Recombinant infectious bronchitis coronavirus Beaudette with the spike protein gene of the pathogenic M41 strain remains attenuated but induces protective immunity

Abstract

We have replaced the ectodomain of the spike (S) protein of the Beaudette strain (Beau-R; apathogenic for Gallus domesticus chickens) of avian infectious bronchitis coronavirus (IBV) with that from the pathogenic M41 strain to produce recombinant IBV BeauR-M41(S). We have previously shown that this changed the tropism of the virus in vitro (R. Casais, B. Dove, D. Cavanagh, and P. Britton, J. Virol. 77:9084-9089, 2003). Herein we have assessed the pathogenicity and immunogenicity of BeauR-M41(S). There were no consistent differences in pathogenicity between the recombinant BeauR-M41(S) and its apathogenic parent Beau-R (based on snicking, nasal discharge, wheezing, watery eyes, rales, and ciliostasis in trachea), and both replicated poorly in trachea and nose compared to M41; the S protein from the pathogenic M41 had not altered the apathogenic nature of Beau-R. Both Beau-R and BeauR-M41(S) induced protection against challenge with M41 as assessed by absence of recovery of challenge virus and nasal exudate. With regard to snicking and ciliostasis, BeauR-M41(S) induced greater protection (seven out of nine chicks [77%]; assessed by ciliostasis) than Beau-R (one out of nine; 11%) but less than M41 (100%). The greater protection induced by BeauR-M41(S) against M41 may be related to the ectodomain of the spike protein of Beau-R differing from that of M41 by 4.1%; a small number of epitopes on the S protein may play a disproportionate role in the induction of immunity. The results are promising for the prospects of S-gene exchange for IBV vaccine development.

FIG. 1.

Comparison of the pathogenicity of IBV strains M41 (donor of the S-protein ectodomain gene sequence), Beau-R (receiver of the S-protein ectodomain gene sequence), and BeauR-M41(S) (Beau-R with the S-protein ectodomain gene sequence of M41). Groups of 20 (experiment 1, panels A, C, E, and G) and 30 (experiment 2, panels B, D, F, and H) 8-day-old chicks were inoculated intranasally or by eye drop with 3.0 log₁₀ CD₅₀ of virus. Mock-infected chicks were inoculated with buffer only. Clinical signs were recorded from days 3 to 7 (experiment 1) and from days 3 to 12 (experiment 2). In experiment 1 there were two additional groups, inoculated with either Beaudette-CK (the strain from which the full-length clone of Beau-R had been derived) or H120, a commercial vaccine strain. The observations were made on individual birds, except for snicking, where birds were observed as a group. (A and B) Titer of infectious virus in tracheal scrapings, mean of three tracheas; (C and D) ciliostasis in tracheal rings, mean of three tracheas; (E and F) snicking (recorded on a group basis); (G and H) nasal discharge (mean of all chicks). M41 was significantly more pathogenic than the other strains (P < 0.05) except with respect to nasal discharge (G).

FIG. 2.

Histopathology of tracheas from chickens on day 4 after inoculation. Transverse sections were stained with hematoxylin-eosin. (A) Mock infected; (B to E) infected with (B) Beau-R, (C) H120, (D) BeauR-M41(S), and (E) M41. In contrast to panels A to D, panel E shows complete deciliation and marked monocytic infiltration in the lamina propria. The H120 strain (C) caused mild deciliation and some monocytic infiltration and vascular congestion. Magnification, ×1,000. (E) Composite of three photographs of the same section.

FIG. 3.

Comparison of the growth curves in TOCs of IBV strains M41 (donor of the S-protein ectodomain gene sequence), Beau-R (receiver of the S-protein ectodomain gene sequence), and BeauR-M41(S) (Beau-R with the S-protein ectodomain gene sequence of M41). Groups of five TOCs were inoculated with 1.7 log₁₀ CD₅₀ of virus in 0.5 ml of medium. After 1 h at 37°C the medium was removed, the TOCs were washed three times, and incubation was continued with 1 ml of medium. At selected time points, medium from three tubes of each virus was titrated in CK cells (37).

FIG. 4.

Comparison of the induction of protection by IBV strains M41 (donor of the S-protein ectodomain gene sequence), Beau-R (receiver of the S-protein ectodomain gene sequence), and BeauR-M41(S) (Beau-R with the S-protein ectodomain gene sequence of M41). The birds of experiment 2 (Fig. 1B, D, F, and H) were challenged, 3 weeks after the initial inoculation, with 3.0 log₁₀ CD₅₀ of M41, applied intranasally and by eye drop. Mock-infected chicks were inoculated with buffer only. Clinical signs were recorded from days 3 to 7. The observations were made on individual birds, except for snicking, for which birds were observed as a group. (A) Titer of infectious challenge virus recovered from the tracheae of three birds at 4 days after challenge; virus was detected in only one group, mock:M41; (B) ciliostasis in tracheal rings (mean of three tracheae collected at 4, 5, and/or 6 days after challenge, as indicated at the top; a low percentage is indicative of protection); (C) nasal discharge (mean of 15 to 18 birds examined individually); (D) snicking (15 to 18 birds examined as a group).