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The serum antibody responses of cattle with respiratory coronavirus infections during the pathogenesis of shipping fever pneumonia were analyzed with different bovine coronavirus antigens, including those from a wild-type respiratory bovine coronavirus (RBCV) strain (97TXSF-Lu 15-2) directly isolated from lung tissue from a fatally infected bovine, a wild-type enteropathogenic bovine coronavirus (EBCV) strain (Ly 138-3), and the highly cell culture-adapted, enteric prototype strain (EBCV L9-81). Infectivity-neutralizing (IN) and hemagglutinin-inhibiting (HAI) activities were tested. Sequential serum samples, collected during the onset of the respiratory coronavirus infection and at weekly intervals for 5 weeks thereafter, had significantly higher IN and HAI titers for antigens of RBCV strain 97TXSF-Lu15-2 than for the wild-type and the highly cell culture-adapted EBCV strains, with P values ranging from

Respiratory bovine coronaviruses (RBCV) emerged as an infectious agent most frequently isolated from respiratory tract samples of cattle with acute respiratory tract diseases. Infectivity-neutralizing (IN) and hemagglutinin-inhibiting (HAI) antibodies induced by RBCV infections were monitored in sequential serum samples collected from cattle during a naturally evolving and experimentally monitored epizootic of shipping fever pneumonia (SFP). Cattle nasally shedding RBCV at the beginning of the epizootic started with low levels of serum IN and HAI antibodies. An increase in serum IN antibody after day 7 led to reduction of virus shedding in nasal secretions by the majority of the cattle between days 7 and 14. A substantial rise in the serum HAI antibody was observed during the initial phase among the sick but not the clinically normal cattle which were infected with RBCV. The RBCV isolation-positive cattle that developed fatal SFP had minimal serum IN and HAI antibodies during the course of disease development. Cattle that remained negative in RBCV isolation tests entered this epizootic with high levels of serum IN and HAI antibodies, which dramatically increased during the next two weeks. Protection against SFP was apparently associated with significantly higher levels of serum IN antibodies at the beginning of the epizootic. The RBCV-neutralizing activity is associated with serum immunoglobulin G (IgG), particularly the IgG2 subclass, while RBCV-specific HAI antibody is related to both serum IgG and IgM fractions.

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.

Objective—To identify cytocidal viruses and Pasteurella spp that could be isolated from cattle involved in 2 natural outbreaks of shipping fever. Design—105 and 120 castrated male 4- to 8-monthold feedlot cattle involved in 1997 and 1998 outbreaks, respectively. Animals—Nasal swab specimens and blood samples were collected, and cattle were vaccinated on arrival at an order-buyer barn from 4 local auction houses. Four days later, they were transported to a feedlot, and additional nasal swab specimens and blood samples were collected. Nasal swab specimens were submitted for virus isolation and bacterial culture; blood samples were submitted for measurement of respiratory bovine coronavirus (RBCV) hemagglutinin inhibition titers. Results—93 of 105 cattle and 106 of 120 cattle developed signs of respiratory tract disease during 1997 and 1998, respectively, and RBCV was isolated from 81 and 89 sick cattle, respectively, while at the orderbuyer's barn or the day after arrival at the feedlot. During the 1997 outbreak, bovine herpesvirus 1 was isolated from 2 cattle at the order-buyer's barn and from 5 cattle 7 and 14 days after arrival at the feedlot, and parainfluenza virus 3 was isolated from 4 cattle 14 days after arrival at the feedlot. During the 1998 outbreak, bovine herpesvirus 1 was isolated from 2 cattle at the order-buyer's barn and on arrival at the feedlot and from 5 cattle 7 and 14 days after arrival at the feedlot, and parainfluenza virus 3 was isolated from 1 animal the day of, and from 18 cattle 7 and 14 days after, arrival at the feedlot. Pasteurella spp was cultured from 4 and 6 cattle at the order-buyer's barn and from 92 and 72 cattle on arrival at the feedlot during the 1997 and 1998 outbreaks, respectively. Conclusion and Clinical Relevance—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. (J Am Vet Med Assoc 2000;216:1599–1604)

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.

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.

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 BCV-L9, implying that the S glycoprotein can promote hemagglutination of chicken erythrocytes in addition to the HE glycoprotein.

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.

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.

