Recombinant live attenuated avian coronavirus vaccines with deletions in the accessory genes 3ab and/or 5ab protect against infectious bronchitis in chickens

Abstract

Avian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens, causing severe economic losses in poultry industry worldwide. Live attenuated viruses are widely used in both the broiler and layer industry because of their efficacy and ability to be mass applied. Recently, we established a novel reverse genetics system based on targeted RNA recombination to manipulate the genome of IBV strain H52. Here we explore the possibilities to attenuate IBV in a rational way in order to generate safe and effective vaccines against virulent IBV (van Beurden et al., 2017). To this end, we deleted the nonessential group-specific accessory genes 3 and/or 5 in the IBV genome by targeted RNA recombination and selected the recombinant viruses in embryonated eggs. The resulting recombinant (r) rIBV-Δ3ab, rIBV-Δ5ab, and rIBV-Δ3ab5ab could be rescued and grew to the same virus titer as recombinant and wild type IBV strain H52. Thus, genes 3ab and 5ab are not essential for replication in ovo. When administered to one-day-old chickens, rIBV-Δ3ab, rIBV-Δ5ab, and rIBV-Δ3ab5ab showed reduced ciliostasis as compared to rIBV H52 and wild type H52, indicating that the accessory genes contribute to the pathogenicity of IBV. After homologous challenge with the virulent IBV strain M41, all vaccinated chickens were protected against disease based on reduced loss of ciliary movement in the trachea compared to the non-vaccinated but challenged controls. Taken together, deletion of accessory genes 3ab and/or 5ab in IBV resulted in mutant viruses with an attenuated phenotype and the ability to induce protection in chickens. Hence, targeted RNA recombination based on virulent IBV provides opportunities for the development of a next generation of rationally designed live attenuated IBV vaccines.

Keywords: Accessory genes; Chicken; Coronavirus; Infectious bronchitis virus; Live attenuated virus; Recombinant vaccine.

Fig. 1

Schematic overview of targeted RNA recombination principle and donor plasmids. (A) Schematic overview of step 2 in the targeted RNA recombination method for generating recombinant (r)IBV wild type (wt) . IBV sequences are represented in blue, MHV sequences in red. pIBV-derived synthetic donor RNA is indicated by a black composite line above which the parts derived from specific sub-plasmids are indicated. PCR amplicons used to confirm the recombination (set C) and the status of gene 3ab (set E) and gene 5ab (set I) are depicted as black bars drawn to scale above the rIBV-wt genome, with encircled letters referring to the primer sets in Table 2. (B) Schematic layout of the donor plasmids p-IBV-Δ3ab, -Δ5ab and -Δ3ab5ab used in targeted RNA recombination to generate rIBV-Δ3ab, -Δ5ab and -Δ3ab5ab, respectively. (C) Nucleotide sequences of the gene and plasmid junctions are marked with numbers corresponding to the numbers in black circles in the schematic donor plasmid layout in (A) and (B). Transcription regulatory sequences are in bold. Nucleotide sequences are indicated for wild type IBV and donor plasmids p-IBV, p-IBV-Δ3ab and p-IBV-Δ5ab. Start codons are highlighted in green, stop codons in red, and start-stop overlaps in yellow. ORFs are underlined, overlapping ORFs are underlined with a bold line, and ORF translations are indicated as amino acids below the nucleotide sequences if applicable. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Immunohistochemistry of CAMs after rescue of rIBV-Δ3ab, rIBV-Δ5ab and rIBV-Δ3ab5ab. Embryonated chicken eggs were inoculated with mIBV-infected LR7 cells that had been transfected with transcripts from donor plasmids p-IBV-Δ3ab, -Δ5ab or -Δ3ab5ab by electroporation. mIBV-infected non-transfected LR7 cells served as controls. Formalin-fixed and paraffin-embedded CAMs were stained using a monoclonal antibody against IBV-S2.

Fig. 3

Genetic characterization of rIBVs. PCR was performed on plasmid DNA (p) and on cDNA templates of viral RNA extracted from AF of ECEs inoculated with mIBV-infected and donor plasmid transcript-transfected LR7 cells (0), and subsequent passages (1–4) of rIBV-wt, rIBV-Δ3ab, rIBV-Δ5ab and rIBV-Δ3ab5ab. Primer set letters correspond to the respective locations in Fig. 1, and include primer sets that span the genomic area of recombination (C), accessory genes 3 (E) and 5 (I). Expected amplicon sizes are indicated in the right column, indicating for primer sets E and I the length in the presence and absence of genes 3ab or 5ab, respectively. M: molecular weight marker.

Fig. 4

In ovo characteristics of rIBVs. (A) Quantitative RT-qPCR analysis of RNA extracted from AF of ECEs collected at 12 h intervals after inoculation with IBV H52 BI, rIBV-wt, rIBV-Δ3ab, rIBV-Δ5ab, and rIBV-Δ3ab5ab was performed. Data points represent means with standard deviations of five eggs per condition, with all samples run and analyzed in triplicate using a ten-fold dilution series of IBV H52 BI as reference to determine virus quantity as EID₅₀/ml equivalents ; (B) Embryonic death is indicated as a percentage of all remaining animals at each time point.

Fig. 5

Ciliostasis after vaccination with IBV H52 BI and rIBVs, followed by challenge with IBV M41 in chickens. Plotted are tracheal ciliostasis scores per individual animal and means per experimental group. Maximal ciliostasis score per animal is 40, which indicates complete ciliostasis in all 10 transversal tracheal sections examined. (A) Ciliostasis in one-day-old chickens determined seven days after vaccination with IBV H52 BI, rIBV-wt, rIBV-Δ3ab, rIBV-Δ5ab, rIBV-Δ3ab5ab, or non-vaccinated controls; (B) Ciliostasis in vaccinated and non-vaccinated animals determined seven days after challenge with IBV M41. A non-vaccinated non-challenged control group served as control.