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
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.
CavanaghD, CasaisR, ArmestoM, HodgsonT, IzadkhastiS et al. Manipulation of the infectious bronchitis coronavirus genome for vaccine development and analysis of the accessory proteins. Vaccine2007; 25:5558–5562 [View Article][PubMed]
CasaisR, DaviesM, CavanaghD, BrittonP. Gene 5 of the avian coronavirus infectious bronchitis virus is not essential for replication. J Virol2005; 79:8065–8078 [View Article][PubMed]
de HaanCA, MastersPS, ShenX, WeissS, RottierPJ. The group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host. Virology2002; 296:177–189 [View Article][PubMed]
HaijemaBJ, VoldersH, RottierPJ. Live, attenuated coronavirus vaccines through the directed deletion of group-specific genes provide protection against feline infectious peritonitis. J Virol2004; 78:3863–3871 [View Article][PubMed]
HodgsonT, BrittonP, CavanaghD. Neither the RNA nor the proteins of open reading frames 3a and 3b of the coronavirus infectious bronchitis virus are essential for replication. J Virol2006; 80:296–305 [View Article][PubMed]
ShenS, WenZL, LiuDX. Emergence of a coronavirus infectious bronchitis virus mutant with a truncated 3b gene: functional characterization of the 3b protein in pathogenesis and replication. Virology2003; 311:16–27 [View Article][PubMed]
van BeurdenSJ, BerendsAJ, Krämer-KühlA, SpekreijseD, ChénardG et al. A reverse genetics system for avian coronavirus infectious bronchitis virus based on targeted RNA recombination. Virol J2017; 14:109 [View Article][PubMed]
van BeurdenSJ, BerendsAJ, Krämer-KühlA, SpekreijseD, ChenardG et al. Recombinant live attenuated avian coronavirus vaccines with deletions in the accessory genes 3ab and/or 5ab protect against infectious bronchitis in chickens. Vaccine2018; 36:1085–1092 [View Article][PubMed]
YounS, LeibowitzJL, CollissonEW. In vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication. Virology2005; 332:206–215 [View Article][PubMed]
DedeurwaerderA, OlyslaegersDA, DesmaretsLM, RoukaertsID, TheunsS et al. ORF7-encoded accessory protein 7a of feline infectious peritonitis virus as a counteragent against IFN-α-induced antiviral response. J Gen Virol2014; 95:393–402 [View Article][PubMed]
KoetznerCA, KuoL, GoebelSJ, DeanAB, ParkerMM et al. Accessory protein 5a is a major antagonist of the antiviral action of interferon against murine coronavirus. J Virol2010; 84:8262–8274 [View Article][PubMed]
ThorneN, ShenM, LeaWA, SimeonovA, LovellS et al. Firefly luciferase in chemical biology: a compendium of inhibitors, mechanistic evaluation of chemotypes, and suggested use as a reporter. Chem Biol2012; 19:1060–1072 [View Article][PubMed]
KintJ, LangereisMA, MaierHJ, BrittonP, van KuppeveldFJ et al. Infectious bronchitis coronavirus limits interferon production by inducing a host shutoff that requires accessory protein 5b. J Virol2016; 90:7519–7528 [View Article][PubMed]
KintJ, DickhoutA, KutterJ, MaierHJ, BrittonP et al. Infectious bronchitis coronavirus inhibits STAT1 signaling and requires accessory proteins for resistance to type I interferon activity. J Virol2015; 89:12047–12057 [View Article][PubMed]
KintJ, Fernandez-GutierrezM, MaierHJ, BrittonP, LangereisMA et al. Activation of the chicken type I interferon response by infectious bronchitis coronavirus. J Virol2015; 89:1156–1167 [View Article][PubMed]
ZhaoJ, FalcónA, ZhouH, NetlandJ, EnjuanesL et al. Severe acute respiratory syndrome coronavirus protein 6 is required for optimal replication. J Virol2009; 83:2368–2373 [View Article][PubMed]
KuoL, GodekeGJ, RaamsmanMJ, MastersPS, RottierPJ. Retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier. J Virol2000; 74:1393–1406 [View Article][PubMed]
SchwarzH, HarlinO, OhnemusA, KaspersB, StaeheliP. Synthesis of IFN-beta by virus-infected chicken embryo cells demonstrated with specific antisera and a new bioassay. J Interferon Cytokine Res2004; 24:179–184 [View Article][PubMed]
CavanaghD, ElusMM, CookJK. Relationship between sequence variation in the S1 spike protein of infectious bronchitis virus and the extent of cross-protection in vivo. Avian Pathol1997; 26:63–74 [View Article][PubMed]
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