Abstract
BackgroundAvian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens that causes severe economic losses in the poultry industry worldwide. Major advances in the study of the molecular biology of IBV have resulted from the development of reverse genetics systems for the highly attenuated, cell culture-adapted, IBV strain Beaudette. However, most IBV strains, amongst them virulent field isolates, can only be propagated in embryonated chicken eggs, and not in continuous cell lines.MethodsWe established a reverse genetics system for the IBV strain H52, based on targeted RNA recombination in a two-step process. First, a genomic and a chimeric synthetic, modified IBV RNA were co-transfected into non-susceptible cells to generate a recombinant chimeric murinized (m) IBV intermediate (mIBV). Herein, the genomic part coding for the spike glycoprotein ectodomain was replaced by that of the coronavirus mouse hepatitis virus (MHV), allowing for the selection and propagation of recombinant mIBV in murine cells. In the second step, mIBV was used as the recipient. To this end a recombination with synthetic RNA comprising the 3′-end of the IBV genome was performed by introducing the complete IBV spike gene, allowing for the rescue and selection of candidate recombinants in embryonated chicken eggs.ResultsTargeted RNA recombination allowed for the modification of the 3′-end of the IBV genome, encoding all structural and accessory genes. A wild-type recombinant IBV was constructed, containing several synonymous marker mutations. The in ovo growth kinetics and in vivo characteristics of the recombinant virus were similar to those of the parental IBV strain H52.ConclusionsTargeted RNA recombination allows for the generation of recombinant IBV strains that are not able to infect and propagate in continuous cell lines. The ability to introduce specific mutations holds promise for the development of rationally designed live-attenuated IBV vaccines and for studies into the biology of IBV in general.
Highlights
Avian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens that causes severe economic losses in the poultry industry worldwide
Generation and antigenic characterization of Murinized IBV (mIBV) and Recombinant IBV (rIBV)-wt Viral RNA of IBV H52 BI and RNAs transcribed from plasmids p-mIBV and p-IBV-N were co-transfected into BHK-21 cells and seeded onto monolayers of LR7 cells
IF staining of LR7 cells infected with mIBV showed positive staining with both anti-IBV and anti-mouse hepatitis virus (MHV) sera, indicating the chimeric nature of mIBV (Fig. 2a)
Summary
Avian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens that causes severe economic losses in the poultry industry worldwide. Avian coronavirus infectious bronchitis virus (IBV) primarily infects the upper respiratory epithelium of chickens, causing a respiratory disease that is frequently complicated by secondary bacterial pathogens [1]. The 5′ two-third of the viral genome comprises gene 1, divided into two large open reading frames 1a and 1b, which code for 15 nonstructural proteins primarily involved in RNA replication and transcription. Interspersed between the structural genes, coronaviruses carry a variable number of genus specific accessory genes [6] Most of their gene products are nonstructural, and their expression is not essential for virus replication in vitro [7,8,9,10,11,12]. An open reading frame located in the intergenic region was identified between genes 4 and 5 [13]
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