Infectious bronchitis virus (IBV) is a coronavirus which affects chickens of all ages. IBV mainly causes respiratory disease but can also result in reduced weight gain, reduced egg production, increased frequency of abnormal eggs and increased rates of mortality. Vaccination is the most important way to control the disease. Nevertheless, novel strains of infectious bronchitis (IB) continue to emerge in the field. In order to respond promptly, combinations of existing IB vaccines are frequently tested to see whether they can provide cross-protection. The efficacy of a combination of vaccines based on Massachusetts, Dutch and QX-like IB strains against emerging IB Israel variant 2 and IB 793B strains was assessed by means of four challenge studies. At least 80% of the birds vaccinated with IB H120 (Mass type) combined with IB D274 (Dutch type) followed by a QX-like IB vaccine booster or vaccinated with a combination of IB H120, IB D274 and QX-like IB were protected against a challenge with IB 793B. In addition, IB 1263 (Mass type) boosted by QX-like IB showed an 85% protection following challenge with IB 793B. A combination of IB H120 and IB D274 boosted by QX-like IB vaccine conferred 70% protection whilst H120 and IB D274 combination on its own showed 61.1% protection against Israel variant 2 challenge. IB 1263 boosted by a QX-like IB vaccine showed 50% protection against IB Israel variant 2. Therefore, it can be concluded that a combination of the IB H120, IB D274 and QX-like IB confers broad protection against different non-related virulent IB strains.

The Gammacoronavirus infectious bronchitis virus (IBV) is a highly contagious global pathogen prevalent in all types of poultry flocks. IBV is responsible for economic losses and welfare issues in domestic poultry resulting in a significant risk to food security. IBV vaccines are currently generated by serial passage of virulent field isolates of IBV through embryonated hen’s eggs. The different patterns of genomic variation accumulated during this process means the exact mechanism of attenuation is unknown and presents a risk for reversion to virulence. Additionally, the passaging process adapts the virus to replicate in chicken embryos increasing embryo lethality. Vaccines produced in this manner are therefore unsuitable for in ovo application. We have developed a reverse genetics system based on the pathogenic IBV strain M41, in order to identify genes that can be targeted for rational attenuation. During the development of this reverse genetics system, we identified four amino acids located in non-structural proteins (Nsps) 10, 14, 15 and 16 that resulted in attenuation in vivo and in ovo . Further investigation highlighted a role for amino acid changes Pro85Leu in Nsp 10 and Val393Ile in Nsp 14 in the attenuated in vivo phenotype observed. This study provides evidence that mutations in the nsps offer a promising mechanism for the development of rationally attenuated live vaccines against IBV, which have the potential for in ovo application. Importance The Gammacoronavirus infectious bronchitis virus (IBV) is the aetiological agent of infectious bronchitis, an acute highly contagious economically important disease of poultry. Vaccination is achieved using a mixture of live attenuated vaccines for young chicks and inactivated vaccines as boosters for layers. Live attenuated vaccines are generated through serial passage in embryonated hens’ eggs, an empirical process to achieve attenuation but with retention of immunogenicity. However, there is a risk of reversion to virulence and vaccines are lethal to the embryo. In this study we identified amino acids in the replicase gene that attenuated IBV strain M41, both in vivo and in ovo . Stability assays indicate that the attenuating amino acids are stable and are unlikely to revert. The data in this study provides evidence that specific modifications in the replicase gene offer a promising direction for IBV live attenuated vaccine development, with the potential for in ovo application.

The attenuation of infectious bronchitis (IB) QX-like virus strain L1148 is described. The virus was passaged multiple times in embryonated specific pathogen free (SPF) chicken eggs, and at different passage levels samples were tested for safety for the respiratory tract and kidneys in 1-day-old SPF chickens. There was a clear decrease in pathogenicity for the respiratory tract and kidneys when the virus had undergone a large number of passages. Passage level 80 was investigated for safety for the reproductive tract in 1-day-old and 7-day-old SPF chickens. In 1-day-old chickens, 12.5% of the vaccinated birds had macroscopic lesions. No lesions were observed if the chickens had been vaccinated at 7 days of age. Passage level 80 was investigated for its ability to spread from vaccinated to non-vaccinated chickens and for dissemination in the body. The virus was able to spread from vaccinated chickens to groups of non-vaccinated chickens, and in the vaccinated birds the virus was found frequently in oro-pharyngeal and cloacal swabs. A fragment of the hypervariable region of the S1 protein of passage level 80 was sequenced and revealed nucleotide changes resulting in two amino acid substitutions. Passage level 80 was given additional passages to levels 82 and 85. Both passage levels were tested for efficacy in SPF chickens and passage level 85 was tested for efficacy in commercial chickens with maternally derived antibodies (MDA) against a challenge with QX-like strain IB D388. In both SPF chickens and chickens with MDA, the vaccines based on strain IB L1148 were efficacious against challenge.