Attenuation of Infectious Bronchitis Virus in Eggs Results in Different Patterns of Genomic Variation across Multiple Replicates.

Michael S Oade,John A Hammond,Paul Britton,Richard J Orton,Erica Bickerton,Sarah Keep,Graham L Freimanis,Tom Gallagher

doi:10.1128/jvi.00492-19

Michael S Oade, John A Hammond + Show 6 more

Open Access

https://doi.org/10.1128/jvi.00492-19

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Abstract

The gammacoronavirus infectious bronchitis virus (IBV) causes an acute, highly contagious respiratory disease of poultry. Live attenuated vaccines are traditionally generated by serial passage of a virulent strain in embryonated chicken eggs; however, the molecular mechanism of attenuation is unknown. M41-CK, a virulent lab-adapted strain of IBV, was egg passaged over 100 times in four parallel independent replicates. All four final egg-passaged viruses were attenuated in vivo and exhibited similar growth phenotypes in adult chicken kidney cells and ex vivo tracheal organ cultures. The virus populations were sequenced by 454 pyrosequencing at the end of passaging, and the results showed that overall sequence diversity in the IBV population increased but the four replicates only had between 11 and 17 consensus-level single nucleotide polymorphisms (SNPs). Although hot spots of variation were identified in spike and nucleocapsid structural proteins as well as the 3' untranslated region, each attenuated virus possessed a different pattern of genomic variation. Overall, only a small number of consensus-level SNPs were acquired during egg passage, leaving a potentially short route back to virulence. These results highlight the unpredictable nature of attenuation by serial egg passage and the need to develop mechanisms to rationally attenuate IBV for the next generation of effective vaccines.IMPORTANCE Infectious bronchitis remains a major problem in the global poultry industry, despite the existence of many different vaccines. IBV vaccines are currently developed by serial passage of a virulent strain on embryonated hen's eggs until attenuation; however, little is known about the evolution of the viral population during the process of attenuation. High-throughput sequencing of four replicates of a serially egg-passaged IBV revealed a different pattern of genomic variation in each attenuated replicate and few consensus-level SNPs. This raises concerns that only a small number of genomic mutations are required to revert to a virulent phenotype, which may result in vaccine breakdown in the field. The observed hot spots of variation in the attenuated viruses have the potential to be used in the rational attenuation of virulent IBV for next-generation vaccine design.

Highlights

IMPORTANCE Infectious bronchitis remains a major problem in the global poultry industry, despite the existence of many different vaccines
A pathogenic strain of infectious bronchitis virus (IBV) belonging to the Massachusetts serotype lineage GI-1 [23], M41-chicken kidney (CK), was used to inoculate four embryonated eggs to establish four separate virus lineages (A, B, C, and D), each starting from the same point of origin
After a total of 106 egg passages (EP), pathogenicity was assessed for resultant EP106 viruses using in vivo and in vitro methods, with a further stock, EP107, grown solely for the purposes of 454 sequencing

Summary

Introduction

IMPORTANCE Infectious bronchitis remains a major problem in the global poultry industry, despite the existence of many different vaccines. For an RNA virus, nsp exhibits 3=-to-5= exoribonuclease activity [5] which, in conjunction with nsp, an RNA-dependent RNA polymerase (RdRp), forms part of a proofreading system that facilitates a high-fidelity replication of both genomic and subgenomic RNAs. The extent to which the proofreading capability of IBV impacts genetic variability, and the role of genetic diversity within an IB viral population, remains undefined despite having a discernible in vivo effect [6]. The extent to which the proofreading capability of IBV impacts genetic variability, and the role of genetic diversity within an IB viral population, remains undefined despite having a discernible in vivo effect [6] Both live attenuated and inactivated vaccines are used as the primary control against IB, live attenuated virus vaccines offer easy application and greater immunogenicity/protection against disease [3]. The replicase [10, 11], structural [12], and accessory [13] genes have all been implicated in IBV pathogenicity

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