Bovine Respiratory Syncytial Virus Strain Research Articles

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 μl (TCID 50/100 μl). 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.

Restriction enzyme analysis of RT-PCR amplicons as a rapid method for detection of genetic diversity among bovine respiratory syncytial virus isolates

Our current knowledge of antigenic variability of the bovine respiratory syncytial virus (BRSV) is quite limited and is mainly dependent on the use of monoclonal antibodies (mAb). In this study, we present not only analysis of the antigenic, but also of the genetic variability of BRSV. Using a panel of BRSV-specific mAb we distinguished five main reactivity patterns, three of which corresponded to the previously established subgroups A, B and AB. A single viral strain yielded the fourth pattern, while four viral strains did not react with any of the used mAbs forming the fifth pattern. To investigate the genetic basis for the antigenic heterogeneity of the BRS virus G protein, DNA of 11 BRSV isolates was directly sequenced. The comparison of the obtained nucleotide or amino acid sequences to those BRSV strains present in the GenBank revealed 88.1-99.4% and 77.7-98.4% similarity, respectively. These results supported the previously stated suggestion to type BRSV isolates according to their genetic relationship. In order to introduce a rapid and simple method to study the genetic variability of BRSV, we utilized the restriction enzyme analysis of RT-PCR products derived from mRNAs corresponding to the most variable region of the BRSV glycoprotein G ectodomain. Using this restriction enzyme analysis we were able to identify genetic variability among BRSV isolates. The detected non-synonymous mutations led frequently to a change in digestion pattern and were predominantly located in two mucin-like regions of the G protein gene. A correlation has been found between grouping of isolates in the phylogenetic tree and their restriction patterns clustering together isolates with the same restriction profiles. However, viruses placed distant in the tree sharing the same restriction patterns were detected supposing that phylogenetic analysis should be necessary for BRSV typing. Thus, we propose to use DNA restriction polymorphism for a rapid detection of genetic variants among BRSV isolates circulating in cattle population and as a preliminary tool for their typing.

Detection of Brazilian bovine respiratory syncytial virus strain by a reverse transcriptase-nested-polymerase chain reaction in experimentally infected calves

A reverse transcriptase (RT)-nested-polymerase chain reaction (PCR) was standardised to detect bovine respiratory syncytial virus (BRSV), using a Brazilian isolate, in three experimentally infected calves. This followed initial tests in infected chicken embryo related (CER) cells. One animal had lesions, characterized by interstitial multifocal pneumonia, severe interstitial and subpleural emphysema, and lung consolidated areas. Lung and tracheal tissues collected 6 days after infection were analysed by RT-nested-PCR. Primers, specific for the BRSV G and F glycoproteins genes, yielded amplification fragments of 371 and 481 bp, respectively, from the RNA of the cell-propagated virus. Using RNA extracted from organs of infected calves, RT-nested-PCR amplified the fragment of the G gene in all tracheal samples, but in only two of three lung samples analysed. These results suggest that RT-nested-PCR could be a promising assay for diagnosis and epidemiological analysis of BRSV in Brazil.

Rescue of bovine respiratory syncytial virus from cloned cDNA: entire genome sequence of BRSV strain A51908.

Infectious bovine respiratory syncytial virus (BRSV) was produced by intracellular co-expression of five plasmid borne cDNAs, each under the control of a T7 RNA polymerase promoter. These separately encoded a full-length, genetically-marked copy of BRSV antigenome along with either BRSV or human respiratory syncytial virus (HRSV) support plasmids, which express N, P, L and M2-1 proteins. HEp2 cells were used in transfection and recombinant vaccinia virus (MVA-T7) provided T7 RNA polymerase to drive the transcription. The recovery of recombinant BRSV (rBRSV) was confirmed by immunological staining of plaques, restriction enzyme digestion and nucleotide sequencing of PCR fragments carrying the genetic markers from the rescued virus. The rBRSV was indistinguishable from its parental wild-type virus in its growth characteristics in cell culture. The present work has completed the entire genome sequence of BRSV strain A51908 (15,140 nt) and has also identified changes in sequence and growth characteristics in cell culture from the original BRSV strain A51908 laboratory isolate.

Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter.

