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Bovine respiratory syncytial virus (BRSV), a member of the genus Pneumovirus of the family Paramyxoviridae, is closely related to human respiratory syncytial virus (HRSV). It is a major cause of lower respiratory tract disease in calves between 1 and 3 months old (12). Seroepizootiologic studies have demonstrated that exposure of cattle to BRSV is widespread in many countries (1, 5). The envelope of BRSV contains two major glycoproteins, the attachment (G) protein and the fusion (F) protein. The G protein is involved in viral attachment (6). The F protein causes fusion of viral and cellular membranes and fusion of infected cells to surrounding cells. The F protein is synthesized as a precursor, F0 (68 kDa), which is proteolytically cleaved to yield two subunits, F1 (46 kDa) and F2 (20 kDa), that are disulfide linked (2). In BRSV-infected calves, the most immunogenic and protective viral antigen is the F glycoprotein, which evokes a strong antibody response and is a major target for cytotoxic T cells (7, 9). Antibodies to this protein can neutralize virus infectivity and prevent cell fusion (15). Therefore, there is a need for a source of the F protein free from other BRSV proteins to analyze its immunogenicity.

The baculovirus expression system provides a method for the production of large quantities of biologically active and antigenic eukaryotic proteins for both research and diagnostic applications. The F protein appears to be an ideal antigen for diagnostic purposes, as sera from BRSV-infected calves contain high levels of antibody to the F protein (11). Although the F protein of BRSV has been expressed by using a baculovirus vector (3), the detailed antigenic property and utility of the recombinant F protein as a diagnostic antigen had not yet been examined. Therefore, we expressed the F protein of BRSV strain A51908 in a baculovirus system and report the immunogenicity and utility of the recombinant F protein in a diagnostic enzyme-linked immunosorbent assay (ELISA).

Construction and characterization of the recombinant baculovirus F protein. The construction and characterization of the recombinant baculovirus encoding the F protein of BRSV strain A51908 were accomplished by established methods (10, 14). A full-length cDNA of the BRSV F gene was originally cloned in the BamHI and XbaI sites of plasmid vector pGEM-7Z⁺ (Promega) (8). The F gene fragment was excised from a plasmid and was inserted into the BamHI and XbaI sites of the baculovirus transfer vector pVL1393 so that the F gene was under the control of the AcNPV (Autographa californica nuclear polyhedrosis virus) polyhedrin promoter. Transfer vector-recombinant DNA and linearized BaculoGold baculovirus DNA (PharMingen) were used to cotransfect Sf9 (Spodoptera frugiperda) cells according to the procedure described by the manufacturer. Briefly, Grace's insect medium was replaced with 1 ml of transfection buffer A in a 60-mm tissue culture plate seeded with 3 × 10⁶ Sf9 cells. A mixture of 0.5 µg of linearized BaculoGold baculovirus DNA and 2 µg of recombinant plasmid transfer vector in 1 ml of transfection buffer B was added dropwise to the insect cells. Following 4 to 6 h of incubation at 27°C, the buffer was removed, the cell monolayer was washed, and fresh Grace's insect medium supplemented with 10% fetal bovine serum was added. After 4 days, the extracellular virus was harvested, passaged three times, plaque purified, and used as a stock virus.

View larger version (48K): [in this window] [in a new window]   FIG. 1.   Expression of F protein by recombinant baculovirus and confirmation of the authenticity of recombinant F protein. Lanes: A, molecular weight standards; B, recombinant baculovirus-infected cell lysate immunoprecipitated with BRSV-specific antiserum; C, BRSV-infected BTu cell lysate immunoprecipitated with BRSV antiserum; D, recombinant baculovirus-infected cell lysate immunoprecipitated with baculovirus-expressed F-specific antiserum; E, recombinant baculovirus-infected cell lysate; F, BRSV-infected BTu cell lysate; G, wild-type AcNPV-infected cell lysate; H, molecular weight standards. Numbers are molecular weights, in thousands.

