Recombinant bovine respiratory syncytial virus with deletion of the SH gene induces increased apoptosis and pro-inflammatory cytokines in vitro, and is attenuated and induces protective immunity in calves

Abstract

Bovine respiratory syncytial virus (BRSV) causes inflammation and obstruction of the small airways, leading to severe respiratory disease in young calves. The virus is closely related to human (H)RSV, a major cause of bronchiolitis and pneumonia in young children. The ability to manipulate the genome of RSV has provided opportunities for the development of stable, live attenuated RSV vaccines. The role of the SH protein in the pathogenesis of BRSV was evaluated in vitro and in vivo using a recombinant (r)BRSV in which the SH gene had been deleted. Infection of bovine epithelial cells and monocytes with rBRSVΔSH, in vitro, resulted in an increase in apoptosis, and higher levels of TNF-α and IL-1β compared with cells infected with parental, wild-type (WT) rBRSV. Although replication of rBRSVΔSH and WT rBRSV, in vitro, were similar, the replication of rBRSVΔSH was moderately reduced in the lower, but not the upper, respiratory tract of experimentally infected calves. Despite the greater ability of rBRSVΔSH to induce pro-inflammatory cytokines, in vitro, the pulmonary inflammatory response in rBRSVΔSH-infected calves was significantly reduced compared with that in calves inoculated with WT rBRSV, 6 days previously. Virus lacking SH appeared to be as immunogenic and effective in inducing resistance to virulent virus challenge, 6 months later, as the parental rBRSV. These findings suggest that rBRSVΔSH may be an ideal live attenuated virus vaccine candidate, combining safety with a high level of immunogenicity.

Fig. 1.

Replication of rBRSV and rBRSVΔSH in (a) calf testes cells and (b) MDBK cells. Triplicate cell monolayers in 6-well plates were infected at an m.o.i. of 0.1. Cells were harvested at daily intervals and stored at −70 °C. Values are the mean log₁₀(p.f.u. ml⁻¹)±sd of triplicate wells.

Fig. 2.

BRSV lacking the SH gene induces apoptosis in MDBK cells and bovine monocytes. (a) MDBK cells were infected with WT rBRSV or rBRSV lacking the SH gene (ΔSH) at an m.o.i. of 3. (b) Bovine monocytes were infected with WT or ΔSH rBRSV at an m.o.i. of 1 or 3. As a control, cells were exposed to mock-infected tissue culture cell lysate. The proportions of apoptotic cells were determined 24 and 48 h p.i. of MDBK cells, or 24 h p.i. of monocytes, using a TUNEL assay and flow cytometry. Results are expressed as the mean percentage apoptotic cells±sd of triplicate samples. *, P<0.0001; **, P<0.01.

Fig. 3.

BRSV lacking the SH gene induces secretion of high levels of TNF-α and IL-1β. Bovine monocytes were infected with WT rBRSV or rBRSV lacking the SH gene (ΔSH) at an m.o.i. of 1 (a, b); and MDBK cells were infected with WT or ΔSH rBRSV at an m.o.i. of 3 (c). As controls, cells were exposed to mock-infected tissue culture cell lysate (Con). At 24 h post-infection, levels of TNF-α (a) and IL-1β (b, c) in the supernatant were determined by ELISA. Results are expressed as the mean±sd of triplicate samples. *, WT rBRSV induced significantly higher levels of TNF-α or IL-1β than controls, P<0.03; **, rBRSVΔSH induced significantly higher levels of TNF-α or IL-1β than WT rBRSV, P<0.0001.

Fig. 4.

Replication of SH deletion mutant of BRSV in the respiratory tract of calves. Two-to-three-week-old gnotobiotic calves were inoculated i.n. and i.t. with 5×10⁶ p.f.u. WT rBRSV (n = 4) or rBRSV lacking the SH gene (ΔSH) (n = 3). (a) Nasopharyngeal excretion of virus expressed as the mean log₁₀(p.f.u. ml⁻¹)±sd. (b) Mean titre of virus [log₁₀(p.f.u.)±sd] in BAL cells, or homogenates of samples from the right apical (RA), right cardiac (RC) and left cardiac (LC) lobes of the lung, 6 days after infection.

Fig. 5.

rBRSV lacking the SH protein induces less pulmonary pathology than WT rBRSV. (a) Macroscopic lung lesions in calves infected 6 days previously with 5×10⁶ p.f.u. rBRSV lacking the SH gene (ΔSH) were significantly reduced compared with those in calves infected with WT rBRSV (P<0.02). (b) The number of neutrophils in BAL (mean±sd), 6 days after infection. The number of neutrophils in BAL from calves infected with rBRSVΔSH was significantly reduced compared with that in calves infected with WT virus (P<0.05).

Fig. 6.

Histopathological changes in tracheal epithelium from BRSV-infected gnotobiotic calves. Vacuoles containing cell debris (arrows) were detected in tracheal epithelium from calves inoculated i.n. and i.t., 6 days previously, with WT rBRSV (b) or rBRSVΔSH (c), but not in the epithelium from calves inoculated with control Vero cell lysate (a). Bar represents 100 µM.

Fig. 7.

BRSV-specific antibody responses induced by mucosal vaccination of calves with rBRSV. Calves were inoculated i.n. and i.t. with 5×10⁶ p.f.u. WT rBRSV (n = 5) or rBRSV lacking the SH gene (ΔSH) (n = 4). Calves were challenged 24 weeks after vaccination with 1×10⁴ p.f.u. of the Snook strain of BRSV in BAL. BRSV-specific IgG antibody responses in sera (a) and BRSV-specific IgA antibody in nasal secretions, 2 weeks after vaccination (b) were determined by ELISA. (c) BRSV-specific serum neutralizing antibody responses were determined by a plaque reduction assay. Results are expressed as the geometric mean titre (log₁₀) ±sd.

Fig. 8.

Recombinant BRSV lacking SH protects against challenge with virulent BRSV. Calves were inoculated i.n. and i.t. with 5×10⁶ p.f.u. WT rBRSV (n = 5) or rBRSV lacking the SH gene (ΔSH) (n = 4). As controls, calves were inoculated i.n. and i.t with control tissue culture cell lysate. Calves were challenged 24 weeks after vaccination with 1×10⁴ p.f.u. of the Snook strain of BRSV in BAL and killed 6 days after challenge. (a) Percentage of the lung showing macroscopic lung lesions, 6 days after challenge. (b) Peak virus titres in the nasopharynx (solid symbols) and titre of virus in BAL cells, 6 days after challenge (open symbols).