In order to generate recombinant bovine respiratory syncytial virus (BRSV), the genome of BRSV strain A51908, variant ATue51908, was cloned as cDNA. We provide here the sequence of the BRSV genome ends and of the entire L gene. This completes the sequence of the BRSV genome, which comprises a total of 15,140 nucleotides. To establish a vaccinia virus-free recovery system, a BHK-derived cell line stably expressing T7 RNA polymerase was generated (BSR T7/5). Recombinant BRSV was reproducibly recovered from cDNA constructs after T7 RNA polymerase-driven expression of antigenome sense RNA and of BRSV N, P, M2, and L proteins from transfected plasmids. Chimeric viruses in which the BRSV leader region was replaced by the human respiratory syncytial virus (HRSV) leader region replicated in cell culture as efficiently as their nonchimeric counterparts, demonstrating that all cis-acting sequences of the HRSV promoter are faithfully recognized by the BRSV polymerase complex. In addition, we report the successful recovery of a BRSV mutant lacking the complete NS2 gene, which encodes a nonstructural protein of unknown function. The NS2-deficient BRSV replicated autonomously and could be passaged, demonstrating that NS2 is not essential for virus replication in cell culture. However, growth of the mutant was considerably slower than and final infectious titers were reduced by a factor of at least 10 compared to wild-type BRSV, indicating that NS2 provides a supporting factor required for full replication capacity.

Genetic and antigenic analysis of the G attachment protein of bovine respiratory syncytial virus strains.

Antigenic and genetic studies of bovine respiratory syncytial virus (BRSV) were made on isolates obtained from three continents over 27 years. Antigenic variation between eight isolates was initially determined using protein G-specific monoclonal antibodies. Four distinct reaction patterns were observed, two of which corresponded to the previously established subgroups A and AB. A third pattern was produced by five Scandinavian strains and a fourth was observed from a single Dutch isolate. The genetic diversity of 27 strains of BRSV was investigated by comparative nucleotide sequence analysis of a 731 nucleotide fragment in the G protein gene. Nine of the BRSV strains were analysed by direct sequencing of RT-PCR amplicons whereas sequences of 18 BRSV and three human respiratory syncytial virus (HRSV) strains were obtained from GenBank. The analysis revealed similarities of 88-100% among BRSV strains and 38-41% between BRSV and HRSV. A phylogenetic tree created for BRSV revealed two main branches, one of which divided into five further lineages, each representing a geographic cluster. A correlation was evident between the positions of some strains in the phylogenetic tree and their antigenic pattern. For HRSV strains, a genetic similarity of only 62% allowed the distinction of two antigenic subgroups, A and B, a pattern which was not seen for BRSV. This study showed that genetic analysis was an accurate method for discriminating BRSV strains and that these viruses should be regarded as a single genetic and antigenic group, within which variants can be distinguished.

A bovine respiratory syncytial virus strain with mutations in subgroup-specific antigenic domains of the G protein induces partial heterologous protection in cattle

Bovine respiratory syncytial virus (BRSV) strains are tentatively divided in subgroups A, AB and B, based on antigenic differences of the G protein. A Dutch BRSV strain (Waiboerhoeve: WBH), could not be assigned to one of the subgroups, because the strain did not react with any monoclonal antibody against the G protein. We describe here that the WBH strain has accumulated critical mutations in subgroup-specific domains of the G protein gene, which also occur but then independently in G protein genes of BRSV subgroup A or B strains. Although the comparison of nucleotide residues 256–792 of the G gene of the WBH strain with those of subgroup A and B strains showed that the G gene of the WBH strain is different from that of BRSV subgroup A and B strains, the sequence divergence was not more than observed within the G genes of human respiratory syncytial virus subgroup A or B strains. The WBH strain did not induce severe disease after experimental infection of calves, and induced partial protection against a heterologous challenge. Despite the dissimilarity of the conserved central regions of the G protein of the WBH strain and that of the challenge strain, a secondary antibody response against this region was induced in WBH-infected calves after challenge. We conclude that complete BRSV virus can partially protect against a BRSV infection with a strain that contains an antigenic dissimilar G protein.

Immune response of calves experimentally infected with non-cell-culture-passaged bovine respiratory syncytial virus.

The immune response of calves was studied following infection with non-cell-passaged Bovine respiratory syncytial virus (BRSV). Two groups of 6 specific pathogen free (SPF) calves were housed in separate isolation rooms. One group was inoculated intranasally with a non-cell-passaged BRSV strain and the control group was mock-infected. A BRSV specific antibody response was observed for all the BRSV infected calves. These antibodies were shown to have neutralizing activity. No lymphocyte proliferation response was detected in the mock-infected group whereas three animals in the infected group were positive three weeks after the infection. All BRSV-infected calves, except one, produced interferon-gamma (IFN-gamma) one week post-infection and IFN-gamma was observed in all six infected calves after three weeks. The control group showed no IFN-gamma synthesis. In spite of the limits of the BRSV infection model, humoral and cellular immune responses were actively developed by all the calves against this pathogen.