Summary The early events in the infection of human rectal tumor cells by bovine coronavirus were investigated by colloidal gold-mediated immunoelectron microscopy and by analysis of the effect of lysosomotropic weak bases on virus yield. Electron microscopic studies revealed sites of fusion between the virus envelope and the plasmalemma but fusion events along intracellular membranes were not observed despite extensive searches. Virion-antibody-colloidal gold complexes were, in fact, endocytosed by synchronously infected cells. These complexes were apparently non-infectious, and they accumulated in vacuoles that resembled secondary lysosomes. Exposure of cells to ammonium chloride or to methylamine during the first hour of infection had little inhibitory effect on the production of infectious virus. Chloroquine treatments were inhibitory but this effect depended on relatively late events in the infectious process. The chloroquine inhibitory step blocked infection of virus adsorbed to cells that were exposed to buffers in the pH range of 4.4 to 8.4. These findings indicate that BCV penetrates its host cell by direct fusion with the plasmalemma and does not require an acidic intracellular compartment for infectious entry.

Summary The ecology of cytopathic expression of bovine coronavirus (BCV) in HRT-18 cells was analyzed within virus-induced plaques by scanning electron microscopy. Virus replication was cytocidal for many HRT-18 cells, a function enhanced in the presence of trypsin. A monolayer of cells remained that imparted a characteristic turbidity to the plaque. These structurally normal, lysisresistant cells did not stain with fluorescent antibodies specific for BCV antigens, failed to adsorb virus particles or mouse erythrocytes in contrast to the susceptible cells. The survival of cells in the plaque interior reflects a non-productively infected population with evidence of viral persistence.

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 31028 nucleotides and included 13 open reading frames (ORFs) flanked by 5′- and 3′-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 5′-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.

Over the last few years numerous respiratory bovine coronaviruses (RBCV) were isolated from respiratory infections of cattle including two shipping fever epizootics (Storz et al., 1996; Storz et al., 2000). The RBCV strain 98TXSF-110-LU (LU) and the enteric strain 98TXSF-110-ENT (ENT) were isolated from lung and intestinal samples, respectively, from an animal experiencing fatal respiratory disease during a 1998 shipping fever epizootic. We obtained and compared the entire genomic sequences of the LU and ENT isolates to delineate nucleotide (nt) and amino acid (aa) differences between them. In addition, we analyzed the leader-mRNA junction sequences of the S and 12.7 kDa mRNAs specified by different BCV strains.

A monoclonal antibody-based capture enzyme-linked immunosorbent assay (ELISA) was developed to detect respiratory bovine coronavirus (RBCV) antigens in nasal swabs collected from cattle showing signs of respiratory tract disease following shipping. These samples had been previously tested for RBCV by inoculation of G clone cultures of human rectal tumor cells (HRT-18G) and for bovine herpes virus 1, parainfluenza virus 3, bovine adenovirus, bovine respiratory syncytial virus, and bovine viral diarrhea virus on other specifically permissive cell cultures. RBCV has not previously been recognized as an important etiological factor in the bovine respiratory disease complex of feedlot cattle. Thirty of 100 samples tested positive for RBCV antigen by capture ELISA in contrast to 38 of 100 samples that yielded RBCV isolates in G clone cells. Samples yielding other bovine respiratory viruses in the absence of RBCV were negative in the capture ELISA, which was based on the use of a single monoclonal antibody that recognizes one RBCV epitope on the S glycoprotein with the broadest reactivity with different strains of RBCV tested. Some RBCV strains may not be detected by this ELISA, which may account for the higher percentage of RBCV-infected cattle detected by RBCV isolation. However, the ELISA was simple to perform, sensitive, and specific and was more rapid than virus isolation. This assay will be useful for processing large numbers of field samples in future epidemiologic and diagnostic studies of RBCV infections of cattle.

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. © 1994 Wiley‐Liss, Inc.

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.

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 degrees C the lysate induced an HA pattern which completely disappeared upon raising of the temperature to 37 degrees 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 degrees 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 HAI titres ranging from 1:16 to 1:128. On the other hand, MAbs directed against glycoprotein HE had similar HAI titres, but they inhibited elution of 8 HA units of BCV at titres of up to 1:65,000.

Chlamydial isolates of bovine origin were serotyped by a plaque reduction method. Of the two major serotypes observed, type 1 included isolates from bovine abortion and enteric infections, whereas type 2 isolates were associated with polyarthritis or encephalomyelitis. These two serotypes were identical to those with a similar disease distribution previously observed in isolates of ovine origin. The two groups did not cross-react and they were serologically unrelated to chlamydiae of avian origin. Thus, it appears that many chlamydial isolates causing intestinal infections or abortion in sheep or cattle are closely related antigenically, as are those producing polyarthritis, encephalomyelitis, and conjunctivitis, and that the two groups are distinct.

