Reverse genetics system for the avian coronavirus infectious bronchitis virus - PubMed (original) (raw)
Reverse genetics system for the avian coronavirus infectious bronchitis virus
R Casais et al. J Virol. 2001 Dec.
Abstract
Major advances in the study of the molecular biology of RNA viruses have resulted from the ability to generate and manipulate full-length genomic cDNAs of the viral genomes with the subsequent synthesis of infectious RNA for the generation of recombinant viruses. Coronaviruses have the largest RNA virus genomes and, together with genetic instability of some cDNA sequences in Escherichia coli, this has hampered the generation of a reverse-genetics system for this group of viruses. In this report, we describe the assembly of a full-length cDNA from the positive-sense genomic RNA of the avian coronavirus, infectious bronchitis virus (IBV), an important poultry pathogen. The IBV genomic cDNA was assembled immediately downstream of a T7 RNA polymerase promoter by in vitro ligation and cloned directly into the vaccinia virus genome. Infectious IBV RNA was generated in situ after the transfection of restricted recombinant vaccinia virus DNA into primary chick kidney cells previously infected with a recombinant fowlpox virus expressing T7 RNA polymerase. Recombinant IBV, containing two marker mutations, was recovered from the transfected cells. These results describe a reverse-genetics system for studying the molecular biology of IBV and establish a paradigm for generating genetically defined vaccines for IBV.
Figures
FIG. 1
Schematic diagram for the assembly of the full-length IBV cDNA in the vaccinia virus genome. The positions of the IBV genes within the IBV-derived cDNAs are shown. The strategy used for the assembly of the full-length IBV cDNA and insertion into the vaccinia virus genome is shown with the final orientation of the cDNA in the vaccinia genome. “AP” indicates that the restriction site on the particular cDNA was dephosphorylated. Rep, S, 3, M, 5, and N refer to the IBV genes; T7ψ, HδR, and T7φ refer to the positions of the T7 promoter, hepatitis delta antigenome ribozyme, and T7 terminator sequences, respectively. The three extra G residues at the 5′ end of the IBV sequence forming part of the T7 promoter sequence are indicated. The terminal _Sal_I sites shown represent the 5.7-kb _Sal_I fragment normally containing the 534-bp vaccinia virus tk gene. The right-hand _Sal_I site is ∼1.1 kb from the tk gene, and the left-hand _Sal_I site is ∼4.1 kb from the tk gene. The non-IBV regions are not drawn to scale. Arrowheads indicate the positions of the 14 oligonucleotides used to generate RT-PCR or PCR products from the IBV genome or IBV cDNA in vNotI/IBVFL. The sizes and annotations (A to G) of the fragments are shown.
FIG. 2
Analysis of the in vitro assembly of IBV-derived cDNAs for the generation of a full-length cDNA. Samples of the ligation mixtures for assembly of IBV cDNAs FRAG-2 and FRAG-3 (A) and assembly of the full-length IBV cDNA by ligation of FRAG-1 to the intermediary 21.6-kb FRAG-2–FRAG-3 fragment (B) were analyzed by pulsed-field gel electrophoresis in 0.8% agarose gels. The cDNAs are as outlined in Fig. 1. Lanes 1 contained DNA markers (8.3 to 48.5 kb), and lanes 2 contained samples of the ligation mixtures.
FIG. 3
Analysis of DNA isolated from the recombinant vaccinia virus vNotI/IBVFL containing the full-length IBV cDNA. (A) Vaccinia virus genomic DNA was analyzed by restriction digestion. Lane 1 contained vNotI/tk DNA digested with _Sal_I, lane 2 contained vNotI/IBVFL DNA digested with _Sal_I, and lane 3 contained vNotI/IBVFL DNA digested with _Sal_I and _Asc_I. The DNA samples were analyzed by pulsed-field gel electrophoresis with 1% agarose gels. The 32-kb vaccinia virus-IBV _Sal_I fragment containing the full-length IBV cDNA (derived from the _Sal_I site following the T7 termination sequence in the IBV cDNA and a _Sal_I site downstream of the vaccinia virus tk gene; Fig. 1) and the 27.9-kb _Asc_I-_Sal_I IBV cDNA fragment are marked by arrows. The _Asc_I site was incorporated upstream of the T7 promoter during the construction of FRAG-1 and is unique in vNotI/IBVFL. The 32-kb _Sal_I fragment contains the 27.9-kb IBV-containing cDNA and the 4.6-kb vaccinia virus-derived DNA containing the tk gene. The 4.6-kb _Asc_I-_Sal_I vaccinia virus-derived DNA comigrates with the smallest vaccinia virus _Sal_I fragment shown at the bottom of the gel. The lane marked M contained DNA markers of 8.3 to 48.5 kb. (B) PCR analysis of DNA extracted from vNotI/IBVFL. Lanes 1 to 7 represent PCR products A to G respectively, as indicated in Fig. 1. Each lane consisted of three tracks in which the first track represented PCR products derived from the appropriate IBV cDNA in pFRAG-1, pFRAG-2, or pFRAG-3; the second track represented PCR products derived from vNotI/IBVFL DNA; and the third track represented PCR products derived from water. The PCR fragments were analyzed by agarose gel electrophoresis with 0.7% agarose. Lane M contained DNA markers with the sizes of the smaller fragments indicated.
