Sequence conservation in the attachment glycoprotein and antigenic diversity among bovine respiratory syncytial virus isolates.

Partial nucleotide sequences were determined from the coding regions of the attachment glycoprotein (G) mRNAS of eight isolates of bovine respiratory syncytial virus (BRSV). The antigenic characteristics of 18 field and reference isolates were analyzed using the reactivity patterns of monoclonal antibodies (MAbs) directed against the human respiratory syncytial virus (HRSV) and BRSV G. fusion protein (F), nucleoprotein (N), and phosphoprotein (P), by radioimmunoprecipitation and immunofluorescence assays. The MAb reaction patterns demonstrated some random antigenic differences among the isolates, but for the most part were cross-reactive to the viral protein epitopes, especially on the F protein. Structural differences in the F and P proteins were observed among BRSV isolates; the P protein migrated at three different apparent molecular weights on PAGE gels. Antigenic and structural variation occurs among isolates, however, the structural differences in the P protein did not correlate with the antigenic differences among the F, N and P proteins. The G mRNA nucleotide sequence identities were high, ranging from 94.1 to 99.9%, and the predicted amino acid sequence identities ranged from 89.9 to 99.6%. Variance was due to substitution point mutations. The G protein ectodomains contained areas of sequence divergence flanking a highly conserved region, with four cysteine residues, which is analogous to the putative HRSV receptor binding domain. The high sequence and amino acid identities and random antigenic diversity among the isolates indicates that the BRSV isolates analyzed belong in a monophyletic group.

Bovine herpesvirus-1 infection reduces bronchial epithelial cell migration to extracellular matrix proteins.

Repair of airway epithelium after viral infection involves migration of epithelial cells to cover injured, denuded areas. We determined whether viral infection reduces the capability of bronchial epithelial cells to migrate and to attach to extracellular matrix proteins. Inoculation of bovine bronchial epithelial cells in vitro with bovine herpesvirus-1 reduced their ability to migrate in two different assays of cell migration. When attachment assays were performed, fewer cells attached to both control wells and matrix protein-precoated wells, suggesting that general mechanisms of adherence to substrates were altered by viral infection. Focal contact points of epithelial cells with the underlying matrix were evaluated with epifluorescence microscopy and monoclonal antibodies to vinculin and alpha v, an integrin chain. Disruption of focal contact points was seen early after infection and was prevented by an inhibitor of viral DNA polymerase, phosphonoacetic acid. Cycloheximide did not cause similar disruptions of focal contacts at early time points. Viral infection thus has marked effects on the interactions of bronchial epithelial cells with extracellular matrix and the organization of matrix to cytoskeleton links. The effects appear to be dependent in part on viral replication in the cells and are not simply due to reductions in host cell protein synthesis.

Viral infection of bovine bronchial epithelial cells induces increased neutrophil chemotactic activity and neutrophil adhesion.

1. Acute bronchitis secondary to viral infection is associated with an influx of neutrophils. We hypothesized that bronchial epithelial cells are capable of releasing neutrophil chemotactic activity in response to viral infection. 2. To test this hypothesis, primary cultures of bovine bronchial epithelial cells were inoculated with a bovine respiratory pathogen, bovine herpes virus-1. 3. Supernatants collected from inoculated cells, before signs of toxicity, demonstrated significant neutrophil chemotactic activity using a blind well chamber neutrophil chemotaxis assay. Lipoxygenase inhibitors markedly reduced the amount of neutrophil chemotactic activity released after bovine herpes virus-1 inoculation. Analysis of arachidonic acid metabolites in cell supernatants by reverse-phase h.p.l.c. confirmed that leukotriene B4, a potent neutrophil chemoattractant, was released. 4. We also confirmed that adhesion of neutrophils to bovine herpes virus-1-inoculated bronchial epithelial cells was increased and mediated in part by the neutrophil integrin, LFA-1. 5. Thus, virally infected airway epithelial cells release leucocyte chemoattractants and hence adhesive interactions, functions that are likely to be important in the inflammatory acute response to viral infection.

