Sarah Keep

A Recombinant Avian Infectious Bronchitis Virus Expressing a Heterologous Spike Gene Belonging to the 4/91 Serotype

We have shown previously that replacement of the spike (S) gene of the apathogenic IBV strain Beau-R with that from the pathogenic strain of the same serotype, M41, resulted in an apathogenic virus, BeauR-M41(S), that conferred protection against challenge with M41 [1]. We have constructed a recombinant IBV, BeauR-4/91(S), with the genetic backbone of Beau-R but expressing the spike protein of the pathogenic IBV strain 4/91(UK), which belongs to a different serogroup as Beaudette or M41. Similar to our previous findings with BeauR-M41(S), clinical signs observations showed that the S gene of the pathogenic 4/91 virus did not confer pathogenicity to the rIBV BeauR-4/91(S). Furthermore, protection studies showed there was homologous protection; BeauR-4/91(S) conferred protection against challenge with wild type 4/91 virus as shown by the absence of clinical signs, IBV RNA assessed by qRT-PCR and the fact that no virus was isolated from tracheas removed from birds primarily infected with BeauR-4/91(S) and challenged with IBV 4/91(UK). A degree of heterologous protection against M41 challenge was observed, albeit at a lower level. Our results confirm and extend our previous findings and conclusions that swapping of the ectodomain of the S protein is a precise and effective way of generating genetically defined candidate IBV vaccines.

Identification of a Noncanonically Transcribed Subgenomic mRNA of Infectious Bronchitis Virus and Other Gammacoronaviruses

ABSTRACT Coronavirus subgenomic mRNA (sgmRNA) synthesis occurs via a process of discontinuous transcription involving transcription regulatory sequences (TRSs) located in the 5′ leader sequence (TRS-L) and upstream of each structural and group-specific gene (TRS-B). Several gammacoronaviruses including infectious bronchitis virus (IBV) contain a putative open reading frame (ORF), localized between the M gene and gene 5, which is controversial due to the perceived absence of a TRS. We have studied the transcription of a novel sgmRNA associated with this potential ORF and found it to be transcribed via a previously unidentified noncanonical TRS-B. Using an IBV reverse genetics system, we demonstrated that the template-switching event during intergenic region (IR) sgmRNA synthesis occurs at the 5′ end of the noncanonical TRS-B and recombines between nucleotides 5 and 6 of the 8-nucleotide consensus TRS-L. Introduction of a complete TRS-B showed that higher transcription levels are achieved by increasing the number of nucleotide matches between TRS-L and TRS-B. Translation of a protein from the sgmRNA was demonstrated using enhanced green fluorescent protein, suggesting the translation of a fifth, novel, group-specific protein for IBV. This study has resolved an issue concerning the number of ORFs expressed by members of the Gammacoronavirus genus and proposes the existence of a fifth IBV accessory protein. We confirmed previous reports that coronaviruses can produce sgmRNAs from noncanonical TRS-Bs, which may expand their repertoire of proteins. We also demonstrated that noncanonical TRS-Bs may provide a mechanism by which coronaviruses can control protein expression levels by reducing sgmRNA synthesis.

Modification of the avian coronavirus infectious bronchitis virus for vaccine development

Infectious bronchitis virus (IBV) causes an infectious respiratory disease of domestic fowl that affects poultry of all ages causing economic problems for the poultry industry worldwide. Although IBV is controlled using live attenuated and inactivated vaccines it continues to be a major problem due to the existence of many serotypes, determined by the surface spike protein resulting in poor cross-protection, and loss of immunogenicity associated with vaccine production. Live attenuated IBV vaccines are produced by the repeated passage in embryonated eggs resulting in spontaneous mutations. As a consequence attenuated viruses have only a few mutations responsible for the loss of virulence, which will differ between vaccines affecting virulence and/or immunogenicity and can revert to virulence. A new generation of vaccines is called for and one means of controlling IBV involves the development of new and safer vaccines by precisely modifying the IBV genome using reverse genetics for the production of rationally attenuated IBVs in order to obtain an optimum balance between loss of virulence and capacity to induce immunity.

Extensive coronavirus-induced membrane rearrangements are not a determinant of pathogenicity.