Analysis of the bovine respiratory syncytial virus fusion protein (F) using monoclonal antibodies

Seven monoclonal antibodies (MAbs) directed against bovine respiratory syncytial virus (BRSV) fusion (F) protein were produced and characterized by radioimmunoprecipitation and immunofluorescence assays. These seven MAbs together with the previously described MAbs (Beeler and Van Wyke Coelingh, 1989) to the F protein of human respiratory syncytial virus (HRSV) were used to study the antigenic variation of 12 strains of ungulate RSV. All except one MAbs specific for the HRSV-F protein reacted with ungulate RSV strain less efficiently, indicating that some epitopes are conserved, and others are not conserved on the F proteins of HRSV and BRSV strains. Three MAbs specific to the BRSV-F protein neutralized virus infectivity and reacted with all the ungulate RSV strains, suggesting that these epitopes are well conserved. Based on the reactivity of three other MAbs specific to the BRSV-F protein, ungulate RSVs could be grouped into two subgroups. The results indicated that there are antigenic variations in the F protein among ungulate RSV strains.

Role of individual N-linked oligosaccharide chains and different regions of bovine respiratory syncytial virus fusion protein in cell surface transport.

The role of N-linked glycosylation and various domains of the fusion (F) protein of bovine respiratory syncytial virus strain A51908 during transport to the cell surface were determined by mutating the four glycosylation sites and by constructing various truncation mutants. The cell surface expression of various mutant proteins was detected by surface immunofluorescence and cell surface biotinylation assay. We found that the glycosylation at positions Gly1, Gly2, Gly3, and a truncation mutant with a cytoplasmic domain deletion play little or no role in transport to cell surface. However, glycosylation at position Gly4, and the truncation mutants having deletion in the signal peptide, the F2 + signal peptide domain, and the membrane anchor domain, had impaired transport to the cell surface. This indicated the importance of these glycosylation sites and domains in facilitating the transport of the F protein to the cell surface.

Antigenic and molecular analyses of the variability of bovine respiratory syncytial virus G glycoprotein.

Antigenic variation among eight bovine respiratory syncytial virus (BRSV) isolates was determined using monoclonal antibodies (MAbs) specific for the attachment (G) protein. Two major (and one intermediate) subgroups were identified, as well as one strain that did not fit any pattern. The subgroups could also be differentiated on the basis of the Mr of the F protein cleavage product, F2. The nucleotide sequence of the G gene of seven of the BRSV strains was determined and compared with published G gene sequences. Subgroups A and A/B were more closely related in protein sequence than subgroups A and B or subgroups A/B and B. These results could not be correlated with those obtained by the determination of the Mr of the F2 polypeptide. Multiple sequence alignments showed a high level of amino acid identity at the inter-subgroup level (85% identity between subgroup A and subgroup B strains), similar to the intra-subgroup human (H)RSV identity, suggesting that the BRSV isolates form a continuum rather than distinct subgroups. However, unusual variability was observed within the immunodominant domain (amino acids 174-188) in contrast with the situation in HRSV strains belonging to the same subgroup.

Subgrouping of bovine respiratory syncytial virus strains detected in lung tissue

Bovine respiratory syncytial virus is an important respiratory pathogen in cattle. Recently, subgroups of BRSV have been identified, based on antigenic differences. However, little is known about subgroups of BRSV that circulate in the cattle population. Therefore, we determined the reactivity of monoclonal antibodies (mAbs), directed against the G, F, or P protein of BRSV, with lung tissue from 47 calves, that suffered from severe respiratory disease. Fourteen animals (30%) proved to be infected with BRSV, because they all reacted with mAbs against the P or F protein, as detected by fluorescent antibody tests. Monoclonal antibodies against the G protein were able to discriminate between the BRSV-positive specimens: 7 strains were identified as subgroup A strains, and 5 strains as subgroup AB, which is introduced as BRSV subgroup in this paper. Two strains could not be identified unambiguously. It is concluded that BRSV subgroup A and AB were associated with severe respiratory disease, and that strains belonging to either subgroup circulated concurrently in the cattle population in the Netherlands.

Assembly of enveloped respiratory syncytial virus particles within the cytoplasm of infected Vero cells.

