A recombinant avian infectious bronchitis virus expressing a heterologous spike gene belonging to the 4/91 serotype - PubMed (original) (raw)
A recombinant avian infectious bronchitis virus expressing a heterologous spike gene belonging to the 4/91 serotype
Maria Armesto et al. PLoS One. 2011.
Abstract
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. 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.
Conflict of interest statement
Competing Interests: This work was supported in part by Intervet Schering-Plough UK. There were no consultancy payments or products in development from the submitted work. This does not alter the authors' adherence to all PLoS ONE policies on sharing data and materials.
Figures
Figure 1. Schematic diagram for the construction of the chimaeric 4/91 S gene.
Two DNA fragments of 2042 and 2300-nucleotides were generated from the IBV 4/91(UK) S gene sequence that overlapped at an internal _Alw_NI site. The 2042-nucleotide cDNA, corresponding to the 5′ half of the 4/91 S gene was digested with _Pac_I and _Alw_NI and ligated to the _Alw_NI-_Bsp_HI fragment generated from the 2300-nucleotide cDNA corresponding to the 3′ half of the 4/91 S gene. The 4/91-derived 3389-nucleotide _Pac_I-_Bsp_HI cDNA was then used to replace the corresponding M41-CK S gene sequence in pGPT-M41S generating a new chimaeric S gene sequence with the ectodomain derived from 4/91(UK) and the cytoplasmic domain from Beau-R in pGPT-4/91S. Plasmid pGPT-4/91S was used to insert the 4/91 chimaeric S gene into the Beau-R genome by homologous recombination utilising the Beau-R replicase and gene 3 sequences, 5′ and 3′ to the S gene sequence, respectively, using rVV VV-BeauR-ΔS by TDS.
Figure 2. Comparison of the amino acid sequences of the S glycoproteins of IBV strains Beau-R, 4/91(UK) and M41-CK.
The sequences were aligned using ClustalX 2.1 and compared using GeneDoc Multiple Sequence Alignment Editor and Shading Utility version 2.7.001 (
http://www.nrbsc.org/gfx/genedoc
). Amino acids shaded in black represent identical amino acid residues found in each sequence; non-highlighted residues represent differing amino acids. The transition sites between the IBV S glycoprotein domains are marked with an arrow; the signal sequence after position 18, the S1/S2 junction is after position 539, the TM domain starts at position 1096 and the cytoplasmic tail starts at position 1121. The ectodomain is composed of amino acids 19–1095 and the endodomain is composed of amino acids 1096–1164.
Figure 3. Assessment of clinical signs associated with BeauR-4/91(S)-infected chicks and following challenge with IBV 4/91(UK) or M41-CK.
The groups of chickens were assessed for the clinical signs snicking (A) and wheezing (B) following challenge with IBV 4/91(UK) or M41-CK. The levels of these clinical signs were significantly reduced in the BeauR-4/91(S):4/91 and BeauR-4/91(S):M41 groups when compared with the mock:4/91 group (p<0.05). Ciliary activity of the trachea was significantly higher in the BeauR-4/91(S):4/91 group when compared with the control group mock:4/91, indicative of homologous protection (C). Clinical signs were recorded from days 3 to 7 post-challenge. The birds were observed individually except for snicking, where they were observed as a group.
Figure 4. Ciliary activity levels displayed by TOCs infected with trachea-derived suspensions taken from birds infected with BeauR-4/91(S) and challenged with 4/91(UK) or M41-CK compared with non-vaccinated but challenged birds.
Ciliary activity levels were significantly higher in BeauR-4/91(S):4/91 and BeauR-4/91(S):M41 groups when compared with mock:4/91 group on each day (p<0.05). Ciliary activity was observed on days 4 (A), 5 (B) and 7 (C) following infection of the TOCs. Each data point represents the average of the readings of TOCs infected with tracheal samples from three birds taken on days 4, 5 and 6 after challenge (average percentage of six TOCs per bird).
References
- Cavanagh D. Coronaviruses in poultry and other birds. Avian Pathol. 2005;34:439–448. - PubMed
- Cavanagh D, Gelb J., Jr . Infectious Bronchitis. In: Saif YM, editor. Diseases of Poultry. 12th ed. Iowa: Blackwell Publishing; 2008. pp. 117–135.
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