Use of different cell lines for in vitro cultures of bovine respiratory syncytial virus.

This study compared the use of different cell lines for in vitro cultures of bovine respiratory syncytial virus (BRSV). The BRSV 375 strain and 3 nasal swabs obtained from Simmental calves were used for this study. The culture was performed on 3 cell lines: bovine kidney cells (LLC-PK1), bovine tracheal cells (TBTR) and primary chicken embryo-related cells (CER). A comparative analysis of titres was performed using a microplate agglutination test with human group O erythrocytes and bovine erythrocytes. The presence of BRSV in all cell lines was confirmed using the reverse transcriptase polymerase chain reaction (RT-PCR) method. The first small refractile changes in the LLC-PK1 cells occurred at 48h after infection. Syncytial changes were noted 4 days after incubation. Large refractile cell changes were observed on day 3 of growth in the TBTR culture. Syncytia were observed on the second day after infection in subsequent passages. The cytopathic effect in the CER cells occurred 24h after infection, and syncytia appeared after 3 passages. Changes in syncytia indicate an adaptation of the virus for the infection of cells other than tracheal cells in primary and secondary cultures. The highest viral titre was obtained using the TBTR line. The titres obtained in the LLC-PK1 and CER cultures averaged 10(1.86)/ml. The low virus titres in all culture types suggest the need for research aimed at the optimisation of culture conditions.

The probability of extinction in a bovine respiratory syncytial virus epidemic model.

Backward bifurcation is a relatively recent yet well-studied phenomenon associated with deterministic epidemic models. It allows for the presence of multiple subcritical endemic equilibria, and is generally found only in models possessing a reasonable degree of complexity. One particular aspect of backward bifurcation that appears to have been virtually overlooked in the literature is the potential influence its presence might have on the behaviour of any analogous stochastic model. Indeed, the primary aim of this paper is to investigate this possibility. Our approach is to compare the theoretical probabilities of extinction, calculated via a particular stochastic formulation of a deterministic model exhibiting backward bifurcation, with those obtained from a series of stochastic simulations. We have found some interesting links in the behaviour between the deterministic and stochastic models, and are able to offer plausible explanations for our observations.

Differential sensitivity of differentiated epithelial cells to respiratory viruses reveals different viral strategies of host infection.

To address the initiation of virus infection in the respiratory tract, we established two culture systems for differentiated bovine airway epithelial cells (BAEC). Filter-grown BAEC differentiated under air-liquid interface (ALI) conditions to generate a pseudo-stratified mucociliary epithelium. Alternatively, precision-cut lung slices (PCLS) from the bovine airways were generated that retained the original composition and distribution of differentiated epithelial cells. With both systems, epithelial cells were readily infected by bovine parainfluenza virus 3 (BPIV3). Ciliated cells were the most prominent cell type affected by BPIV3. Surprisingly, differentiated BAEC were resistant to infection by bovine respiratory syncytial virus (BRSV), when the virus was applied at the same multiplicity of infection that was sufficient for infection by BPIV3. In the case of PCLS, infection by BRSV was observed in cells located in lower cell layers but not in epithelial cells facing the lumen of the airways. The identity of the infected cells could not be determined because of a lack of specific antibodies. Increasing the virus titer 30-fold resulted in infection of the ALI cultures of BAEC, whereas in PCLS the ciliated epithelium was still refractory to infection by BRSV. These results indicate that differentiated BAEC are readily infected by BPIV3 but rather resistant to infection by BRSV. Disease caused by BRSV may require that calves encounter environmental stimuli that render BAEC susceptible to infection.

Susceptibility of different cell lines to infection with bovine respiratory syncytial virus.

The growth of bovine respiratory syncytial virus (BRSV) was evaluated in six different cell lines. Chicken embryo related cells (CER), a chicken embryo fibroblast/baby hamster kidney hybrid and bovine CRIB cells (a bovine viral diarrhea virus-resistant clone of MDBK cells) showed to be the most appropriate for virus multiplication. Both cells provided infectious virus titres of up to 10(5.5) 50% tissue culture infective doses per 100 microl (TCID(50)/100 microl). One-step growth curves revealed no significant differences in the growth of BRSV in these two cell lines. Furthermore, they proved to be susceptible to infection with three different BRSV strains. It was concluded that both CER and CRIB cells may be used for laboratory multiplication of BRSV with optimal results.