Electron microscopic examination of syncytia induced by a bovine respiratory syncytial virus strain in Vero cell cultures revealed the presence in the cytoplasm of assembled enveloped virus particles within inclusions of variable sizes. Moreover, budding virus particles were shown occasionally in the intracytoplasmic vesicles. These particles were filamentous in form, about 80-120 nm in diameter, variable in length, containing 12 nm diameter nucleocapsids, and looked like the extracellular particles budding at the plasma membranes. This is the first report on assembly of intracellular virus particles in cells infected by a member of the family Paramyxoviridae. Vero cell-dependent variations appear to be the factor leading to the defect in the late virus replication cycle.

Infected Cell Types in Ovine Lung Following Exposure to Bovine Respiratory Syncytial Virus

Sixteen adult sheep (ten females, six males obtained from a closed flock at National Animal Disease Center, Ames, IA) were experimentally infected with bovine respiratory syncytial virus strain 375 (BRSV), and lung tissues were stained for viral antigen. Two infected sheep were euthanatized at each of the following post-inoculation times: 12, 24, 36, 48, 72, 96, 144, and 192 hours. Lung, nasal turbinates, trachea, right cranial bronchial and mediastinal lymph nodes, liver, and spleen were collected for histologic evaluation. An indirect immunoperoxidase technique was performed on routine paraffin-embedded sections of lung tissue, trachea, turbinates, and bronchial and mediastinal lymph nodes to determine the location of the BRSV antigen. For lung tissue from each sheep 400 light microscopic fields at 160x magnification were examined for staining for BRSV antigen. Lung tissue was also collected for virus and bacterial isolation. Daily serum samples were taken for determination of anti-BRSV titers. Severe respiratory disease was not produced in any sheep. Bovine respiratory syncytial virus was isolated from lung tissue collected from all sheep up through 144 hours post-inoculation. At 12 hours post-inoculation (case No. 2) respiratory syncytial virus antigen was detected in bronchiolar epithelium and a mononuclear cell within an alveolar space. Lung tissue from the sheep necropsied between 24 and 144 hours post-inoculation (case Nos. 3-14) contained BRSV antigen in bronchiolar epithelium, type I pneumocytes, type II pneumocytes, alveolar macrophages, and mononuclear cells within alveolar spaces. Macrophages staining for viral antigen were rare. Bronchiolar and type I epithelial cells comprised the majority of infected cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and sequence comparison of bovine respiratory syncytial virus fusion protein

The fusion (F) proteins of 10 strains of bovine respiratory syncytial virus (BRSV) were compared by radioimmunoprecipitation with fractionation on SDS-polyacrylamide gels. Two different molecular weights (15 kDa and 20 kDa) of the F2 proteins were demonstrated among the BRSV strains tested. To delineate the molecular basis for differences in the molecular weights of F2 subunits among the BRSV strains, the nucleotide sequences of the F genes of FS1 and VC464 strains were determined from cDNA clones. The deduced amino acid sequences were then compared to those of BRSV strains RB94, 391−2 and A51908. The F gene was highly conserved (> 95%) among BRSV strains. Comparison of the deduced F2 amino acid sequences showed that the strain with F2 subunits of 20 kDa had three N-linked glycosylation sites, whereas the strains with F2 subunits of 15 kDa had two N-linked glycosylation sites. Analysis of F2 subunits in their deglycosylated forms indicated that the difference in the molecular weights of the F2 subunits was due to the difference in the extent of glycosylation.

Sequence analysis of M2 mRNA of bovine respiratory syncytial virus obtained from an F-M2 dicistronic mRNA suggests structural homology with that of human respiratory syncytial virus

The nucleotide sequences of the F and M2 mRNAs of strain A51908 of bovine respiratory syncytial virus (BRSV) were determined by sequencing cDNA of an intracellular dicistronic mRNA. Comparison of the F mRNA sequence with those of other BRSV strains showed that there was extensive sequence identity at both the nucleotide (95% identity) and amino acid (94% identity) levels. Alignment of the nucleotide and encoded amino acid sequences of M2 mRNA of BRSV with those of human respiratory syncytial virus (HRSV) M2 mRNA showed 69% identity at the nucleotide level and 80% identity at the amino acid level. The general features of BRSV F and M2 proteins are similar to those described previously for the HRSV proteins. The M2 mRNA of BRSV also contained a second internal, overlapping open reading frame (ORF) similar to one reported for HRSV. The predicted products of the second ORFs of BRSV and HRSV shared 43% amino acid identity. As described for HRSV, the 3'-terminal end of the M2 mRNA overlaps with the 5' end of the L gene by 68 nucleotides. The identity between the N-terminal regions of the L proteins of BRSV and HRSV is 75%. In addition, the intergenic sequence of the F-M2 gene junction of BRSV was determined.

