Multiple novel non-canonically transcribed sub-genomic mRNAs produced by avian coronavirus infectious bronchitis virus

Fig. 1.. Identification of an mRNA located between N gene and the poly(A) tail for Beau-R but not M41-CK. (a) Northern blot analysis of Beau-R infected whole-cell lysate. A clearly distinguishable band, designated 7, is present at the bottom of the gel at relatively low abundance. The location of this transcript likely corresponds to the area of the genome between gene 6 (N) and the 3′-UTR. A faint band designated as 2* is also observed between genes 2 and 3. (b) Leader-body PCR analysis of Beau-R and M41-CK infected CK cell whole-cell lysate. Amplification of sgmRNAs by PCR produces two bands in the instance of Beau-R after separation by agarose gel electrophoresis. The larger sized product (~2000 bp) corresponds to the expected band size for gene 6 (N protein) transcripts. A smaller product (~500–650 bp) is also produced in Beau-R, but does not occur in M41-CK infected cell lysate. (c) Sequencing of leader-body PCR products. Sequencing of this smaller band confirms a match to the 3′ end of the virus genome (Beau-R NCBI GenBank accession number AJ311317), but the sequence also possesses a leader sequence corresponding to the 5′ end of the genome and, hence, has the characteristics of a…

Fig. 2.. A gene 7 transcript is produced by diverse strains of IBV. (a) Leader-body PCR analysis of infected CK cell whole-cell lysate. Amplification of sgmRNAs by PCR and subsequent separation by agarose-gel electrophoresis produces two bands in the instance of Beau-CK, D1466, QX, H120 and CR88, and three bands in the instance of Italy-02. All six viruses produce bands corresponding to gene 6 (N protein,~2000 bp) and gene 7 (~500–600 bp). The larger band in Italy-02 relates to gene 5. (b) Northern blot analysis of infected whole-cell lysate. A clearly distinguishable band is present at the bottom of the gel for H120, Beau-R, Beau-CK and CR88 that corresponds to sgmRNA 7. Confirmatory to Fig. 1(b), M41-CK does not have a band corresponding with sgmRNA 7. All five viruses have bands corresponding to the other sgmRNAs as anticipated. However, there is a consistent pattern of bands between genes 6 and 7 across the IBVs that cannot be explained. (c) Sequence analysis of leader-body PCR products. All sequences are indicative of sgmRNAs; however, the TRS-B and the site of leader-body switch is not consistent. Genome positions for each IBV strain are indicated on the left (NCBI GenBank accession…

Fig. 3.. A gene 7 transcript is also present in TCoV. (a) Leader-body PCR analysis of European TCoV strain 080385d RNA harvested from infected turkey embryos. Separation of PCR-amplified TCoV sgmRNAs by agarose gel electrophoresis produces a ~2000 bp band and a~500–600 bp band corresponding to the expected sizes of gene 6 and gene 7, respectively. The gene 7 band has notably reduced intensity versus gene 6 as seen in IBV. (b) Sequencing of leader-body PCR products. The TCoV 080385d sequence is available (NCBI GenBank accession number KR822424.1); however, the 5′ extremities including the leader sequence have not been fully resolved. A sequence comparison versus the TCoV strain MG10 RefSeq (NCBI GenBank accession number NC_010800.1) confirms the smaller band matches the 5′ leader TCoV sequence (orange) with this exception of 1 nucleotide (purple). Reads confirm that TCoV 080385d gene 7 utilizes a TAACA CS. Red, N stop codon; blue, matches to the canonical CS (CTTAACA); green, prospective ORF 7 start codon; grey, ambiguity code (Ns) in sequence; orange, nucleotide matches between TCoV 080385d gene 7 leader sequence and TCoV MG10 NCBI RefSeq (NCBI GenBank accession number NC_010800.1);…

Fig. 4.. Template switch between the TRS-L and TRS-B occurs between positions 3 and 4 of the TRS-L. Sequence analysis of sgmRNA 7 transcripts expressed in CK cells infected with rIBV BeauR-l-CTGAACAA. The virus genome sequence is shown at the bottom, the sequence of sgmRNA 7 in the middle, and the virus genome leader sequence at the top. TRSs are coloured in blue, with the mutated nucleotide at position 3 of the TRS-L coloured in orange. The stop codon for N is highlighted in red and the start codon for ORF 7 in green. The sgmRNA 7 includes the G at position 3, which has originated from the leader sequence, indicating that the template switch has occurred between positions 3 and 4 of the TRS-L. Genome positions according to NCBI GenBank accession number AJ311317 are indicated on the left.

