The cloning strategy for a full-length IBV-Beaudette construct is illustrated in Fig. 1A. Five RT-PCR fragments were produced that cumulatively amplified the entire genome of the Vero cell adapted Beaudette IBV strain. Amplicons, overlapping by about 10 nucleotides, were designated A through E and the fragment lengths were as follows; A, 2269 nts; B, 6356 nts; C, 6895 nts; D, 5030 nts; and E, 7058 nts (Fig. 1B). However, consistent with the report that these regions were toxic or unstable in bacteria, difficulties arose in cloning the B and C fragments into bacterial plasmid based cloning vectors (Casais et al., 2001). Therefore, the B and C fragments were each cloned as two smaller fragments B1 and B2, and C1 and C2. Fragments A through C2 were cloned into the pSMART vector and fragments D through E2 were cloned into pCR-XL-TOPO cloning vectors. In order to determine the consensus sequence of the genome, two to five independent clones of each amplicon were sequenced using a panel of internal primers. Clones with consensus sequences were used for in vitro assembly of full-length cDNA template. There were several sequence differences, distributed throughout the entire genome of IBV, between our standard cell adapted IBV strain and the published Beaudette sequence (Table 1 ). The standard parental strain of IBV Beaudette used for these studies had a five nucleotide insertion at position 25,414 between the M and 5a ORFs, resulting in a genome size of 27,613, instead of 27,608 nt. The sequence differences were confirmed by sequencing three independent clones and direct sequencing of RT-PCR products. A nucleotide insertion was found in an intergenic sequence of our strain compared to the original published sequence. In addition, an E amplicon with a single unique nucleotide change of A to G at position 25,793 nt, resulting in amino acid change from Q to R, was used as a marker for the molecular clone. Full-length cDNA was in vitro assembled with amplicons conforming with our established consensus sequence. To increase full-length in vitro ligation efficiency, ordered ligation reactions were used. The appropriately sized ligation reaction products were purified from 1% agarose gels before further ligating with neighboring inserts (Fig. 1C).Fig. 1

SapI and BsmBI restriction enzyme sites were used for in vitro assembly of the cDNA for the full-length IBV genome. (A) The 5′ end of the A fragment was amplified with primers that incorporated the T7 RNA polymerase promoter and similarly, a 19 nucleotide polyadenylated tail was inserted at the 3′ end of E. Gray boxes represent ORF of the IBV genome. Open boxes represent genome fragments amplified by RT-PCR and black boxes represent 5′ and 3′ untranslated regions. (B) DNA amplicons used for in vitro ligation. Each amplicon was digested with the appropriate restriction enzyme and separated from the plasmid vectors using agarose gel electrophoresis. Lane M represents the 1 kb DNA ladder; lane 1, fragment A prepared by XhoI digestion, CIP treatment and then SapI restriction enzyme digestion; lane 2, fragment B1 prepared by BsmBI and SapI; lane 3, fragment B2 prepared by digestion with BsmBI and SapI restriction enzyme; lane 4, fragment C1 prepared by BsmBI and SapI digestion; lane 5, fragment C2 prepared by BsmBI digestion; lane 6, fragment D prepared by BsmBI digestion; lane 7, fragment E1 prepared by EcoRI digestion followed by BsmBI digestion, and lane 8, the E2 fragment. (C) In vitro orderly ligation of the generated cDNA fragments. The inserts were ligated stepwise as follows. Lane M represents the 1-kb DNA extension ladder; lane 1, ligation products of A and B1; lane 2, ligation products of B2 and C1; lane 3, purified ligation product of C2 and D; lane 4, purified ligation product of E1 and E2; lane 5, ligation products of lanes 1 and 2; lane 6, ligation products of lanes 3 and 4; lane 7, the final ligation product of lane 5 and 6 (shown by the arrow to the right).

A full-length genomic transcript of IBV was produced in vitro using T7 RNA polymerase with the T7 RNA promoter, incorporated at the 5′ end of fragment A. The final transcript product was verified by Northern blot analysis using a probe against 3′ UTR (data not shown). Any transcript of a size comparable to viral genomic RNA with a detectable 3′ UTR presumably contained the entire genome of IBV because only the A fragment had a T7 RNA promoter and thus could initiate transcription.