Summary Bovine Herpesvirus Type 4: Studies on the Biology and Spread in Cattle Herds and in Insemination Bulls A serological survey with approx. 3,000 bovine sera revealed that 18.4% of the animals tested from West Germany and 8.7% from the USA harboured antibodies against bovine herpesvirus-4. A large number of BHV-4 reactors was found among healthy bulls in different German bull studs. In one stud with almost 70% seropositive animals an incidence rate of ca. 5% was observed. Virus could not be isolated from the semen of some of these animals. No correlation between the number of BHV-4 positive animals and respiratory or genital distress, Infectious Bovine Rhinotracheitis or Malignant Catarrhal Fever was detectable. The virus has a comparatively long replication cycle in tissue culture and a wide host cell range. Eight isolates were indistinguishable by serological means and no cross-reactions with BHV-1, —2, —3 and —6 were detectable, when tested by indirect fluorescence assays. Using this test, virus specific antigens could be stained in the nucleus of infected cells from 10 hours p. i. onwards and four hours later also in the cytoplasm. Zusammenfassung Eine serologische Untersuchung mit etwa 3000 Rinderseren zeigte, das in der Bundesrepublik 18,4%, in den USA 8,7% aller getesteten Tiere Antikorper gegen das bovine Herpesvirus-4 besasen. Auffallig viele Reagenten befanden sich dabei unter den gesunden Bullen verschiedener deutscher Besamungsstationen. In einer Station mit fast 70% Reagenten traten in einem Jahr knapp 5% Neuinfektionen auf. Versuche, aus dem Sperma dieser Tiere Virus zu isolieren, blieben erfolglos. Zusammenhange zwischen der Anzahl BHV-4-positiver Tiere und dem Auftreten von Erkrankungen des Respirations- oder Genitaltraktes, infektioser boviner Rhinotracheitis und bosartigem Katarrhalfieber liesen sich nicht ermitteln. In vitro zeichnet sich dieses Virus durch eine relativ langsame Replikation und ein breites Wirtszellspektrum aus. Wahrend 8 untersuchte Isolate serologisch einheitlich reagierten, liesen sich Kreuzreaktionen mit BHV-1, —2, —3 und —6 im indirekten Fluoreszenzantikorpertest nicht feststellen. Mit Hilfe dieses Testes waren virusspezifische Antigene in infizierten Zellen ab 10 Stunden p.i. im Kern, 4 Stunden spater auch im Cytoplasma nachweisbar.

Summary Polykaryon formation in bovine fetal spleen (BFS) cells infected with bovine coronavirus L9 occurred only in media supplemented with trypsin. A single 1 to 2 h trypsin treatment 10 h and later after infection induced formation of polykaryons. Trypsin treatment at pH 7.5 and 8.0 induced polykaryons while treatments at lower or higher pH levels did not. Cell fusion activity was partially suppressed by the presence of antibody.

The growth characteristics of Chlamydia psittaci serotypes 1 and 2 (ovine and bovine origin) were studied in mouse L cells. Formation of inclusions and yield of infectious progeny for serotype 1 were maximal when host cells were treated with cycloheximide and the pH in the cell culture medium was 7.2-7.4. The number of cells that contained inclusions and the infectivity yield for serotype 2 were maximal when the pH was 6.6-7.0. Treatments with diethylaminoethyl dextran and cycloheximide increased inclusion formation in serotype 2 but decreased the yield per infected cell. For both serotypes, centrifugation of the chlamydiae onto monolayers infected 50-1,200 times the number of cells infected with use of stationary adsorption. An enumeration method for L-cell infectious units was developed that gave higher titers than the chicken embryo 50% lethal dose after yolk sac inoculation.

