively. The predicted HE protein of HCV-OC43 was 95% identical to the HEs of BRCV-G95 and other bovine coronaviruses, and 60% identical to the HEs of mouse hepatitis viruses. Phylogenetic analysis suggests that the HE genes of coronaviruses and influenza C virus have a common ancestral origin, and that bovine coronaviruses and HCV-OC43 are closely related.

Antibody responses to respiratory coronavirus infections of cattle during shipping fever pathogenesis.

Summary. Antibody responses against respiratory bovine coronavirus (RBCV) infections were monitored in cattle from the onset of a naturally occurring severe shipping fever (SF) epizootic to complete recovery of affected cattle or fatal outcomes. The infection with RBCV was detected in nasal secretions of 86 cattle, and 81 of them developed acute respiratory tract disease, including fatal pneumonia. Cattle nasally shedding RBCV at the beginning of the epizootic experienced characteristic primary immune responses with specific antibodies for hemagglutinin-esterase (HE) and spike (S) glycoproteins. Virus shedding in nasal secretions of the majority of the cattle ceased between days 7 and 14 with the appearance of HE- and S-specific antibodies. Nasal samples and lung tissues from 9 of the 10 fatal cases had high titers of RBCV, but these cattle had only IgM responses to RBCV infections. Cattle remaining negative in RBCV isolation tests entered this epizootic with antibodies against HE and S. Protection against respiratory tract disease was apparently associated with high level of opsonic and virus-neutralizing IgG2. The HE and S glycoproteins were recognized earliest by the bovine immune system while the N protein induced antibody responses during the later stage of initial infection and the early stage of reinfection. The membrane (M) glycoprotein was the least immunogenic of the major viral structural proteins.

Comparison of hemagglutinating, receptor-destroying, and acetylesterase activities of avirulent and virulent bovine coronavirus strains

SummaryHemagglutinating 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 5×105 to 4.5×106 plaque forming units per 50 µl 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 3h at 37 and 42°C. It was inactivated within 30 min at 56°C while the hemagglutinin function of this strain was stable for 3 h at 37, 42, and 56°C, but it was inactivated at 65°C within 1 h.

Comparison of the S genes and the biological properties of respiratory and enteropathogenic bovine coronaviruses

SummaryThe nucleotide sequence of the S gene of the bovine respiratory coronavirus (BRCV) strain G95, which was isolated from nasal swabs of a calf suffering from respiratory disorders, was determined and compared with the S gene of the enteropathogenic bovine coronavirus (BECV) strain LY138. Sequence analysis revealed 98.7% nucleotide and 98.3% deduced amino acid identities between the S genes of BRCV-G95 and BECV-LY138 without any deletions or insertions. Nucleotide substitutions were distributed randomly throughout the gene. Five monoclonal antibodies specific for the S protein distinguished BRCV-G95 from BECV-L9, but failed to differentiate it from BECV-LY138 in Western blots under denatured and native conditions. BRCV-G95 induced cytopathic changes in cell cultures that were similar to BECV-LY138 but different from BECV-L9. These results suggest that strain BRCV-G95 is more closely related to the virulent strain BECV-LY138 than to the avirulent, cell culture-adapted strain BECV-L9.

Comparison of bovine coronavirus (BCV) antigens: Monoclonal antibodies to the spike glycoprotein distinguish between vaccine and wild-type strains

Abstract Monoclonal antibodies (MAbs) against two major structural proteins of the cell-adapted Mebus strain of bovine coronavirus (BCV-L9) were produced and characterized. Seven MAbs reacted with the peplomeric glycoprotein, gp100/S, while three MAbs reacted with the nucleoprotein p53/N in Western blot analysis of BCV polypeptides. MAbs to gp100/S reacted with discontinuous epitopes of gp100/S in Westerns under mild but not under standard denaturing conditions. In contrast, MAbs to p53/N reacted in both types of Westerns, and those epitopes were thus continuous. MAbs to p53/N failed to neutralize BCV infectivity, while 4 MAbs to gp100/S neutralized BCV effectively. Cross reactivity of MAbs to gp100/S specified by five virulent wild-type strains and two high passage, cell-culture-adapted strains in mildly denaturing Westerns and neutralization assays indicated that two epitopes were conserved in all seven strains, while two epitopes of the avirulent strains were not detected in the wild-type strains. Non-neutralizing MAbs of gp100/S reacted with all seven strains in Westerns with the exception of one MAb that was specific for the highly cell-adapted strain BCV-L9.

The hemagglutinin/esterase glycoprotein of bovine coronaviruses: Sequence and functional comparisons between virulent and avirulent strains

Abstract The entire nucleotide sequences of the hemagglutinin/esterase (HE) genes specified by the highly virulent strain LY138 and the avirulent strain L9 of bovine coronavirus (BCV) were determined. These sequences were compared with recently published sequences of the HE genes of the Quebec and Mebus strains. A large open reading frame of 1272 nt encoding a protein of 424 amino acid residues was predicted. The putative esterase active site was conserved in the virulent and avirulent BCV strains, indicating that this domain is probably not a determinant for BCV virulence. Four amino acid substitutions occurred between the HE proteins of BCV-L9 and BCV-LY138 (Leu to Pro at 5, Leu to Val at 103, Ser to Pro at 367, and Thr to Asn at 379). Monoclonal antibodies specific for the HE glycoprotein inhibited the hemagglutination and acetylesterase activities of BCV-L9, but showed no inhibitory effect on the acetylesterase activity of BCV-LY138. These results suggest that at least one epitope is located proximal to one of the three strain-specific amino acids. Four S-specific monoclonal antibodies inhibited hemagglutination but not acetylesterase activity of BCVL9, implying that the S glycoprotein can promote hemagglutination of chicken erythrocytes in addition to the HE glycoprotein.

Structural proteins of bovine coronavirus strain L9: effects of the host cell and trypsin treatment

SummaryThe polypeptide profile of the cell-adapted strain of bovine coronavirus (Mebus BCV-L9) is remarkably affected by the host cell and trypsin. We compared the structural proteins of virus purified from different cell lines and found cell-dependent differences in the virus structure. BCV was purified from four clones of human rectal tumour cells (HRT-18): 3 F3, D 2, 3 E 3, and 4 B 3. The structural profiles of BCV propagated in clones 3 E 3 and 3 F 3 were identical, consisting of proteins with molecular weights of 185, 160, 140, 125, 110, 100, 52, 46, 37, 31–34, and 26–28 kilodaltons (kd). BCV purified from clone D2 lacked the 100 kd species, and clone 4 B 3 yielded virus lacking the 46 kd protein. We compared the structures of BCV propagated in HRT-18 cells [BCV(HRT-18)] and virus raised in bovine fetal spleen cells [BCV(D 2 BFS)]. The concentration of the 185 kd protein was higher in BCV (D 2BFS), and it also contained a 200 kd species. Protein profiles of in vitro trypsin treated and untreated BCV(HRT-18) differed only under reducing conditions, suggesting that trypsin cleavage sites are located within disulfide-linked regions of affected proteins. Propagation of BCV in D 2 BFS cells in the presence of trypsin resulted in cleavage of the 185 kd protein and a concommitant increase of the 100 kd protein. Activation of the fusion function probably depends on this cleavage process because fusion of BCV-infected D 2 BFS cells is trypsin dependent.