The transcripts from the full-length cDNA (along with the N transcript alone), total cellular RNA from IBV infected Vero cells (positive control), and PBS (negative control) were electroporated into non-permissive BHK-21 cells, which were then cultured with Vero cells. Three days after transfection, typical cytopathic effects (CPE), including syncytia formation, were seen in cells transfected with the positive control. Supernatant from the cells, which were overgrown by 3 days after transfection, were harvested and passaged into fresh Vero cells. One day after passage, the positive control showed CPE. Two days after passage, cells transfected with the transcript of the full-length ligated cDNAs showed CPE. CPE was never observed in the negative control cells. We were unable to rescue virus from full-length IBV RNA transcript without co-transfection of N transcript, confirming the possible essential role of N transcript as described by previous reports (Casais et al., 2001, Yount et al., 2000, Yount et al., 2002). The virus generated by the molecular cDNA clone was referred to as MIBV. The derivation of MIBV from the ligated cDNA was confirmed by RT-PCR sequence identification of the marker mutation of a G at nt 25,793.

MIBV was further characterized after three rounds of plaque purification. MIBV formed plaques which were indistinguishable in size and morphology from standard Beaudette cell adapted viral plaques (Figs. 2A and B). The growth kinetics of MIBV and the standard virus were also compared and found to be similar (Fig. 3 ).Fig. 2

MIBV generated similar sized plaques as the wild type IBV. Vero cells were infected with each virus and then overlaid with media containing 0.8% agarose. Three days after infection, agarose was removed and cells were stained with crystal violet. The MIBV plaques are shown in panel A and standard cell adapted Beaudette IBV, in panel B.

The reverse genetic strategy we adapted for IBV was used to evaluate whether a small group-specific gene was necessary for the survival of IBV in cell culture. Furthermore, the replacement with a foreign ORF would be a first step in developing constructs of IBV as expression vectors. Because it has been shown that the expression level of coronavirus genes can be modified by their transcription regulatory sequences (TRS), rather than add an additional TRS, a reporter gene was used to replace most of the 5a ORF, thus maintaining the 5a TRS sequence. Based on results with MHV, we hypothesized that IBV group-specific genes would not be necessary for viral replication in cell culture. The 5a ORF was used rather than the 3a, 3b region which contains an internal ribosomal entry site (IRES) needed for expression of the essential structural, E protein (Le et al., 1994). The EGFP ORF was used to replace the 5a ORF, beginning at the start codon, while the 3′ end of 5a ORF, that included the stop codon, was kept intact because this region overlaps with the 5b ORF.

PCR mutagenesis and the “no see'm” methodology were used to replace ORF5a with EGFP. The cloning strategy used to replace most of the 5a ORF with EGFP (Δ5a/EGFP) is illustrated in Fig. 4A. Three regions, including the EGFP ORF (Fig. 4B, lane 1), and sequences between a region upstream of 5a, including ORF M (Fig. 4B, lane 2), a region downstream of 5a through the 3′ UTR (Fig. 4B, lane 3), were PCR amplified using primers that incorporated the BsmBI restriction enzyme recognition sequence (Table 2). These three PCR products were cloned into the pSTBlue cloning vector and the inserts were excised from the vectors with the BsmBI restriction enzyme.Fig. 4

Construction of EGFP/Δ5a amplicon. (A) The 5a ORF was replaced with the EGFP ORF. Open boxes represent ORF of the IBV genome. Black lines represent PCR products. Arrows indicate the restriction enzyme sites inserted into the PCR products. (B) PCR products containing EGFP in place of ORF 5a. M represents the 1-kb DNA molecular size marker; lane 1, PCR product of EGFP, denoted as F in (A); lane 2, PCR product of M ORF and junction of 5′ of ORF 5a, denoted as G in (A); lane 3; PCR product encompassing 3′ end of 5a ORF and downstream of the ORF through 3′ UTR of IBV, denoted as H in (A). (C) Underlined are IBV sequences and bold are EGFP sequences.