Indirect enzyme-linked immunosorbent assays were applied to differentiate Chlamydia (C.) psittaci -specific IgG1 and IgG2 levels in 143 individual serum samples from cattle with naturally occurring chlamydial infections and in 190 sequential serum samples from 26 experimentally infected pregnant cows, calves, and a bull. The mean IgG1:IgG2 ratio of naturally infected cattle was 1:4 indicating a significant (p C. psittaci and Coxiella (C.) burnetii simultaneously. The predominance of C. psittaci -specific IgG2, in contrast to the predominance of C. burnetii -specific IgG1 detected in these same individual serum samples under identical conditions, indicates that the ability to preferentially produce either IgG1 or IgG2 was not limited in these individual cattle. A transient yet significant IgG1 response was also developed in cows following chlamydia-induced abortions (immunotype 1) or in cattle infected with the polyarthritis-serositis-encephalomyelitis agents (immunotype 2). IgG1 levels decreased faster than IgG2 levels. These findings have diagnostic implications and identify the need for determining the immunoglobulin classes and subclasses of the humoral immune responses of animals and man to chlamydial infections.

Summary Biological properties and restriction enzyme patterns of the slowly replicating herpesviruses isolated from cattle affected with different diseases in North America and Europe were analysed. These virus isolates induced identical plaques that developed within 7 to 9 days in bovine foetal spleen cells and within 5 days in actively growing Georgia bovine kidney cells. These virus isolates were found to be antigenically related when tested in the indirect immunofluorescence test, and antigenic relationships with bovine herpesvirus 1 (BHV-1), BHV-2, BHV-3 or BHV-6 were not detected. The genomes of these strains were shown to have virtually identical cleavage sites when treated with restriction enzymes EcoRI, BamHI, SstII, SphI and HindIII. The resulting restriction enzyme patterns differed strikingly from those of BHV-1, BHV-2, BHV-3 and BHV-6. Because the herpesviruses tested become enveloped on the nuclear as well as on endoplasmic membranes, a process through which they induce cytoplasmic vesicles filled with enveloped viral particles, and because of the unique cytoplasmic inclusions that are induced, we classify them tentatively as bovine cytomegaloviruses.

Antigens of the immunotype 1 strain B-577 of Chlamydia psittaci, which were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes were used to probe sequential serum samples of cattle with experimentally induced or naturally occurring chlamydial infections. Applying IgGl- and IgG2-specific markers in an enzyme immunoassay procedure, a predominance of IgG2 reactions with different proteins was determined. The interaction of IgGl with antigens such as the genus-specific lipopolysaccharide and the major outer membrane protein was usually limited to periods immediately following overt clinical disease. Some other antigens like the 60,000 and 62,000 D proteins, for example, were recognized by both subclasses over the entire period of investigation. This indicates that it may be possible to determine the phase of infection through analysis of the IgGl and IgG2 responses with the Western blot technique. The different IgGl and IgG2 responses of cattle infected with different strains of Chlamydia psittaci as well as the diverse reactions of cattle from different herds with naturally occurring chlamydial infections further indicate that it may be feasible to distinquish the strains causing these chlamydial infections using different antigens in the Western blot technique. The results obtained by this method may have implications for the production of a subunit vaccine as well as for serodiagnostic purposes.

One-step growth curves of a bovine enterovirus and the virus of bovine diarrhea (BVD) in singly and doubly infected bovine embryonic kidney (BEK) and bovine embryonic spleen (BES) cells were determined. In BEK cells, bovine enterovirus infectivity increased exponentially in both tissue culture fluid and cells 4 to 10 hours after inoculation and then levelled off. In BES cells, the exponential increase lasted 14 to 17 hours. In the growth curve of the BVD virus in BEK cells, the extracellular and cell-associated viruses increased exponentially until hour 12, increasing gradually thereafter. In BES cells, BVD virus infectivity increased exponentially 24 hours after infection and then levelled off. The peak infectivity in BES cells was reached 24 hours before that in BEK cells. Growth of bovine enterovirus was studied in cells of BEK and BES monolayers preinfected with the BVD virus for 20 hours. The infectivity titer in preinfected BEK cells was approximately 2 logs less than in preinfected BES cells. A decreased yield in infectivity was observed compared to the bovine enterovirus one-step growth curve.

In assessing the host cell range of bovine parvoviruses, these viruses were found to replicate optimally in actively dividing bovine fetal lung and spleen cells. Other primary bovine fetal cells supported growth to a lesser extent, but bovine line cells and line cells of other animal species tested did not. Minimal infectivity remained after passage of bovine parvovirus in cells from chicken embryos and guinea pig fetuses. During bovine parvovirus replication in bovine fetal lung and spleen cells, production kinetics of infectious virus and hemagglutinins were determined. An eclipse period of 16 h occurred, and viral release from cells was not detected until 30 h after inoculation of bovine fetal lung cells and 36 h after inoculation of bovine fetal spleen cells. Cell-associated virus titers were always higher than extracellular virus titers. Hemagglutinins were detected in parallel to infectious virus.