FIG. 4
Detection of IBV in infected Vero cells by indirect immunofluorescence. Vero cells at 60% confluency were infected with IBV, fixed after 18 h with 50% methanol-acetone, analyzed by indirect immunofluorescence with rabbit anti-IBV polyclonal sera, followed by FITC-labeled goat anti-rabbit antibody, and then stained with propidium iodide to visualize the nuclear DNA. (a) Vero cells that had been infected with Beau-R and were analyzed with preimmune rabbit serum. The remaining panels show Vero cells, analyzed with rabbit anti-IBV serum, infected with Beau-US, exhibiting syncytium formation (b and e); Beau-CK (c); or Beau-R (d and f). Magnifications: a to d, ×16; e and f, ×63.
FIG. 5
Analysis of IBV-specific RNAs after transfection of _Asc_I-restricted vNotI/IBVFL DNA into CK cells infected with rFPV-T7. CK cells infected with rFPV-T7 were transfected with _Asc_I-restricted vNotI/IBVFL DNA (P0 cells), and at 84 h posttransfection the cell medium was filtered to remove any rFPV-T7. Potential IBV (V1) in the filtered medium was used to infect CK cells (P1). This was repeated up to passage 5 (P5) by using any recovered IBV (V2 to V4) in the cell medium. The total cellular RNA was extracted from the transfected (P0) or infected (P1 to P5) CK cells, electrophoresed in denaturing formaldehyde-agarose gels, and Northern blotted, and IBV-derived RNAs were detected nonisotopically with a 309-bp IBV 3′-UTR probe (13). Lane 1, RNA from mock-infected CK cells; lanes 2 to 7, RNA from P0 to P5 CK cells potentially infected with recovered IBV; lane 8, RNA from CK cells infected with Beau-US. The IBV-specific RNAs (indicated by gRNA and mRNAs 2 to 6) represent the IBV genomic RNA and sg mRNAs 2 to 6. The RNAs detected between sg mRNAs 4 and 5 and below sg mRNA 6 are routinely observed from all strains of IBV, as originally identified by (36), and are of unknown origin.
FIG. 6
Analysis of the marker mutations in recombinant IBV Beau-R. (A) _Bst_BI digestion of the 1,544-bp RT-PCR products generated by using oligonucleotides BG-68 and BG-132, corresponding to the region of the _Bst_BI site present in the Beau-CK and Beau-US genomes. Lanes 1, 2, and 3, correspond to the _Bst_BI-restricted RT-PCR products amplified from RNA isolated from CK cells infected with Beau-US, Beau-R, and Beau-CK, respectively. Lane M contained DNA markers. (B) Sequence analysis of IBV genomic RNA, derived from CK cells infected with either Beau-US or Beau-R, representing the _Bst_BI site that contained the C19666→U point mutation in Beau-R. (C) Sequence analysis of IBV genomic RNA, analyzed from CK cells infected with Beau-US and Beau-R, representing the region at the 3′ end of the N gene sequence corresponding to the silent A27087→G point mutation in the Beau-R sequence. The point mutations are marked with an asterisk, and the positions of the mutations within the cDNA sequence are shown. The genomic sequence derived from Beau-CK (6) is the same as that determined for the Beau-US sequence.
FIG. 6
Analysis of the marker mutations in recombinant IBV Beau-R. (A) _Bst_BI digestion of the 1,544-bp RT-PCR products generated by using oligonucleotides BG-68 and BG-132, corresponding to the region of the _Bst_BI site present in the Beau-CK and Beau-US genomes. Lanes 1, 2, and 3, correspond to the _Bst_BI-restricted RT-PCR products amplified from RNA isolated from CK cells infected with Beau-US, Beau-R, and Beau-CK, respectively. Lane M contained DNA markers. (B) Sequence analysis of IBV genomic RNA, derived from CK cells infected with either Beau-US or Beau-R, representing the _Bst_BI site that contained the C19666→U point mutation in Beau-R. (C) Sequence analysis of IBV genomic RNA, analyzed from CK cells infected with Beau-US and Beau-R, representing the region at the 3′ end of the N gene sequence corresponding to the silent A27087→G point mutation in the Beau-R sequence. The point mutations are marked with an asterisk, and the positions of the mutations within the cDNA sequence are shown. The genomic sequence derived from Beau-CK (6) is the same as that determined for the Beau-US sequence.
References
- Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K. Current protocols in molecular biology. New York, N.Y: John Wiley & Sons, Inc.; 1987.
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