During infection, the influenza A virus RNA polymerase produces both full-length and aberrant RNA molecules, such as defective viral genomes (DVGs) and mini viral RNAs (mvRNAs). Subsequent innate immune activation involves the binding of host pathogen receptor retinoic acid–inducible gene I (RIG-I) to viral RNAs. However, it is not clear what factors determine which influenza A virus RNAs are RIG-I agonists. Here, we provide evidence that RNA structures, called template loops (t-loops), stall the viral RNA polymerase and contribute to innate immune activation by mvRNAs during influenza A virus infection. Impairment of replication by t-loops depends on the formation of an RNA duplex near the template entry and exit channels of the RNA polymerase, and this effect is enhanced by mutation of the template exit path from the RNA polymerase active site. Overall, these findings are suggestive of a mechanism involving polymerase stalling that links aberrant viral replication to the activation of the innate immune response.

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 Gammacoronavirus infectious bronchitis virus (IBV) is a highly contagious economically important respiratory pathogen of domestic fowl. Reverse genetics allows for the molecular study of pathogenic determinants to enable rational vaccine design. The recombinant IBV (rIBV) Beau-R, a molecular clone of the apathogenic Beaudette strain, has previously been investigated as a vaccine platform. To determine tissues in which Beau-R could effectively deliver antigenic genes, an in vivo study in chickens, the natural host, was used to compare the pattern of viral dissemination of Beau-R to the pathogenic strain M41-CK. Replication of Beau-R was found to be restricted to soft tissue within the beak, whereas M41-CK was detected in beak tissue, trachea and eyelid up to seven days post infection. In vitro assays further identified that, unlike M41-CK, Beau-R could not replicate at 41 °C, the core body temperature of a chicken, but is able to replicate a 37 °C, a temperature relatable to the very upper respiratory tract. Using a panel of rIBVs with defined mutations in the structural and accessory genes, viral replication at permissive and non-permissive temperatures was investigated, identifying that the Beau-R replicase gene was a determinant of temperature sensitivity and that sub-genomic mRNA synthesis had been affected. The identification of temperature sensitive allelic lesions within the Beau-R replicase gene opens up the possibility of using this method of attenuation in other IBV strains for future vaccine development as well as a method to investigate the functions of the IBV replicase proteins.