Effect of macrophages and in vitro infection with parainfluenza type 3 and respiratory syncytial viruses on the mitogenic response of bovine lymphocytes

Summary Bovine blood lymphocytes, depleted of macrophages by absorption on plasma-gelatin coated plastic flasks, followed by passage through Sephadex G-10 columns, failed to respond to pokeweed mitogen stimulation Adherent monocytes or alveolar macrophages added to purified lymphocyte preparations at 10% or less were able to restore the transformation response. Exposure of alveolar macrophages or purified lymphocytes to 2 bovine respiratory syncytial virus strains for 24 hours substantially reduced the transformation response when mixed with uninfected lymphocytes or macrophages. Exposure of alveolar macrophages or purified lymphocytes to 2 bovine parainfluenza type 3 virus strains produced a similar reduction in activity after 48 hours. Heat inactivation of the viruses removed their inhibitory ability. Immunofluorescence studies revealed that both alveolar macrophages and lymphocytes were permissive for parainfluenza type 3 virus, whereas only a small number of alveolar macrophages and lymphocytes were infected with respiratory syncytial virus. The results suggest that both viruses are capable of adversely affecting the interaction between macrophages and lymphocytes, although the mechanisms by which this is achieved may be different.

Preparation and Characterization of Monoclonal Antibodies Directed against Five Structural Components of Human Respiratory Syncytial Virus Subgroup B

Mouse hybridomas producing antibodies against the structural proteins of strain WV4843, a subgroup B strain of respiratory syncytial (RS) virus, were produced by fusion of Sp2/0 myeloma cells with spleen cells from BALB/c mice immunized with purified preparations of the virus. After immunoprecipitation test with [35S]methionine-labelled extracellular virions, 35 clones found to produce antibodies against the fusion (F) protein, six against the member (M) protein, 21 against the nucleocapsid (NP) and eight against the phospho- (P) protein were further characterized. Immunoprecipitation with [3H]glucosamine-labelled intracellular virus polypeptides detected nine hybridoma cell lines producing antibodies against the large glyco- (G) protein of the virus. By competitive binding ELISA tests with monoclonal antibodies against each of the structural components, a minimum of two, 24, four, 15 and three epitopes were detected on the G, F, M, NP and P proteins, respectively. Eleven monoclonal antibodies directed against nine epitopes of the F protein could neutralize the infectivity of the virus. In contrast, none of the nine monoclonal antibodies against G could neutralize the infectivity of the virus. In order to find out more about the antigenic relationship between human and bovine RS virus strains all monoclonal antibodies were reacted with subgroup A RS virus and also with three different strains of bovine RS virus and one strain of caprine RS virus in immunofluorescence, ELISA and immunoprecipitation tests. In addition, 31 previously developed monoclonal antibodies against subgroup A virus were reacted with the bovine and caprine strains. The numbers of monoclonal antibodies of subgroup B specific for the B type of the two human subgroups were 9/9, 3/35, 0/6, 0/21, 0/8, for the G, F, M, NP and P proteins, respectively. No antigenic variations were found between the three bovine strains and the caprine strain. They did not react with the nine monoclonal antibodies against the G protein of subgroup B, nor did they react with nine monoclonal antibodies against subgroup A. Most but not all of the monoclonal antibodies against the other structural proteins of the two human RS virus subgroups reacted with the four strains. All 11 monoclonal antibodies against the F protein of subgroup B that could neutralize the infectivity of subgroup B also reacted with the bovine strains and neutralized their infectivity. It is concluded that although the bovine strains share many epitopes with the two human subgroups, they are antigenically distinct from the human viruses.

Differences in fluorescent antibody staining of bovine respiratory syncytial virus-infected cells by ovine and bovine sera

In indirect fluorescent antibody tests in which sera from cattle and sheep with respiratory disease problems were used to stain foetal bovine lung cells infected with a bovine respiratory syncytial virus strain, differences were noted in the pattern of fluorescence produced by some sheep sera and that produced by positive bovine sera. In serum neutralisation tests, also using a bovine respiratory syncytial virus strain, 4 of 7 sera giving this atypical pattern of fluorescence had very low neutralising antibody titres (highest 1 4 ), and 3 were negative. It is suggested that two related but antigenically distinguishable respiratory syncytial virus types are present in sheep, one of which is similar to bovine strains.