Live-cell characterization and analysis of a clinical isolate of bovine respiratory syncytial virus, using molecular beacons.

Understanding viral pathogenesis is critical for prevention of outbreaks, development of antiviral drugs, and biodefense. Here, we utilize molecular beacons to directly detect the viral genome and characterize a clinical isolate of bovine respiratory syncytial virus (bRSV) in living cells. Molecular beacons are dual-labeled, hairpin oligonucleotide probes with a reporter fluorophore at one end and a quencher at the other; they are designed to fluoresce only when hybridizing to a complementary target. By imaging the fluorescence signal of molecular beacons, the spread of bRSV was monitored for 7 days with a signal-to-noise ratio of 50 to 200, and the measured time course of infection was quantified with a mathematical model for viral growth. We found that molecular beacon signal could be detected in single living cells infected with a viral titer of 2 x 10(3.6) 50% tissue culture infective doses/ml diluted 1,000 fold, demonstrating high detection sensitivity. Low background in uninfected cells and simultaneous staining of fixed cells with molecular beacons and antibodies showed high detection specificity. Furthermore, using confocal microscopy to image the viral genome in live, infected cells, we observed a connected, highly three-dimensional, amorphous inclusion body structure not seen in fixed cells. Taken together, the use of molecular beacons for active virus imaging provides a powerful tool for rapid viral infection detection, the characterization of RNA viruses, and the design of new antiviral drugs.

Respiratory syncytial virus (RSV) nonstructural (NS) proteins as host range determinants: a chimeric bovine RSV with NS genes from human RSV is attenuated in interferon-competent bovine cells.

Bovine respiratory syncytial virus (BRSV) escapes from cellular responses to alpha/beta interferon (IFN-alpha/beta) by a concerted action of the two viral nonstructural proteins, NS1 and NS2. Here we show that the NS proteins of human RSV (HRSV) are also able to counteract IFN responses and that they have the capacity to protect replication of an unrelated rhabdovirus. Even combinations of BRSV and HRSV NS proteins showed a protective activity, suggesting common mechanisms and cellular targets of HRSV and BRSV NS proteins. Although able to cooperate, NS proteins from BRSV and HRSV showed differential protection capacity in cells from different hosts. A chimeric BRSV with HRSV NS genes (BRSV h1/2) was severely attenuated in bovine IFN competent MDBK and Klu cells, whereas it replicated like BRSV in IFN-incompetent Vero cells or in IFN-competent human HEp-2 cells. After challenge with exogenous IFN-alpha, BRSV h1/2 was better protected than wild-type BRSV in human HEp-2 cells. In contrast, in cells of bovine origin, BRSV h1/2 was much less resistant to exogenous IFN than wild-type BRSV. These data demonstrate that RSV NS1 and NS2 proteins are major determinants of host range. The differential IFN escape capacity of RSV NS proteins in cells from different hosts provides a basis for rational development of attenuated live RSV vaccines.

Recombinant bovine respiratory syncytial virus with deletions of the G or SH genes: G and F proteins bind heparin.

Bovine respiratory syncytial virus (BRSV) encodes three transmembrane envelope glycoproteins, namely the small hydrophobic (SH) protein, the attachment glycoprotein (G) and the fusion glycoprotein (F). The BRSV reverse genetics system has been used to generate viable recombinant BRSV lacking either the G gene or the SH gene or both genes. The deletion mutants were fully competent for multicycle growth in cell culture, proving that, of the BRSV glycoprotein genes, the SH and G genes are non-essential. Virus morphogenesis was not impaired by either of the deletions. The deletion mutants were used to study the role of the F glycoprotein and the contributions of SH and G with respect to virus attachment. Attachment mediated by the F protein alone could be blocked by soluble heparin, but not by chondroitin sulphate. Heparin affinity chromatography revealed that both the BRSV G and F glycoproteins have heparin-binding activity, with the affinity of the F glycoprotein being significantly lower than that of G. Therefore, the roles of the BRSV glycoproteins in virus attachment and receptor binding have to be reconsidered.

Bovine respiratory syncytial virus nonstructural proteins NS1 and NS2 cooperatively antagonize alpha/beta interferon-induced antiviral response.