To confirm the stability of the recombinant GIBV, the virus was subsequently passaged in Vero cells. EGFP expression was detected in each of seven sequential passages. GIBV was further characterized by comparing plaque morphology and one step growth curves. Compared to our standard virus and the virus from the cloned MIBV, GIBV-generated plaques were much smaller in size (Fig. 6A). The growth of GIBV was similar to the standard IBV with maximum growth at 14 h post-infection. However, GIBV grew to a 10-fold lower titer compared with the standard Beaudette IBV or MIBV (Fig. 3).Fig. 6

EGFP deletion revertant of GIBV showed comparable plaques to standard IBV or MIBV. After six serial passages of GIBV, heterogeneous-sized plaques were observed. The arrow indicates typical GIBV plaques with the smaller size compared to MIBV and the arrow head indicates a larger plaque from the GIBV stock that was comparable to the standard cell adapted IBV, and plaques purified from the larger plaques in A are shown in panel B.

A V5ero cell-adapted strain of IBV Beaudette US, from American Type Culture Collection (Manassas, VA) was plaque purified three times before use. The virus was propagated in an African green monkey kidney Vero cell line, obtained from ViroMed Laboratory (Minnetonka, MN) and maintained in Dulbecco's modified eagle medium (DMEM) containing 5% fetal bovine serum (FBS) supplemented with penicillin G (100 units/ml) and streptomycin (100 μg/ml). The baby hamster kidney cell line, BHK-21, was also obtained from ViroMed and maintained in DMEM containing 10% FBS supplemented with antibiotics as described for Vero cells.

Total cellular RNA was extracted from IBV-Beaudette infected Vero cells with Trizol reagent (Invitrogen, Carlsbad, CA), according to the manufacturer's directions. Reverse transcription was performed with Superscript II (Invitrogen) and reverse direction primers P1R, P2R, P3R, BEAUSSR, and GNRT (Table 2 ), containing BsmBI and SapI restriction endonuclease recognition sequences. The IBV sequence, as reported in GenBank (accession number M95169), was used for primer design and nucleotide sequencing. Each DNA fragment was amplified from cDNA templates by PCR using Expand Long polymerase (Boehringer Mannheim Biochemical). In order to transcribe RNA using T7 RNA polymerase, the T7 RNA promoter sequence was incorporated into the T7P1F primer. PCR primer pairs used to amplify genomic regions are listed in Table 2. PCR amplification of cDNA fragments was performed using the following conditions; denaturation at 94 °C for 2 min, 10 cycles at 94 °C for 30 s, 55 °C for 30 s, and 68 °C for 2–6 min depending on the size of the fragment and 18 cycles of 94 °C for 30 s, 55 °C for 30 s, and 68 °C for 2–6 min (with an additional 20 s for each subsequent cycle). The PCR products were isolated from agarose gels and cloned into pCR-XL-TOPO (Invitrogen) or pSMART (Lucigen, Middleton, WI) vectors, according to the manufacturer's directions.Table 2

Primer pairs used for cloning and mutagenesis of the IBV and GIBV amplicons

Two to four independent clones of each amplicon were isolated and sequenced by using specific primers and the ABI dye termination sequencing method. A consensus sequence was determined by comparison of direct PCR product sequencing from each independent clone and amplicons, encoding the consensus sequence of each region of IBV, were cloned using standard recombinant DNA techniques (Sambrook et al., 1989).

The entire IBV N ORF, including the 3′ UTR, was amplified by RT-PCR from total cellular RNA extracted from IBV infected Vero cells, and the RT-PCR product was cloned into a transcription vector, pGEM-3Zf(+) (Promega, Madison, WI).