Tritiated uridine was selectively incorporated into the cytoplasm but not the nucleus of BEK cells infected with bovine enterovirus. This was accompanied by complete shutdown of cellular RNA synthesis. Bovine embryonic spleen cells infected with the BVD virus incorporated3H-uridine into the nucleus and cytoplasm, while uninfected cells showed only nuclear and nucleolar incorporation. Bovine embryonic spleen cells that were preinfected with BVD virus for 20 hours and then superinfected with bovine enterovirus showed complete absence of nuclear and nucleolar labelling in the early stages of superinfection with the bovine enterovirus, but nuclear labelling returned in reduced amounts 8 to 10 hours after superinfection and persisted throughout the remainder of the double infection ending in cell death.

Five identical viral isolates were recovered from bovine fetal spleen cells maintained in culture for 30 to 67 days. During this time the cells were sub-cultured 3 to 8 times. Cells cultured from other organs of the 3 donor fetuses did not evidence lytic viral infections. The viral isolates were identified serologically, morphologically, culturally and by characterization of the virus DNA as the herpesvirus of infectious bovine rhinotracheitis. The available data indicate latent infection of the donor fetuses as the source of these viral isolates.

A hemadsorbing enteric virus (HADEN), isolated from the gastrointestinal tract of cattle and previously classified as a bovine enterovirus, was found to induce Feulgen-positive intranuclear inclusions and to be inhibited in its replication by FUDR, BUDR and actinomycin D, indicating DNA as genetic material. Because of this fact and the other known properties of HADEN virus it should be considered a bovine parvovirus.

Eight chlamydial isolates of ovine origin were tested in a plaque reduction system using homologous and heterologous rooster antisera. The eight isolates could be separated into two separate immunotypes. Type 1 included isolates associated with ovine abortion and one agent recovered from the feces of an apparently normal sheep. Type 2 isolates were associated with polyarthritis and conjunctivitis. These two serotypes were not cross-reactive with several chlamydiae of avian origin. Further application of the plaque reduction test may provide a useful means of typing chlamydiae.

Cornonaviruses cause severe and economically important intestinal infections in newborn calves which become manifest as enteritis with diarrhea and additional clinical consequences. Studies concerning the pathogenesis of enteropathogenic bovine coronavirus (EBC-virus) enteritis have led to a better understanding of morphological alterations of the intestinal tract as well as of the individual cells, helping to explain the functional disorders observed with this disease (for references see Storz and Rott, 1980). Relatively little is known, however, about structural details of the virus and about the biochemical events of EBC-virus replication, principally a result of the difficulties encountered in growing the virus in tissue culture. After we succeeded in adapting EBC-virus strain L-9 isolated by Mebus et al. (1973) to grow in bovine fetal thyroid or brain cells in reasonable amounts it became possible to study some of the many unanswered questions. As a beginning, efforts were made to determine optimal growth conditions in order to provide an in vitro source for viral antigen. In addition, biochemical analysis of the virus was undertaken.

Morphogenic features of bovine parvovirus replication and associated cellular alterations in bovine fetal lung and spleen cells were examined by electron microscopy. Morphological evidence of parvoviral replication was detected first at 24 hr after inoculation when empty viral capsids were found in association with the smooth endoplasmic reticulum, in the lumina of nuclear pores, and in the nucleus. At 36 hr empty capsids were found in the parafibrosa of the nucleolus. Few complete virions were seen in the nucleus at 24 and 36 hr postinfection. The ratio of complete to empty virions increased and complete virions predominated 48, 60, and 72 hr after infection. Empty capsids and complete virions measured 20–22 nm in diameter. At 48 and 60 hr complete virions accumulated within the nucleus near nuclear pores, and similar 20–22 nm particles were seen on the cytoplasmic side of the nuclear membrane. The cytoplasm was severely degenerated in cells 72 hr after inoculation, the nuclear membranes were fragmented, and complete virions replaced the heterochromatin. Crystals composed either of empty capsids or complete virions were occasionally seen in the nuclei of different infected cells.

