Gammacoronavirus infectious bronchitis virus (IBV) causes an economically important respiratory disease of poultry. Protective immunity is associated with the major structural protein, spike (S) glycoprotein, which induces neutralising antibodies and defines the serotype. Cross-protective immunity between serotypes is limited and can be difficult to predict. In this study, the ability of two recombinant IBV vaccine candidates, BeauR-M41(S) and BeauR-4/91(S), to induce cross-protection against a third serotype, QX, was assessed. Both rIBVs are genetically based on the Beaudette genome with only the S gene derived from either M41 or 4/91, two unrelated serotypes. The use of these rIBVs allowed for the assessment of the potential of M41 and 4/91 S glycoproteins to induce cross-protective immunity against a heterologous QX challenge. The impact of the order of vaccination was also assessed. Homologous primary and secondary vaccination with BeauR-M41(S) or BeauR-4/91(S) resulted in a significant reduction of infectious QX load in the trachea at four days post-challenge, whereas heterologous primary and secondary vaccination with BeauR-M41(S) and BeauR-4/91(S) reduced viral RNA load in the conjunctiva-associated lymphoid tissue (CALT). Both homologous and heterologous vaccination regimes reduced clinical signs and birds recovered more rapidly as compared with an unvaccinated/challenge control group. Despite both rIBV BeauR-M41(S) and BeauR-4/91(S) displaying limited replication in vivo, serum titres in these vaccinated groups were higher as compared with the unvaccinated/challenge control group. This suggests that vaccination with rIBV primed the birds for a boosted humoral response to heterologous QX challenge. Collectively, vaccination with the rIBV elicited limited protection against challenge, with failure to protect against tracheal ciliostasis, clinical manifestations, and viral replication. The use of a less attenuated recombinant vector that replicates throughout the respiratory tract could be required to elicit a stronger and prolonged protective immune response.

Evidence suggests that susceptibility to avian influenza A virus in chickens is influenced by host genetics, but the mechanisms are poorly understood. A previous study demonstrated that inbred line 0 chickens are more resistant to low-pathogenicity avian influenza (LPAI) infection than line CB.12 birds based on viral shedding, but the resistance was not associated with higher AIV-specific IFNγ responses or antibody titres. In this study, we investigated the proportions and cytotoxic capacity of T-cell subpopulations in the spleen and the early immune responses in the respiratory tract, analysing the innate immune transcriptome of lung-derived macrophages following in vitro stimulation with LPAI H7N1 or the TLR7 agonist R848. The more susceptible C.B12 line had a higher proportion of CD8αβ+ γδ and CD4+CD8αα+ αVβ1 T cells, and a significantly higher proportion of the CD8αβ+ γδ and CD8αβ+ αVβ1 T cells expressed CD107a, a surrogate marker of degranulation. Lung macrophages isolated from line C.B12 birds expressed higher levels of the negative regulator genes TRIM29 and IL17REL, whereas macrophages from line 0 birds expressed higher levels of antiviral genes including IRF10 and IRG1. After stimulation with R848, the macrophages from line 0 birds mounted a higher response compared to line C.B12 cells. Together, the higher proportion of unconventional T cells, the higher level of cytotoxic cell degranulation ex vivo and post-stimulation and the lower levels of antiviral gene expression suggest a potential role of immunopathology in mediating susceptibility in C.B12 birds.

Synonymous recoding of RNA virus genomes is a promising approach for generating attenuated viruses to use as vaccines. However, such recoding typically hinders virus growth. A potential solution to this is CpG dinucleotide enrichment. CpGs are recognised by cellular zinc-finger antiviral protein (ZAP), leading to viral RNA degradation and/or type I interferon stimulation and consequently reduced viral yields. Therefore, in principle, removing the ZAP CpG sensor from a virus propagation system will reverse attenuation of a CpG-enriched virus. Using influenza A viruses (IAVs) engineered for increased CpG content in different genome regions, we show that ZAP mediated virus attenuation is dependent on the viral segment to which CpGs are added. In ZAP-expressing human cells, CpG introduction invariably impaired transcript and protein production, but ZAP-sensitivity did not convey type I interferon activation. Importantly for vaccine development, CpG-enriched viruses were genetically stable during serial passage. Furthermore, the ZAP-sensitive virus was fully replication competent in MDCK cells and in embryonated hens’ eggs, both of which are used to propagate live attenuated influenza vaccines. Thus, ZAP-sensitive CpG enriched viruses that are defective in human systems can yield high titre in vaccine propagation systems, providing a realistic, economically viable platform for live attenuated vaccine development.