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.

DNA sequences coding for 81% of the ompA gene from 24 chlamydial strains, representing all chlamydial species, were determined from DNA amplified by polymerase chain reactions. Chlamydial strains of serovars and strains with similar chromosomal restriction fragment length polymorphism had identical ompA DNA sequences. The ompA sequences were segregated into 23 different ompA alleles and aligned with each other, and phylogenetic relationships among them were inferred by neighbor-joining and maximum parsimony analyses. The neighbor-joining method produced a single phylogram which was rooted at the branch between two major clusters. One cluster included all Chlamydia trachomatis ompA alleles (trachoma group). The second cluster was composed of three major groups of ompA alleles: psittacosis group (alleles MN, 6BC, A22/M, B577, LW508, FEPN, and GPIC), pneumonia group (Chlamydia pneumoniae AR388 with the allele KOALA), and polyarthritis group (ruminant and porcine chlamydial alleles LW613, 66P130, L71, and 1710S with propensity for polyarthritis). These groups were distinguished through specific DNA sequence signatures. Maximum parsimony analysis yielded two equally most parsimonious phylograms with topologies similar to the ompA tree of neighbor joining. Two phylograms constructed from chlamydial genomic DNA distances had topologies identical to that of the ompA phylogram with respect to branching of the chlamydial species. Human serovars of C. trachomatis with essentially identical genomes represented a single taxonomic unit, while they were divergent in the ompA tree. Consistent with the ompA phylogeny, the porcine isolate S45, previously considered to be Chlamydia psittaci, was identified as C. trachomatis through biochemical characteristics. These data demonstrate that chlamydial ompA allelic relationships, except for human serovars of C. trachomatis, are cognate with chromosomal phylogenies.

Results suggest that RBCV may play a causative role in outbreaks of shipping fever in cattle. More than 80% of the sick cattle shed RBCV at the beginning of 2 outbreaks when the Pasteurella spp infection rate was low.

Respiratory tract infections with viruses andPasteurella spp. were determined sequentially among 26 cattle that died during two severe epizootics of shipping fever pneumonia. Nasal swab and serum samples were collected prior to onset of the epizootics, during disease progression, and after death, when necropsies were performed and lung samples were collected. Eighteen normal control cattle also were sampled at the beginning of the epizootics as well as at weekly intervals for 4 weeks. Respiratory bovine coronaviruses (RBCV) were isolated from nasal secretions of 21 and 25 cattle before and after transport. Two and 17 cattle nasally shed Pasteurella spp. before and after transport, respectively. RBCV were isolated at titers of 1 × 103to 1.2 × 107 PFU per g of lung tissue from 18 cattle that died within 7 days of the epizootics, but not from the lungs of the remaining cattle that died on days 9 to 36. Twenty-five of the 26 lung samples were positive for Pasteurella spp., and their CFU ranged between 4.0 × 105 and 2.3 × 109 per g. Acute and subacute exudative, necrotizing lobar pneumonia characterized the lung lesions of these cattle with a majority of pneumonic lung lobes exhibiting fibronecrotic and exudative changes typical of pneumonic pasteurellosis, but other lung lobules had histological changes consisting of bronchiolitis and alveolitis typical of virus-induced changes. These cattle were immunologically naive to both infectious agents at the onset of the epizootics, but those that died after day 7 had rising antibody titers against RBCV andPasteurella haemolytica. In contrast, the 18 clinically normal and RBCV isolation-negative cattle had high hemagglutinin inhibition antibody titers to RBCV from the beginning, while their antibody responses to P. haemolytica antigens were delayed. Evans' criteria for causation were applied to our findings because of the multifactorial nature of shipping fever pneumonia. This analysis identified RBCV as the primary inciting cause in these two epizootics. These viruses were previously not recognized as a causative agent in this complex respiratory tract disease of cattle.

The complete genome sequences are reported here of two field isolates of bovine coronavirus (BCoV), which were isolated from respiratory and intestinal samples of the same animal experiencing fatal pneumonia during a bovine shipping fever epizootic. Both genomes contained 31 028 nucleotides and included 13 open reading frames (ORFs) flanked by 5h-and 3h-untranslated regions (UTRs). ORF1a and ORF1b encode replicative polyproteins pp1a and pp1ab, respectively, that contain all of the putative functional domains documented previously for the closest relative, mouse hepatitis virus. The genomes of the BCoV isolates differed in 107 positions, scattered throughout the genome except the 5h-UTR. Differences in 25 positions were non-synonymous and were located in all proteins except pp1b. Six replicase mutations were identified within or immediately downstream of the predicted largest pp1a-derived protein, p195/ p210. Single amino acid changes within p195/ p210 as well as within the S glycoprotein might contribute to the different phenotypes of the BCoV isolates.Coronaviruses are important causes of human and animal diseases that include respiratory infection, gastroenteritis, hepatic and neurological disorders as well as immune-mediated disease such as feline infectious peritonitis, and other persistent infections (reviewed in Spaan et al., 1988 ;Wege et al., 1982). We investigated two different epizootics of acute respiratory

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-F15. 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 N-linked 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-F15. 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.

Plaque formation, replication, and related cytopathic functions of the enteropathogenic bovine coronavirus strain L9 in bovine fetal thyroid (BFTy) and bovine fetal brain (BFB) cells were investigated in the presence and absence of trypsin. Plaque formation was enhanced in both cell types. Plaques reached a size with an average diameter of 5 mm within 4 days with trypsin in the overlay, whereas their diameter remained less than 1 mm at this time after plating without trypsin in the overlay. Fusion of both cell types was observed 12 to 18 h after infection when trypsin was present in the medium. Fusion was not observed in infected BFB cell cultures and was rarely observed 48 h after infection of BFTy cells maintained with the trypsin-free medium. The largest polycaryons formed had 15 to 22 nuclei. They then lysed and detached. Cell fusion depended on de novo synthesis of hemagglutinin and infectivity. Fusion from without was not observed. Virus produced under trypsin-enhancing conditions accompanied by cell fusion did not lyse mouse erythrocytes that reacted with L9 coronavirus hemagglutinin. Trypsin-treated, infected BFTy cultures produced coronaviral particles that excluded stain from the envelope confinement. These virions had uniformly shorter surface projections than did the viral forms generated by trypsin-free cell cultures.

A group of twenty-five isolates of Chlamydia psittaci representing at least seven different biotypes of bovine, ovine, caprine, equine, feline, porcine, and guinea pig origin were immunotyped by an indirect microimmunofluorescence test. Different groups of chiamydia-free BALB/c mice received two weekly intravenous inoculations with chicken embryo-propagated, partially purified elementary bodies of each strain. Antisera for immunotyping Were collected 4 days after the first inoculation and 3 to 4 days after the second inoculation and tested for antichlamydial immunoglobulin M and immunoglobulin G antibodies by the indirect microimmunofluorescence test with cell culture-propagated, partially purified homologous and heterologous antigens. Nine immunotypes of C. psittaci were distinguished. The correlation between immunotypes and biotypes was close, and a pattern of either disease or host specificity could be associated with each immunotype. Most immunotypes identified induced cross-reacting antibodies against each other, but no significant cross-reactions were observed with elementary bodies of the mouse pneumonitis strain of C. trachomatis. Findings from this study should provide the necessary background for the rational selection of prototype strains of C. psittaci for further antigenic analysis at the molecular level.