Anti-F serum. To assess the ability of the F protein produced by the recombinant baculovirus to induce antibodies which could react with the F protein present in BRSV-infected cells, an antiserum in rabbits was produced by using the F protein obtained from SDS-polyacrylamide gel electrophoresis-separated infected Sf9 cells. In brief, the infected Sf9 cell lysate was immunoprecipitated with polyclonal antibodies raised against BRSV strain A51908 and the proteins were resolved by SDS-12.5% polyacrylamide gel electrophoresis. After electrophoresis, the proteins were visualized by soaking the gel in ice-cold 100 mM KCl solution. The exact location of the recombinant F protein was determined by comparison with the relative mobility of the F protein of BRSV-infected bovine nasal turbinate (BTu) cell lysate and protein standards (Promega). The protein band was cut from the gel, homogenized in phosphate-buffered saline, and stored at 70°C. Each rabbit received one subcutaneous injection of approximately 200 µg of either F protein or wild-type AcNPV cell protein in Freund's incomplete adjuvant on days 10, 20, and 30. Blood was collected 5 days after the last injection, and serum samples were stored at 20°C. The ability of the antiserum to recognize F protein was tested by immunoprecipitation. Preimmune serum and antiserum to the wild-type AcNPV-infected Sf9 cell proteins did not react with any viral or cellular proteins in BRSV-infected BTu cell lysates, whereas antiserum to the F protein specifically precipitated the BRSV F protein. The anti-F protein serum reacted in an ELISA with 12 BRSV strains and HRSV strain A2 (data not shown). The anti-F protein serum also gave bright immunofluorescence on acetone-fixed BRSV-infected BTu cells but not on uninfected BTu cells, which served as a negative control. This indicated that antiserum to the baculovirus-expressed BRSV F protein could be used as a diagnostic reagent for detection of BRSV antigen.

Neutralization assay using anti-F serum. The ability of anti-F protein serum to neutralize the virus infectivity was tested by a 80% plaque reduction test (Table 1). Briefly, various twofold dilutions (1:2 through 1:1,024) of anti-F protein serum were mixed with 100 µl of tissue culture medium containing approximately 100 PFU of either HRSV strain A2 or BRSV strain FS1, incubated for 2 h at 37°C, and then inoculated onto BTu cells in six-well plates. After an adsorption period of 2 h, the virus-antibody mixture was removed and the cell monolayer was overlaid with minimal essential medium containing 0.8% methylcellulose and 6% fetal bovine serum. Seven days after incubation, virus plaques were counted after staining with crystal violet. Negative and positive control experiments were done with normal rabbit serum and with polyclonal antiserum, respectively. There were averages of 133 BRSV and 131 HRSV plaques in the wells not treated with the anti-F serum. In BRSV-infected wells treated with 1:2 to 1:32 dilutions of anti-F serum, there was more than an 80% reduction of BRSV plaques, whereas in HRSV-infected wells treated with anti-F serum, dilutions of 1:2 to 1:8 gave more than an 80% reduction of HRSV plaques. This indicates that 1:32 and 1:8 dilutions of anti-F serum gave almost complete neutralization (80%) of BRSV and HRSV infectivity, respectively. It appears that the baculovirus-expressed BRSV F protein contained important neutralizing epitopes and can induce antibodies in animals that not only neutralize BRSV infection but also can cross-neutralize HRSV infection to a lesser extent.

Vaccination of calves and serological testing. Since the antibody response to BRSV is predominantly directed against the F protein and the F gene is highly conserved (>95%) among BRSV strains (7), the baculovirus-expressed F protein was used as an antigen in an ELISA for the detection of BRSV antibodies. Ten consecutive serum samples collected on days 0 (day of weaning), 7, 14, 28, 35, 42, 56, 84, 112, and 140 from six calves were tested in this assay. The ELISA results were compared with those of virus neutralization (VN) tests to investigate the consistency of the immunity level as measured by these two methods (Fig. 2). In this study, six purebred Angus calves from the Wye herd (Wye Research and Educational Center, Queenstown, Md.) were used. Calves were weaned at an average age of 205 days. All calves were found to be BRSV free by both virus isolation and VN tests. Four calves (vaccinated group; calves 63, 75, 84, and 90) were inoculated intramuscularly with an attenuated BRSV vaccine strain as recommended by the manufacturer (Pfizer Animal Health). A booster vaccination was given 4 weeks later. The two remaining calves served as nonvaccinated controls. On the day of weaning, the nasal mucosa of each calf was swabbed for virus isolation. Nasal swabs were swirled in a transport medium, and the eluates were inoculated onto BTu cells for virus culturing. The specimens were considered negative if no cytopathic effects (syncytium formation) developed within two subpassages.

View larger version (29K): [in this window] [in a new window]   FIG. 2.   ELISA and VN test results obtained with serum samples from four vaccinated calves. Calves were inoculated intramuscularly on day 0 with an attenuated BRSV vaccine, and a booster vaccination was given after 4 weeks. The ELISA and the VN test were performed as described in the text.