Cotton rats were used to study the replication and pathogenesis of bovine parainfluenza virus type 3 (bPIV3) and to test the efficacy of the F and HN glycoproteins in modulating infection. In vitro cultures of cotton rat lung cells supported the growth of bPIV3 as shown by virus recovery, immunofluorescence, immunoprecipitation, and syncytium induction. Intranasal (i.n.) inoculation of cotton rats with 10 7 PFU resulted in peak recovery of virus after 2 days (8 ؋ 10 4 PFU/g of lung tissue) and significant bronchiolitis with lymphocyte infiltration 5 to 7 days postinfection. Immunohistochemical staining of lungs and trachea demonstrated that virus antigen-positive cells increased in frequency over the course of infection to a maximum on day 5. Serum antibody responses were evaluated by enzyme-linked immunosorbent assays (ELISA), hemagglutination inhibition (HAI), and serum neutralization (SN). Following a single i.n. inoculation, serum antibody levels were 1/40,960, 1/32, and 1/80, as detected by ELISA, HAI, and SN, respectively. When an intramuscular inoculation of 10 7 PFU was administered 10 days prior to the i.n. inoculation, a secondary response which resulted in an ELISA titer of 1/163,000, an HAI titer of 1/640, and an SN titer of 1/512 was induced. IN inoculation of recombinant adenoviruses type 5 containing the bPIV3 F or HN protein or a combination of the two viruses protected cotton rats from bPIV3 challenge. Protection was evaluated serologically by ELISA, HAI, and SN titers, histopathology, immunohistochemistry, and virus recovery.
Abstract. Accurate identification of bovine Parainfluenza type 3 virus in bovine respiratory disease requires dependable, sensitive, and specific techniques for detection in affected animals. Immunohistochemical testing can be a rapid and reliable means of demonstration of virus in tissues from suspect cases; however, this procedure is dependent upon the quality of the antisera directed against the viral antigens. The production of rabbit polyclonal and murine monoclonal antibodies directed against bovine Parainfluenza type 3 virus and techniques for their use in fresh-frozen and formalin-fixed paraffin-embedded tissues in immunofluorescence and immunoperoxidase-based immunohistochemical tests are described.
We constructed replication-competent human adenovirus type 5 (HAd5) recombinants (HAd5-HN and HAd5-F) containing the bovine parainfluenza virus type 3 (BPIV3) hemagglutinin-neuraminidase (HN) or fusion (F) gene under the control of the simian virus 40 (SV40) regulatory sequences. These genes were inserted in the early region 3 (E3) of the HAd5 genome in the E3 parallel orientation. Expression of HN or F in HAd5-HN- or HAd5-F-infected cell extracts, respectively, was observed by immunoprecipitation using a BPIV3-specific polyclonal antiserum. Our results suggest that HN and F expressed by HAd5 recombinants were functionally similar to the native HN and F expressed in BPIV3-infected cells.
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