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J Virol ; 96(6): e0205921, 2022 03 23.
Article in English | MEDLINE | ID: covidwho-1788916


The Gammacoronavirus infectious bronchitis virus (IBV) is a highly contagious global pathogen prevalent in all types of poultry flocks. IBV is responsible for economic losses and welfare issues in domestic poultry, resulting in a significant risk to food security. IBV vaccines are currently generated by serial passage of virulent IBV field isolates through embryonated hens' eggs. The different patterns of genomic variation accumulated during this process means that the exact mechanism of attenuation is unknown and presents a risk of reversion to virulence. Additionally, the passaging process adapts the virus to replicate in chicken embryos, increasing embryo lethality. Vaccines produced in this manner are therefore unsuitable for in ovo application. We have developed a reverse genetics system, based on the pathogenic IBV strain M41, to identify genes which can be targeted for rational attenuation. During the development of this reverse genetics system, we identified four amino acids, located in nonstructural proteins (nsps) 10, 14, 15, and 16, which resulted in attenuation both in vivo and in ovo. Further investigation highlighted a role of amino acid changes, Pro85Leu in nsp 10 and Val393Leu in nsp 14, in the attenuated in vivo phenotype observed. This study provides evidence that mutations in nsps offer a promising mechanism for the development of rationally attenuated live vaccines against IBV, which have the potential for in ovo application. IMPORTANCE The Gammacoronavirus infectious bronchitis virus (IBV) is the etiological agent of infectious bronchitis, an acute, highly contagious, economically important disease of poultry. Vaccination is achieved using a mixture of live attenuated vaccines for young chicks and inactivated vaccines as boosters for laying hens. Live attenuated vaccines are generated through serial passage in embryonated hens' eggs, an empirical process which achieves attenuation but retains immunogenicity. However, these vaccines have a risk of reversion to virulence, and they are lethal to the embryo. In this study, we identified amino acids in the replicase gene which attenuated IBV strain M41, both in vivo and in ovo. Stability assays indicate that the attenuating amino acids are stable and unlikely to revert. The data in this study provide evidence that specific modifications in the replicase gene offer a promising direction for IBV live attenuated vaccine development, with the potential for in ovo application.

Amino Acids , Coronavirus Infections , Infectious bronchitis virus , Poultry Diseases , Viral Nonstructural Proteins , Viral Vaccines , Amino Acids/chemistry , Amino Acids/genetics , Animals , Chick Embryo , Chickens , Coronavirus Infections/prevention & control , Coronavirus Infections/veterinary , Coronavirus Infections/virology , Female , Infectious bronchitis virus/genetics , Poultry Diseases/prevention & control , Poultry Diseases/virology , Vaccines, Attenuated/genetics , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Vaccines/genetics
Methods Mol Biol ; 2203: 67-74, 2020.
Article in English | MEDLINE | ID: covidwho-761347


This chapter reports the high-throughput sequencing protocol for sequencing Coronaviruses and other positive strand viruses to produce a dataset of significant depth of coverage. The protocol describes sequencing of infectious bronchitis virus propagated in embryonated eggs and harvested in the allantoic fluid. The protocol is composed of three main steps-enrichment of the allantoic fluid using ultracentrifugation, extraction of total RNA from allantoic fluid, and library preparation from total RNA to DNA sequencing libraries. The workflow will be suitable for all coronaviruses using high-throughput sequencing platforms.

Coronavirus/genetics , Whole Genome Sequencing/methods , Animals , Chorioallantoic Membrane/virology , Genome, Viral , High-Throughput Nucleotide Sequencing/methods , Infectious bronchitis virus/genetics , Infectious bronchitis virus/isolation & purification , Workflow
J Gen Virol ; 101(10): 1103-1118, 2020 10.
Article in English | MEDLINE | ID: covidwho-688886


Coronavirus sub-genomic mRNA (sgmRNA) synthesis occurs via a process of discontinuous transcription involving complementary transcription regulatory sequences (TRSs), one (TRS-L) encompassing the leader sequence of the 5' untranslated region (UTR), and the other upstream of each structural and accessory gene (TRS-B). Several coronaviruses have an ORF located between the N gene and the 3'-UTR, an area previously thought to be non-coding in the Gammacoronavirus infectious bronchitis virus (IBV) due to a lack of a canonical TRS-B. Here, we identify a non-canonical TRS-B allowing for a novel sgmRNA relating to this ORF to be produced in several strains of IBV: Beaudette, CR88, H120, D1466, Italy-02 and QX. Interestingly, the potential protein produced by this ORF is prematurely truncated in the Beaudette strain. A single nucleotide deletion was made in the Beaudette strain allowing for the generation of a recombinant IBV (rIBV) that had the potential to express a full-length protein. Assessment of this rIBV in vitro demonstrated that restoration of the full-length potential protein had no effect on viral replication. Further assessment of the Beaudette-derived RNA identified a second non-canonically transcribed sgmRNA located within gene 2. Deep sequencing analysis of allantoic fluid from Beaudette-infected embryonated eggs confirmed the presence of both the newly identified non-canonically transcribed sgmRNAs and highlighted the potential for further yet unidentified sgmRNAs. This HiSeq data, alongside the confirmation of non-canonically transcribed sgmRNAs, indicates the potential of the coronavirus genome to encode a larger repertoire of genes than has currently been identified.

Infectious bronchitis virus/genetics , RNA, Messenger/genetics , RNA, Viral/genetics , Regulatory Sequences, Nucleic Acid/genetics , Transcription, Genetic/genetics , 5' Untranslated Regions/genetics , Animals , Base Sequence , Cell Line , Chickens , Chlorocebus aethiops , Coronavirus Infections/veterinary , Coronavirus Infections/virology , Open Reading Frames/genetics , Poultry Diseases/virology , Vero Cells , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Replication/genetics