The functions of bovine respiratory syncytial virus (BRSV) nonstructural proteins NS1 and NS2 were studied by generation and analysis of recombinant BRSV carrying single and double gene deletions. Whereas in MDBK cells the lack of either or both NS genes resulted in a 5,000- to 10,000-fold reduction of virus titers, in Vero cells a moderate (10-fold) reduction was observed. Interestingly, cell culture supernatants from infected MDBK cells were able to restrain the growth of NS deletion mutants in Vero cells, suggesting the involvement of NS proteins in escape from cytokine-mediated host cell responses. The responsible factors in MDBK supernatants were identified as type I interferons by neutralization of the inhibitory effect with antibodies blocking the alpha interferon (IFN-alpha) receptor. Treatment of cells with recombinant universal IFN-alpha A/D or IFN-beta revealed severe inhibition of single and double deletion mutants, whereas growth of full-length BRSV was not greatly affected. Surprisingly, all NS deletion mutants were equally repressed, indicating an obligatory cooperation of NS1 and NS2 in antagonizing IFN-mediated antiviral mechanisms. To verify this finding, we generated recombinant rabies virus (rRV) expressing either NS1 or NS2 and determined their IFN sensitivity. In cells coinfected with NS1- and NS2-expressing rRVs, virus replication was resistant to doses of IFN which caused a 1,000-fold reduction of replication in cells infected with wild-type RV or with each of the NS-expressing rRVs alone. Thus, BRSV NS proteins have the potential to cooperatively protect an unrelated virus from IFN-alpha/beta mediated antiviral responses. Interestingly, BRSV NS proteins provided a more pronounced resistance to IFN in the bovine cell line MDBK than in cell lines of other origins, suggesting adaptation to host-specific antiviral responses. The findings described have a major impact on the design of live recombinant BRSV and HRSV vaccines.

Mutational analysis of the bovine respiratory syncytial virus nucleocapsid protein using a minigenome system: mutations that affect encapsidation, RNA synthesis, and interaction with the phosphoprotein.

The nucleocapsid (N) protein of bovine respiratory syncytial virus (BRSV) is a multifunctional protein that plays a central role in transcription and replication of viral genomic RNA. To investigate the domains and specific residues involved in different N activities, we generated a total of 27 deletion and 12 point mutants of the N protein. These mutants were characterized using an intracellular BRSV-CAT minigenome replication system for the ability to (1) direct minigenome RNA synthesis, (2) direct minigenome encapsidation, and (3) form a complex with the phosphoprotein (P). The mutations tested were defective in synthesis of RNA from the BRSV-CAT minigenome template with the exception of the following: a deletion involving the first N-terminal amino acid and mutations involving conservative substitution at the second amino acid and at certain internal cysteine residues. Micrococcal nuclease enzyme protection assays showed that mutations involving amino acids 1-364 of the 391-amino-acid N protein prevented minigenome encapsidation. Thus the BRSV N protein has a C-terminal, 27-amino-acid tail that is not required for encapsidation. Interestingly, two of the mutations that ablated encapsidation did not greatly affect RNA synthesis; the mutant involving deletion of the N-terminal amino acid and the mutant involving a substitution at position 2. This finding indicates that the formation of a nucleocapsid sufficient to protect the RNA from nuclease is not required for template function. Coimmunoprecipitation of N and P using N- or P-specific antiserum revealed two regions of the N protein that are important for association with the P protein: a central portion of 244-290 amino acids and a C-terminal portion of 338-364 amino acids.

Bovine respiratory syncytial virus replicates minimally in bovine alveolar macrophages.

The interaction between two different bovine respiratory syncytial virus (BRSV) strains and bovine alveolar macrophages (BAMs) was studied in vitro. Bovine respiratory syncytial virus replicated minimally in BAMs and most of the virus produced remained cell-associated. Approximately 1 out of 1,000 BAMs produced infectious virus, a number that further declined during the 7 days of culture. In contrast, BAMs exposed to bovine parainfluenza 3 virus (PI3V) produced high amounts of infectious virus. The number of BAMs that contained BRSV antigen depended on the antigen load of the inoculum and not on the infectivity of the virus. Antibody mediated enhancement of infection was not detected. It is concluded that bovine alveolar macrophages exhibit a high intrinsic resistance to BRSV, but not to PI3V.