Identifying bovine respiratory syncytial virus by reverse transcription-polymerase chain reaction and oligonucleotide hybridizations.

An assay to identify tissue culture cells infected with bovine respiratory syncytial virus (BRSV) that utilizes reverse transcription (RT), the polymerase chain reaction (PCR), and a synthetic oligonucleotide hybridization probe has been developed. The RT-PCR assay uses a BRSV-specific negative-sense oligonucleotide primer to synthesize cDNA from a BRSV fusion protein mRNA template and another BRSV-specific oligonucleotide primer (positive sense) upstream from the negative-sense primer for PCR amplification. In the presence of mRNA templates of BRSV isolates originating from locations throughout the United States, the BRSV RT-PCR assay resulted in amplified products (381 bp) that were specific to BRSV, as demonstrated in hybridizations with a positive-sense oligonucleotide probe complementary to internal sequences and in sequence comparisons with the F protein of BRSV 391-2. In analysis of the BRSV RT-PCR assay with prototype strains of human RSV subgroups A and B, amplification of a similar 381-bp RT-PCR product was not evident, and no RT-PCR product hybridized with the internal probe. We conclude that the specific ability to amplify DNA sequences of BRSV F protein mRNA by RT-PCR and then to demonstrate the presence of the amplified product with a BRSV-specific oligonucleotide probe will greatly add to the speed, sensitivity, and specificity of BRSV diagnostics.

Monitoring bovine viral diarrhea virus vaccines for adventitious virus, using T1 ribonuclease viral RNA oligonucleotide fingerprinting.

Viral RNA oligonucleotide fingerprinting was used to discriminate 3 cytopathic vaccine bovine viral diarrhea viruses (BVDV) grown in medium supplemented with serum contaminated with noncytopathic BVDV from the same 3 viruses grown in cell culture free of BVDV. Oligonucleotide fingerprinting also effectively discriminated between reference Singer BVDV, NADL BVDV, and New York-1 BVDV grown in BVDV-free noncontaminated or BVDV-contaminated cell cultures. Oligonucleotide fingerprint mapping of viral RNA maybe used to determine the purity of virus stocks, as well as that of BVDV vaccines.

Genetic comparison of ovine and bovine pestiviruses.

Viral RNA oligonucleotide fingerprinting was used to compare genetic relationship among pestiviruses originating from ovine or bovine host species. Ovine pestiviruses, including reference border disease virus and 2 border disease isolates originating from natural pestivirus infections of sheep, appeared to have a more distant genetic relationship among themselves than with certain bovine pestiviruses. A closer genetic relatedness was evident between border disease virus and 3 noncytopathic bovine pestiviruses, including Draper bovine viral diarrhea virus (BVDV), a BVDV isolate that originated from aborted bovine fetuses, and a virus that was isolated from the serum of a calf that had a chronic BVDV infection. Four noncytopathic bovine viruses, including Draper BVDV and 3 field isolates, were closely related. Reference Oregon C24V BVDV, a cytopathic virus, was closely related to only 1 of the 7 noncytopathic viruses in this study.

Investigation of bovine viral diarrhea virus infections in a range beef cattle herd.

Bovine viral diarrhea virus (BVDV) infections resulting in clinical disease developed in calves, despite vaccination of dams and high maternal BVDV antibody titers in calves. Eight persistently infected (PI) calves born to immunocompetent dams were identified in the herd. Neutralizing BVDV antibody titers of PI calves had decreased greatly by the time the calves were 1 to 2 months old. Antibody titers of PI calves decreased more rapidly than antibody titers of calves that were not PI. Reduced antibody titers in PI calves allowed detection of BVDV in serum specimens of all PI calves by the time they were 8 weeks old. Persistent infection in suspect calves was detectable serologically and was confirmed by virologic examination of serum specimens 4 months after weaning, when the calves were 9 months old. Growth rates were reduced in viremic calves.