Bovine respiratory syncytial virus

Blood, D. Radostits, O. M, Arundel, J. H, and Gay, C. From WikiVet English. Bovine Respiratory Syncytial Virus. Navigation menu Personal tools Create account Log in. Namespaces Page Discussion. Views Read View source View history.

Related topics. Antigens Cattle Lung Microbiology procedure. Papers overview Semantic Scholar uses AI to extract papers important to this topic. El complejo respiratorio bovino, consiste en una infeccion respiratoria, provocada por la combinacion de diferentes agentes… Expand. In the case of rhabdoviruses and most paramyxoviruses, N, P, and L proteins were found to be sufficient to render full-length antigenome RNAs infectious.

We report here the cDNA cloning of the entire BRSV genome and the establishment of a system allowing genetic manipulation of BRSV in order to provide tools for experimental analysis of pneumovirus molecular biology and for development of defined, attenuated vaccines. Moreover, the established vaccinia virus-free recovery system allowed us to isolate considerably attenuated virus mutants and to provide direct evidence that the NS2 gene is not essential for BRSV replication.

Titrations were carried out in duplicate in microwell plates by the limiting dilution method. An indirect immunofluorescence assay using a bovine serum specific to BRSV was done, and foci of infected cells were counted. The NS1 noncoding region preceding the translation start codon was modified to contain a synthetic Not I tag see Results. Subsequently, the HRSV leader sequence was replaced by the BRSV leader sequence by site-directed mutagenesis 22 in order to generate nonchimeric recombinants.

The locations of transcripts shaded bars and protein-encoding frames open bars are shown relative to the viral RNA vRNA solid bar. In the enlargements, the organizations of recombinant viruses and ATue are compared. Genetic tags Not I and nucleotide deletions in the NS1 noncoding regions shaded of the recombinant viruses are indicated. The relative positions of corresponding nucleotides are given for the gene starts of NS1, NS2, and N and nucleotides flanking the NS2 deletion.

The overall lengths of the vRNAs are shown on the right. The last step for generation of a recombinant full-length construct consisted of the insertion of the NS2 gene.

Inserts generated by PCR were sequenced completely. Two days after transfection, cells were stained with a fluorescein isothiocyanate conjugate recognizing rabies virus N protein Centocor and analyzed by immunofluorescence microscopy not shown. One hour before transfection, cells were washed twice with medium without FCS. Transfection experiments were carried out with a mammalian transfection kit CaPO 4 transfection protocol; Stratagene. Five days after transfection, cells were split at a ratio of Between 7 and 10 days posttransfection, a typical CPE was observed, yielding several foci per dish.

The cells were split every 4 to 5 days, until between days 21 and 28 posttransfection a total CPE was observed. The virus was released by freezing and thawing. To generate DNA probes for hybridization, for cloning of expression plasmids, and for RNA analysis of recombinant virus, RT-PCR was done with avian myeloblastosis virus reverse transcriptase Life Sciences for first-strand synthesis, as described above, and a proofreading thermostable polymerase Pfu ; Stratagene for PCR under conditions recommended by the supplier.

The first was designed to demonstrate the synthetic Not I restriction site following the NS1 gene start. The reverse primer ATue nt to was derived from the NS1 gene sequence. First-strand synthesis was done with the same set of leader-specific primers, whereas a primer derived from the N gene sequence ATue nt to served as reverse primer. Each probe was tested for specificity by sequence analysis and by hybridization with virus RNA before use. Clones covering the entire L gene were obtained by genome walking.

Besides the correctly primed cDNA clones, we found a considerable amount of BRSV-specific clones that were random primed, together covering the entire genome except for the NS2 gene. A consensus sequence was derived from at least three independent cDNA clones. The last 1. In this case, at least six different PCR clones were analyzed to generate a consensus sequence. The total sequence identity was found to be Together, the predicted amino acid sequences of the nine genes contain 97 differences compared to the A sequence, over a total of 2, amino acids.

Since the sequence data obtained differed substantially from the published A sequence, we designated our starting virus BRSV strain ATue The predicted open reading frame codes for a protein of 2, amino acids. No additional open reading frames longer than 30 amino acids were identified. This feature therefore appears to be common in the Pneumovirinae subfamily. The terminal regions are highly conserved.

Compared to HRSV, the first 17 nt are identical and the first 28 nt contain two differences. Numbering of the trailer sequences starts with the first nucleotide downstream of the L gene transcription stop signal. The sequences are shown as DNA positive strands. Only nucleotides differing from the recombinant sequence are indicated.

Gaps are indicated by dots, and the NS1 gene start signal and translation start codon are underlined. This is thus far the longest trailer sequence of a member of the Paramyxoviridae family. The BRSV leader and trailer regions themselves exhibit a terminal complementarity of the extreme 11 nt which is interrupted at position 4, as is also found in TRTV. Three G residues were introduced between the promoter and the leader sequence to facilitate initiation of transcription.

Thus, the NS1 translation stop codon is followed by the gene end signal derived from the NS2 gene. Due to the cloning procedure, 4 nt from the wild-type noncoding sequence between the NS1 translation stop codon and the NS1 gene end signal nt to were deleted.

One of the exchanges is located in the BRSV trailer sequence nt , T to C and the other in the L coding sequence nt , A to C , resulting in an amino acid change from isoleucine to leucine. Most systems for recovery of negative-strand RNA viruses make use of vaccinia helper virus providing T7 RNA polymerase for intracellular transcription of full-length RNAs and expression of support proteins. However, to prevent possible interference with the notoriously very slow BRSV replication or virus assembly and to avoid the necessity of separating vaccinia virus from BRSV, we decided to establish a vaccinia virus-free system.

The present paper contains an updated review on BRSV covering most aspects of the structure, molecular biology, pathogenesis, pathology, clinical features, epidemiology, diagnosis and immunology based on approximately references from international research journals. These references are in PubMed. This may not be the complete list of references from this article. National Center for Biotechnology Information , U. Journal List Acta Vet Scand v.

Acta Vet Scand. Published online Mar 1. Author information Article notes Copyright and License information Disclaimer. Larsen, Email: kd. Corresponding author. Received Feb 10; Accepted Dec This article has been cited by other articles in PMC. Functional diversity of helper T lymphocytes. Experimental infection of lambs with bovine respiratory syncytial virus and Pasteurella haemolytica: clinical and microbiologic studies.

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