The EGFP ORF (F fragment) was PCR amplified from pLEGFP-N1 (Clontech Laboratories, Inc., Palo Alto, CA), using the GFPSF and GFPER primer pair, and cloned into pSTBlue (Table 2). The region upstream of the 5a ORF (G fragment) was PCR amplified using the E amplicon as a template with the 5ASR and BMF primer pair and the product cloned into pSTBlue (Novagen, Darmstadt, Germany), referred to as 5aS. The region downstream of the 5a ORF (fragment H which included the 3′UTR and polyadenylated tail) was PCR amplified using the E amplicon and the 5AEF and GNRT primer pair before cloning into the pCR-XL-TOPO vector (referred to as 5aE). The G fragment was prepared by digesting 5aS with XhoI and BsmBI restriction enzymes. The F fragment was digested from the vector with BsmBI. The G and F fragments were ligated into the 5aE amplicon which was digested with XhoI and BsmBI restriction enzymes. Replacement of 5a sequences with EGFP ORF was confirmed by sequencing of the Δ5a/EGFP amplicon.

Each amplicon, having the consensus sequence, was prepared from an overnight bacterial culture. Plasmid was isolated and digested with the indicated restriction endonucleases according to the manufacturer's directions. Briefly, the IBV A amplicon was digested with XhoI and treated with calf intestine alkaline phosphatase (CIP), before digesting with the SapI enzyme. The IBV E amplicon was digested with EcoRI, CIP treated and digested with BsmBI. The B1, B2 and C1 amplicons were digested with BsmBI at 55 °C and then digested with SapI at 37 °C. The C2 and D amplicons were digested with BsmBI. Inserts of each amplicon were resolved by electrophoresis on 1% agarose gels containing crystal violet and isolated with QIAquick gel extraction kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's directions. One picomole of each cDNA insert was ligated in an ordered reaction (A + B1, B2 + C1, C2 + D, E1 + E2 or EΔ5α/EGFP + E2). The appropriately sized ligation reaction products were purified from 1% agarose gels and further ligated with neighboring inserts for 4 h, pooled and ligated overnight (Fig. 1C). The final ligation product was purified by extracting with a phenol/chloroform/isoamyl alcohol (25:24:1) mixture and precipitated with ethanol before using as a template for in vitro transcription reactions.

Full-length transcripts of the IBV cDNA constructs were generated in vitro using the mMESSAGE mMACHINE T7 Ultra kit (Ambion, Austin, TX) according to the manufacturer's direction with certain modifications. The in vitro transcription reaction was performed at 37 °C for 2 h in 20 μl reaction mixtures, supplemented with 3 μl of 30 mM GTP, resulting in a 1:1 ratio of GTP to capping analog. A similar reaction was performed for the N transcript using a 1:3 ratio of GTP and capping analog. Before electroporation, the transcripts were treated with DNase I and analyzed by 1% denaturing agarose gel (containing 2.2M formaldehyde) electrophoresis. The identity of the full-length in vitro transcript was confirmed by northern blot analysis using a probe against 3′ UTR of IBV.

After BHK-21 cells were grown to subconfluence (80%), treated with trypsin, and washed with cold DEPC treated PBS twice, they were resuspended in DEPC-treated PBS at a concentration of 10⁷ cells/ml. RNA transcripts were added to 400 μl of the BHK-21 cell suspension in microfuge tubes on ice, gently pipetted and transferred to electroporation cuvettes. Three consecutive electrical pulses of 850 V at 25 μF were given, using the electro-cell manipulator 600, BTX (Genetronics, Inc., San Diego, CA). The transfected BHK-21 cells were diluted 1 to 20 with complete DMEM in 100 mm cell culture Petri dishes and co-cultured with 2 × 10⁶ Vero cells/dish.

Triplicate wells of Vero cells in six-well plates were infected with virus at a multiplicity of infection (m.o.i.) of 2 to 3. The cells were harvested every 4 h for 24 h and stored at −80 °C until they were used for quantification by titration. The cells were three times frozen and thawed, and 10-fold serially diluted in DMEM without serum. Two hundred microliters of each dilution were inoculated into six-well plates for an hour, washed with PBS and overlaid with DMEM, containing 2% FBS, 0.8% agarose, before incubating at 37 °C for 3 days. The plaques were counted after removing the agarose overlay from the cells and staining the monolayer with crystal violet.