Temperature Sensitivity: A Potential Method for the Generation of Vaccines against the Avian Coronavirus Infectious Bronchitis Virus

Abstract

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.

The Characterization of chIFITMs in Avian Coronavirus Infection In Vivo, Ex Vivo and In Vitro

“…The absence of replicating Beau-R in the trachea supports what has been seen in previous studies [ 20 , 69 , 71 ]. Infection with Beau-R did not elicit an upregulation of either IFN-α , IFN-β or chIFITMs mRNA in infected trachea ( Figure 1 B–G), strengthening the assumption that Beau-R fails to produce a productive infection in vivo [ 55 ]. In the trachea of chickens infected with M41-CK, the viral copy number was significantly higher when compared to mock-infected and Beau-R-infected samples, peaking at 4 dpi (9.8 × 10 4 ± 1.5 × 10 5 copies/µL; p = 0.017), steadily decreasing until 7 dpi (1.28 × 10 2 ± 2.7 × 10 1 copies/µL) ( Figure 1 A).…”

“…In the first study, mock-infected birds and those infected with rIBV Beau-R, a molecular clone of the pathogenic Beau-CK strain [ 5 ], displayed no IBV-related clinical signs and no reduction in ciliary activity while birds infected with M41-CK displayed clinical signs from day 2 post-inoculation and had a reduced ciliary activity (<2%), as previously described in [ 55 ].…”

“…All animal experiments have been published previously [ 54 , 55 ] and were carried out following the UK Home Office guidelines and the UK Animals (Scientific Procedures) Act 1986. All experiments were performed at The Pirbright Institute animal facilities, license number X24684464.…”

“…On 1, 4, 6 and 7 dpi, 6 randomly selected birds were euthanized by cervical dislocation and trachea tissue harvested. Ciliary activity in the trachea was assessed on 4 and 6 dpi [ 55 ]. Tracheal ciliary activity is used as the gold standard to determine the pathogenicity of an IBV strain [ 65 ], a ciliary activity score of less than 50% is deemed pathogenic.…”

“…Tracheal ciliary activity is used as the gold standard to determine the pathogenicity of an IBV strain [ 65 ], a ciliary activity score of less than 50% is deemed pathogenic. The clinical signs and ciliary activity have previously been reported [ 55 ].…”