Antibodies in human serum against an enteropathogenic bovine coronavirus were detected by double immunodiffusion (DID), neutralization of infectivity, indirect immunofluorescence, and immune electron microscopy. Human sera reacting in the DID test neutralized the infectivity of the bovine coronaviruses to indices of 2.5 to greater than 5. Nineteen of 40 DID-negative, heat-inactivated sera had neutralizing indices of 1 to 3.0. Human serum with neutralizing and DID antibodies produced juxtanuclear and cytoplasmic fluorescence identical to that of bovine immune serum in cells infected with the bovine coronavirus. Antibodies in human and bovine sera interacted with the peplomeres of the bovine coronavirus, matting and bridging them, when present in excess, and facilitated formation of large viral aggregates when present in equivalent concentrations. Complement added to the virus-antibody complexes did not alter specifically the morphology of single, antibody-laden viral particles or viral particles in aggregates. Evidence of the transmission of coronavirus from experimentally inoculated calves to man, with ensuing gastroenteritis, was found by electron microscopic tracing of the coronavirus and its virus-antibody complexes.

Maculopapular, pock-like lesions were detected repeatedly on hands and arms of students having contact with sheep or cattle. Pustular lesions were seen on the mammary gland and vulva of a goat. Biopsy samples of the lesions from 3 students and the goat were cultured on bovine fetal spleen (BFS) cells. Viruses were isolated from all samples. The viral isolates induced similar cytopathic changes in BFS cells. Slowly spreading plaque-like changes consisting of large, round cells developed in BFS monolayers. In a growth curve experiment involving the Shoe strain and BFS cells, virus infectivity increased 8 hr after inoculation. The major portion of the infectivity remained associated with the cell fraction. Maximal titers were found 36 hr after infection. Eosinophilic inclusions of varying size were detected in the cytoplasm 8–18 hr after infection of BFS cells. Later these inclusions enlarged, coalesced, stained basophilically, and remained attached to the pycnotic nuclei. Vacuoles created a ballooning appearance and separated the plasma membrane from the nucleus-viral inclusion complex by 24 hr after infection when the cells detached. Negatively stained preparations of purified virus of the isolates contained ovoid virions covered with diagonally woven bands. The long axis of the virions measured 220 and 250 nm and the short axis was 120–140 nm. These features are characteristic of the parapox virus genus which includes members that induce orf.

Summary The bovine coronavirus strain LY-138 was purified by differential as well as velocity and isopycnic centrifugation in sucrose or CsCl gradients. The substrate for purification was contents of the small intestine of experimentally inoculated calves. This strain is highly enteropathogenic, but it could not yet be propagated in cultured cells. Intact virions had a density of 1.245 g/cm3 in CsCl and 1.185 g/cm3 in sucrose. A spherical core-like structure with an average diameter of 82 nm remaining after treatment with chloroform had a density of 1.299 g/cm3 in CsCl and 1.201 g/cm3 in sucrose. Seven distinct bands of polypeptides and 4 shoulders were detected after electrophoresis of SDS-solubilized virions in polyacrylamide gels. The approximate molecular weights ranged from 110,000 to 36,000. Four of the bands gave a PAS positive reaction. These 4 glycoproteins and an additional protein with an approximate molecular weight of 70,000 were removed by chloroform treatment. The remaining core-like structure contained the 2 polypeptides VP3 and VP7.

Parvovirus replication is dependent upon progression of host cells through S phase of the cell cycle. Tritiated thymidine pulse-labeling was used to monitor host cellular DNA synthesis in order to study the development of nuclear alterations in bovine fetal spleen (BFS) cells infected with bovine parvovirus (BPV). Cultures of uninfected BFS cells synthesized DNA parasynchronously between 18 and 48 hr after planting. Cells were infected between 12 and 18 hr after planting to take advantage of the maximum number of S phase cells. Development of intranuclear inclusions in parasynchronous cells infected with four strains of BPV was studied by light and electron microscopy. Nucleopathic changes consisting of nucleolar stranding, increased creased basophilia and degranulation of the nucleoplasm were detected 12 hr post-infection (PI). A second early alteration was replacement of the nucleoplasm with a fine-grained homogeneous material by 18 hr PI. Cowdry type A and novel multifocal inclusions developed in cells with early changes beginning at 18 to 24 hr PI. Electron microscopic studies revealed discrete foci of empty virus capsids and virions in the nuclei which appeared to correspond with the foci in multifocal inclusions. These foci infer subnuclear compartmentalization of parvovirus replication. The uniqueness and striking morphology of multifocal inclusions have diagnostic significance.