Positive-strand RNA (+RNA) viruses rearrange cellular membranes during replication, possibly in order to concentrate and arrange viral replication machinery for efficient viral RNA synthesis. Our previous work showed that in addition to the conserved coronavirus double membrane vesicles (DMVs), Beau-R, an apathogenic strain of avian Gammacoronavirus infectious bronchitis virus (IBV), induces regions of ER that are zippered together and tethered open-necked double membrane spherules that resemble replication organelles induced by other +RNA viruses. Here we compared structures induced by Beau-R with the pathogenic lab strain M41 to determine whether membrane rearrangements are strain dependent. Interestingly, M41 was found to have a low spherule phenotype. We then compared a panel of pathogenic, mild and attenuated IBV strains in ex vivo tracheal organ culture (TOC). Although the low spherule phenotype of M41 was conserved in TOCs, each of the other tested IBV strains produced DMVs, zippered ER and spherules. Furthermore, there was a significant correlation for the presence of DMVs with spherules, suggesting that these structures are spatially and temporally linked. Our data indicate that virus induced membrane rearrangements are fundamentally linked to the viral replicative machinery. However, coronavirus replicative apparatus clearly has the plasticity to function in different structural contexts.

Spherules and IBV.

Infectious bronchitis virus (IBV) is an economically important virus infecting chickens, causing large losses to the poultry industry globally. While vaccines are available, there is a requirement for novel vaccine strategies due to high strain variation and poor cross-protection. This requires a more detailed understanding of virus-host cell interactions to identify candidates for targeted virus attenuation. One key area of research in the positive sense RNA virus field, due to its central role in virus replication, is the induction of cellular membrane rearrangements by this class of viruses for the assembly of virus replication complexes. In our recent work, we identified the structures induced by IBV during infection of cultured cells, as well as primary cells and ex vivo organ culture. We identified structures novel to the coronavirus family, which strongly resemble replication sites of other positive sense RNA viruses. We have begun to extend this work using recombinant IBVs, which are chimera of different virus strains to study the role of viral proteins in the induction of membrane rearrangements.

The ADRP domain from a virulent strain of infectious bronchitis virus is not sufficient to confer a pathogenic phenotype to the attenuated Beaudette strain

The replicase gene of the coronavirus infectious bronchitis virus (IBV) encodes 15 non-structural proteins (nsps). Nsp 3 is a multi-functional protein containing a conserved ADP-ribose-1″-phosphatase (ADRP) domain. The crystal structures of the domain from two strains of IBV, M41 (virulent) and Beaudette (avirulent), identified a key difference; M41 contains a conserved triple-glycine motif, whilst Beaudette contains a glycine-to-serine mutation that is predicted to abolish ADRP activity. Although ADRP activity has not been formally demonstrated for IBV nsp 3, Beaudette fails to bind ADP-ribose. The role of ADRP in virulence was investigated by generating rIBVs, based on Beaudette, containing either a restored triple-glycine motif or the complete M41 ADRP domain. Replication in vitro was unaffected by the ADRP modifications and the in vivo phenotype of the rIBVs was found to be apathogenic, indicating that restoration of the triple-glycine motif is not sufficient to restore virulence to the apathogenic Beaudette strain.

Reverse Genetics of Avian Coronavirus Infectious Bronchitis Virus

We have developed a reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) in which a full-length cDNA corresponding to the IBV genome is inserted into the vaccinia virus genome under the control of a T7 promoter sequence. Vaccinia virus as a vector for the full-length IBV cDNA has the advantage that modifications can be introduced into the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. Here, we describe the use of transient dominant selection as a method for introducing modifications into the IBV cDNA; that has been successfully used for the substitution of specific nucleotides, deletion of genomic regions, and the exchange of complete genes. Infectious recombinant IBVs are generated in situ following the transfection of vaccinia virus DNA, containing the modified IBV cDNA, into cells infected with a recombinant fowlpox virus expressing T7 DNA dependant RNA polymerase.

Partial purification of IBV and subsequent isolation of viral RNA for next-generation sequencing.

RNA viruses are known for a high mutation rate and rapid genomic evolution. As such an RNA virus population does not consist of a single genotype but is rather a collection of individual viruses with closely related genotypes—a quasispecies, which can be analyzed by next-generation sequencing (NGS). This diversity of genotypes provides a mechanism in which a virus population can evolve and adapt to a changing environment. Sample preparation is vital for successful sequencing. The following protocol describes the process of generating a high-quality RNA preparation from IBV grown in embryonated eggs and then partially purified and concentrated through a 30 % sucrose cushion for NGS.