A Temperature-Sensitive Recombinant of Avian Coronavirus Infectious Bronchitis Virus Provides Complete Protection against Homologous Challenge.

Avian coronavirus infectious bronchitis virus (IBV) is the etiological agent of infectious bronchitis, an acute highly contagious economically relevant respiratory disease of poultry. Vaccination is used to control IBV infections, with live-attenuated vaccines generated via serial passage of a virulent field isolate through embryonated hens' eggs. A fine balance must be achieved between attenuation and the retention of immunogenicity. The exact molecular mechanism of attenuation is unknown, and vaccines produced in this manner present a risk of reversion to virulence as few consensus level changes are acquired. Our previous research resulted in the generation of a recombinant IBV (rIBV) known as M41-R, based on a pathogenic strain M41-CK. M41-R was attenuated in vivo by two amino acid changes, Nsp10-Pro85Leu and Nsp14-Val393Leu; however, the mechanism of attenuation was not determined. Pro85 and Val393 were found to be conserved among not only IBV strains but members of the wider coronavirus family. This study demonstrates that the same changes are associated with a temperature-sensitive (ts) replication phenotype at 41°C in vitro, suggesting that the two phenotypes may be linked. Vaccination of specific-pathogen-free chickens with M41-R induced 100% protection against clinical disease, tracheal ciliary damage, and challenge virus replication following homologous challenge with virulent M41-CK. Temperature sensitivity has been used to rationally attenuate other viral pathogens, including influenza, and the identification of amino acid changes that impart both a ts and an attenuated phenotype may therefore offer an avenue for future coronavirus vaccine development. IMPORTANCE Infectious bronchitis virus is a pathogen of economic and welfare concern for the global poultry industry. Live-attenuated vaccines against are generated by serial passage of a virulent isolate in embryonated eggs until attenuation is achieved. The exact mechanisms of attenuation are unknown, and vaccines produced have a risk of reversion to virulence. Reverse genetics provides a method to generate vaccines that are rationally attenuated and are more stable with respect to back selection due to their clonal origin. Genetic populations resulting from molecular clones are more homogeneous and lack the presence of parental pathogenic viruses, which generation by multiple passage does not. In this study, we identified two amino acids that impart a temperature-sensitive replication phenotype. Immunogenicity is retained and vaccination results in 100% protection against homologous challenge. Temperature sensitivity, used for the development of vaccines against other viruses, presents a method for the development of coronavirus vaccines.

The Genetic Stability, Replication Kinetics and Cytopathogenicity of Recombinant Avian Coronaviruses with a T16A or an A26F Mutation within the E Protein Is Cell-Type Dependent.

The envelope (E) protein of the avian coronavirus infectious bronchitis virus (IBV) is a small-membrane protein present in two forms during infection: a monomer and a pentameric ion channel. Each form has an independent role during replication; the monomer disrupts the secretory pathway, and the pentamer facilitates virion production. The presence of a T16A or A26F mutation within E exclusively generates the pentameric or monomeric form, respectively. We generated two recombinant IBVs (rIBVs) based on the apathogenic molecular clone Beau-R, containing either a T16A or A26F mutation, denoted as BeauR-T16A and BeauR-A26F. The replication and genetic stability of the rIBVs were assessed in several different cell types, including primary and continuous cells, ex vivo tracheal organ cultures (TOCs) and in ovo. Different replication profiles were observed between cell cultures of different origins. BeauR-A26F replicated to a lower level than Beau-R in Vero cells and in ovo but not in DF1, primary chicken kidney (CK) cells or TOCs. Genetic stability and cytopathic effects were found to differ depending on the cell system. The effect of the T16A and A26F mutations appear to be cell-type dependent, which, therefore, highlights the importance of cell type in the investigation of the IBV E protein.

Porcine Respiratory Coronavirus as a Model for Acute Respiratory Coronavirus Disease

In the light of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, we have developed a porcine respiratory coronavirus (PRCV) model for in depth mechanistic evaluation of the pathogenesis, virology and immune responses of this important family of viruses. Pigs are a large animal with similar physiology and immunology to humans and are a natural host for PRCV. Four PRCV strains were investigated and shown to induce different degrees of lung pathology. Importantly, although all four strains replicated equally well in porcine cell lines in vitro and in the upper respiratory tract in vivo, PRCV strains causing more severe lung pathology were also able to replicate in ex vivo tracheal organ cultures as well as in vivo in the trachea and lung. The time course of infection of PRCV 135, which caused the most severe pulmonary pathology, was investigated. Virus was shed from the upper respiratory tract until day 10 post infection, with infection of the respiratory mucosa, as well as olfactory and sustentacular cells, providing an excellent model to study upper respiratory tract disease in addition to the commonly known lower respiratory tract disease from PRCV. Infected animals made antibody and T cell responses that cross reacted with the four PRCV strains and Transmissible Gastroenteritis Virus. The antibody response was reproduced in vitro in organ cultures. Comparison of mechanisms of infection and immune control in pigs infected with PRCVs of differing pathogenicity with human data from SARS-CoV-2 infection and from our in vitro organ cultures, will enable key events in coronavirus infection and disease pathogenesis to be identified.