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 chlamydia-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.

Tissue samples were removed at necropsy from five American bison (Bison bison) with clinical signs of a disease resembling malignant catarrhal fever (MCF). Using cell associated virus techniques, attempts were made to isolate viruses from these tissues by culturing them directly or by co-culture with bovine fetal cells. Among the viruses isolated was one which was syncytiogenic and multiplied in bovine fetal spleen cells and remained highly cell-associated. The presence of reverse transcriptase activity indicated that it was a retrovirus. Also, it had antigenic cross activity with bovine syncytial virus, but not with bovine leukemia or bovine maedi-like retroviruses. We do not attribute a direct causative role of this retrovirus to MCF, but indirect relationships are possible.

Strains of Chlamydia psittaci from cattle, sheep, pigs, mice, guinea pigs, rabbits, cats, and parrots were subdivided based on their biological characteristics. Chlamydiae grown in the yolk sac of chicken embryos were used to infect L cell monolayers. The host cells were infected without further treatment or treated with diethylaminoethyl-dextran, cycloheximide, or both. The following criteria were used for biotyping the strains: the morphology of the inclusions and time after infection at which they appeared, the effect of chlamydial multiplication on the host cell cytoskeleton, and the change in the number of cells infected in response to diethylaminoethyl-dextran and cycloheximide. These properties were determined for 29 strains of C. psittaci. Based on the results, the strains were placed into eight biotypes.

L cells inoculated at multiplicities of infection greater than or equal to 1 inclusion-forming unit of the abortigenic chlamydial strain B577 were destroyed within 10 to 15 days. Upon continued incubation in fresh medium, a few surviving cells repopulated the flasks, and the reemerging cultures remained persistently infected. The persistent state was characterized by cycles of repopulation with a low ratio of infected cells and cycles of extensive cytopathic changes in which greater than 90% of the cells had chlamydial inclusions and which could be delayed or even terminated by penicillin treatment. Immunofluorescence and superinfection during the period of repopulation revealed that the persistently infected cells could adsorb chlamydiae but their multiplication was arrested. This nonpermissive state could be terminated by the specific action of cycloheximide. L cells spontaneously cured from a persistent infection exhibited no change in susceptibility to chlamydiae when compared with normal L cells. However, chlamydiae derived from L cells after 7.5 months of persistence destroyed L-cell monolayers more rapidly and at lower multiplicities of infection than the wild type. This state of chlamydia-host cell interaction could not be established with the arthropathogenic strain LW613 because chlamydial infectivity was lost after the first cytolytic burst of infection in the cell cultures. The persistence described for the strain B577-L-cell system appears to differ from previously described models involving other chlamydial strains.

The major antigen involved in neutralization of BHV-1 was a glycoprotein with an apparent molecular weight of 93,000 in SDS polyacrylamide gels. Although antigen analysis did not allow differentiation between BHV-1, a differentiation into “IBR-like” and “IPV-like” strains was possible using restriction enzyme analysis (HpaI). Eight BHV-1 isolates from normal foetuses belonged to the “IBR-like” viruses. From the data available it was concluded that only “IBR-like” strains have the information for induction of abortion.

Summary A herpesvirus isolated from several organs of an American bison affected with malignant catarrhal fever was cultured in bovine foetal spleen cells and studied by electron microscopy. The fine structural features of the mature virion and the mode of virus morphogenesis were found to be similar to herpesviruses classified in the subgroup cytomegalovirus. The capsids were granular, hexagonal in shape and contained pleomorphic cores in thin sections. Envelopment of the capsids occurred primarily by budding on cytoplasmic membranes which appeared to be formed as extended vesicles of the Golgi apparatus; budding on nuclear membranes was only rarely observed. Cytoplasmic inclusions consisting of granular threads and amorphous electron-dense material were found in association with virions during the late stages of infection. The formation of cytoplasmic inclusions, the morphogenesis and ultrastructure of the virus are all consistent with classification of this virus as a cytomegalovirus.

Summary The 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.

Three chlamydial strains were isolated from the lungs of three different strains of mice that were inbred for over 30 years. These chlamydial strains were resistant to sulfadiazine and did not induce iodine-positive, glycogen-like material in the inclusions of infected L cells which characterizes them as Chlamydia psittaci .

Changes of Levels of Infectivity in Incubated Sub-Cellular Elements from Western-Equine-Encephalitis-Infected and Normal Chicken Embryo Cells