Deletion of accessory genes 3a, 3b, 5a or 5b from avian coronavirus infectious bronchitis virus induces an attenuated phenotype both in vitro and in vivo

Abstract

Avian coronavirus infectious bronchitis virus (IBV) infects domestic fowl, resulting in respiratory disease and causing serious losses in unprotected birds. Its control is mainly achieved by using live attenuated vaccines. Here we explored the possibilities for rationally attenuating IBV to improve our knowledge regarding the function of IBV accessory proteins and for the development of next-generation vaccines with the recently established reverse genetic system for IBV H52 based on targeted RNA recombination and selection of recombinant viruses in embryonated eggs. To this aim, we selectively removed accessory genes 3a, 3b, 5a and 5b individually, and rescued the resulting recombinant (r) rIBV-Δ3a, rIBV-Δ3b, rIBV-Δ5a and rIBV-Δ5b. In vitro inoculation of chicken embryo kidney cells with recombinant and wild-type viruses demonstrated that the accessory protein 5b is involved in the delayed activation of the interferon response of the host after IBV infection. Embryo mortality after the inoculation of 8-day-old embryonated chicken eggs with recombinant and wild-type viruses showed that rIBV-Δ3b, rIBV-Δ5a and rIBV-Δ5b had an attenuated phenotype in ovo, with reduced titres at 6 h p.i. and 12 h p.i. for all viruses, while growing to the same titre as wild-type rIBV at 48 h p.i. When administered to 1-day-old chickens, rIBV-Δ3a, rIBV-Δ3b, rIBV-Δ5a and rIBV-Δ5b showed reduced ciliostasis in comparison to the wild-type viruses. In conclusion, individual deletion of accessory genes in IBV H52 resulted in mutant viruses with an attenuated phenotype.

Recombinant infectious bronchitis coronavirus H120 with the spike protein S1 gene of the nephropathogenic IBYZ strain remains attenuated but induces protective immunity

“…The viral load of the respiratorytype strain, rH120 in the different tissues indicated that the viral levels in the trachea, early following infection were significantly higher than those of rIBYZ, which indicates that the ability of the virus to attached to trachea epithelial cells of the rH120 strain was stronger than that of the rIBYZ strain. However, during the later time points post-infection, the level of the rH120 strain was significantly lower than rIBYZ, which suggests that the virulence of IBV depends both on tissue invasion and viral replication ability, as well as other functions mediated by other viral proteins (e.g., non-structural, structural, and accessory proteins) [6,29,[58][59][60]. The reduction of the viral load at 5 dpi in the group infected with the rIBYZ strain may be associated with the necrosis and shedding of the trachea epithelium.…”

Genetic and biological characteristics of four novel recombinant avian infectious bronchitis viruses isolated in China

“…recombinant viruses. By using reverse genetics, a previous report showed that the replicase gene of IBV is a determinant of pathogenicity (Armesto et al, 2009) and recent studies have shown that IBV pathogenicity is also due to the accessory proteins (Laconi et al, 2018;van Beurden et al, 2018). In order to establish the mechanisms, further study by reverse genetics and animal studies is needed to verify the association between changes in the genome sequence and the pathogenicity of IBV strains.…”

“…However, the results of S1 sequence analysis might not be solely sufficient to explain the changes observed in IBV antigenicity, tissue tropism, and pathogenicity. In recent years, several studies have noted that viral replication, pathogenicity, and immune escape might be modulated by non-structural and accessory viral proteins in IBV and other coronaviruses (Armesto et al, 2009;Chen et al, 2009;Laconi et al, 2018;Zheng et al, 2008;van Beurden et al, 2018). These findings emphasize the importance of studying the characteristics of the complete genomes of coronaviruses and their associations with antigenicity, tissue tropism, and pathogenicity.…”

Successful establishment of a reverse genetic system for QX-type infectious bronchitis virus and technical improvement of the rescue procedure

“…IBV also possesses two accessory genes, Gene 3 and Gene 5, which have been shown to be dispensable for virus replication in cell culture Youn et al, 2005;Hodgson et al, 2006;Bentley et al, 2013). However, it seems that the deletion of accessory genes 3a, 3b, 5a or 5b from IBV could induce an attenuated phenotype both in vitro and in vivo (Laconi et al, 2018;van Beurden et al, 2018).…”

“…Therefore, infectious fulllength clones of coronaviruses have only been established at the beginning of the 21 st century (Casais et al, 2001). Subsequently, reverse genetics was successfully applied to study the role of accessory genes in viral replication Youn et al, 2005;Laconi et al, 2018), the potential of IBV as a vaccine vector (Britton et al, 2012;Bentley et al, 2013), the virulence determinants of the Beaudette and M41 strains (Fang et al, 2007;Maria et al, 2009;Keep et al, 2018), and the relationship between the S gene and tissue tropism of the virus (Casais et al, 2003;Britton et al, 2005;Promkuntod et al, 2013;Shan et al, 2018). However, to the best of our knowledge, all existing reverse genetics systems for IBV were either based on Vero cell-adapted Beaudette strain, which is non-pathogenic in chickens (Geilhausen et al, 1973), or the Mass-type strains, which are genetic quite distant from the prevalent QX-type strains.…”