Infectious bronchitis (IB) is a highly contagious respiratory and urogenital disease of chickens caused by infectious bronchitis virus (IBV). This disease is of considerable economic importance and is primarily controlled through biosecurity and immunization with live attenuated and inactivated IB vaccines of various serotypes. In the present study, we tested the safety and efficacy of an attenuated predominant Chinese QX-like IBV strain. The results revealed that the attenuated strain has a clear decrease in pathogenicity for specific-pathogen-free (SPF) chickens compared with the parent strain. Strain YN-inoculated birds had clinical signs of varying severity with 30% mortality, while the attenuated group appeared healthy, with less tissue damage. The attenuated strain also had relatively low tissue replication rates and higher antibody levels. The superior protective efficacy of the attenuated strain was observed when vaccinated birds were challenged with a homologous or heterologous field IBV strain, indicating the potential of the attenuated YN (aYN) as a vaccine. Producing a vaccine targeting the abundant serotype in China is essential to reducing the economic impact of IB on the poultry industry.

Some coronaviruses (CoVs) have an extra furin cleavage site (RRKR/S, furin-S2′ site) upstream of the fusion peptide in the spike protein, which plays roles in virion adsorption and fusion. Mutation of the S2′ site of QX genotype (QX-type) infectious bronchitis virus (IBV) spike protein (S) in a recombinant virus background results in higher pathogenicity, pronounced neural symptoms and neurotropism when compared with conditions in wild-type IBV (WT-IBV) infected chickens. In this study, we present evidence suggesting that recombinant IBV with a mutant S2′ site (furin-S2′ site) leads to higher mortality. Infection with mutant IBV induces severe encephalitis and breaks the blood–brain barrier. The results of a neutralization test and immunoprotection experiment show that an original serum and vaccine can still provide effective protection in vivo and in vitro. This is the first demonstration of IBV-induced neural symptoms in chickens with encephalitis and the furin-S2′ site as a determinant of neurotropism.

Coronavirus (CoV) nsp15 is an endoribonuclease conserved throughout the CoV family. The enzymatic activity and crystal structure of infectious bronchitis virus (IBV) nsp15 are undefined, and the protein’s role in replication remains unclear. We verified the uridylate-specific endoribonuclease (EndoU) activity of IBV and found that the EndoU active sites were located in the C-terminus of nsp15 and included His223, His238, Lys278 and Tyr334. We further constructed an infectious clone of the IBV-rSD strain (rSD-wild-type [WT]) and EndoU-deficient IBVs by changing the codon for the EndoU catalytic residues to alanine. Both the rSD-WT and EndoU-deficient viruses propagated efficiently in embryonated chicken eggs. Conversely, EndoU-deficient viral propagation was severely impaired in chicken embryonic kidney cells, which was reflected in the lower viral mRNA accumulation and protein synthesis. After infecting chickens with the parental rSD-WT strain and EndoU-deficient viruses, the EndoU-deficient-virus-infected chickens presented reduced mortality, tissue injury and viral shedding. IMPORTANCE Coronaviruses can emerge from animal reservoirs into naive host species to cause pandemic respiratory and gastrointestinal diseases with significant mortality in humans and domestic animals. Infectious bronchitis virus (IBV), a γ-coronavirus, infects respiratory, renal and reproductive systems, causing millions of dollars in lost revenue worldwide annually. Mutating the viral endoribonuclease resulted in an attenuated virus and prevented protein kinase R activation. Therefore, EndoU activity is a virulence factor in IBV infections, thus providing an approach for generating live-attenuated vaccine candidates for emerging coronaviruses.

To explore the critical genes associated with infectious bronchitis virus (IBV) virulence, we compared the genome sequences of virulent YN strain and its attenuated strain aYN. Accumulation of mutations in the S gene and the accessory gene 5a were observed, suggesting a potential role in the loss of viral pathogenicity. Two recombinant IBVs (rIBVs) with replacement of the S gene or 5a with corresponding regions from aYN were rescued to verify this speculation. Embryo mortality time/rate showed that rYN-S-aYN and rYN-5a-aYN had an attenuated phenotype in ovo. Replication assay in ovo and in vitro demonstrated the rIBVs had similar replication patterns with wild-type rIBV. Both rIBVs showed reduced mortality, tissue lesions and tissue virus titers in chicken. In conclusion, S gene and 5a accessory gene are responsible for the attenuation of virulent IBV. Insight into the genes responsible for virus attenuation will facilitate the development of future vaccines against IBV.

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