Fig. 5.. Alignment of ORF 7 prospective proteins in different IBV strains and TCoV using MView. Gene 7 sgmRNA sequences were screened for the presence of an AUG start codon. A 53–77 aa protein sequence was identified in TCoV and all IBVs with exception to Beau-R, which has a highly truncated 10 aa protein sequence. A single T deletion in Beau-R at position 27 141 (BeauR-ORF7rep) results in a frameshift mutation and the potential to generate a 73 aa product.

Fig. 6.. Repairing ORF 7 in Beau-R does not affect viral growth in vitro. (a) Pairwise alignment between Beau-R and BeauR-ORF7rep recombinant virus sequences. A single T deletion in Beau-R at position 27 141 (BeauR-ORF7rep) results in a frameshift mutation and the potential to generate a 73 aa product. Genome positions according to NCBI GenBank accession number AJ311317 are indicated on the left. (b, c) Growth kinetics of Beau-R, BeauR-ORF7rep and BeauR-ORF7rep-HIS viruses in vitro. Confluent CK cells seeded in six-well plates were infected with (b) 1×104 p.f.u. or (c) 1×105 p.f.u. At 1, 24, 48, 72 and 96 h.p.i., supernatant was harvested and assessed for infectious viral progeny by titration in CK cells. Each value represents the mean of three replicates with error bars representing sem. Statistical differences denoted by either * (P<0.05) or ** (P<0.005) were assessed using a two-way ANOVA paired with a Tukey test for multiple comparisons. The only differences identified were between Beau-R and BeauR-ORF7rep B at 48 and 96 h.p.i. (d) Ciliary activity in ex vivo TOCs. In replicates of ten, ex vivo TOCs were infected with 1×105 p.f.u. of either Beau-R, BeauR-ORF7rep A or BeauR-ORF7rep B,…

Fig. 7.. Detection of a transcript-like sequence between genes 2 and 3. (a) Leader-body PCR analysis of Beau-R and Beau-CK infected CK cell whole-cell lysate. Amplification of sgmRNAs by PCR utilizing primer leader 1 and BG139 produces two bands, one relating to gene 2 (S) transcripts and the second sgmRNA 2* transcripts. (b) Sequence analysis of leader-body PCR products identifies a TRS-B of AACAA. TRSs present in the leader, sgmRNA and the genome are highlighted in blue. Highlighted in red, upstream of the genomic TRS-B, is a TRS-like sequence. (c) Genome alignment of Beau-R (AY311317.1) position 21 220 to 21 279 surrounding the TRS-B (highlighted in blue) with other IBV strains. A TRS-like sequence is highlighted in red and percentage identity (pid) is indicated for each sequence compared to Beau-R. Genome positions for each IBV strain are indicated on the left (NCBI GenBank accession numbers: AJ311317, MN548285, MN548286, MN548287, MN548288, MN548289).

Fig. 8.. HiSeq analysis confirms the presence of genes 2* and 7, and indicates potential for other transcripts across the genome. Sequencing coverage of Beau-CK HiSeq reads to Beau-CK genome using three separate inputs. All reads (passing quality filter) were aligned to the Beau-CK using BBMap (blue). Beau-CK reads were also aligned the Beau-CK genome using bwa with reads flagged as chimeric subsequently aligned using BBMap (green). Forward and reverse reads were merged using pear and reads possessing the leader sequence selected. The 5′ end of the read (relative to the genome) up to and including the leader sequence was removed from the read, and the remainder aligned to the genome using bwa (red). The trace for the chimeric read coverage plot (green) indicate positions in the genome covered by reads that align in two places, whereas the trace in the leader-matching (red) coverage plot indicates positions that are covered by reads that also possess the leader sequence. Coverage peaks occur at sites of known transcripts (2, 3, 4, 4b, 5 and 6), with additional peaks corresponding to gene 2* and 7 transcripts also identifiable.

Fig. 9.. The presence of gene 7 varies between strains of the Massachusetts serotype. (a) The nucleotide sequences of different Mass strains were compared over the start of gene 7. Red, N stop codon; blue, nucleotide matches to the canonical TRS; green, prospective ORF 7 start codon. (b) A comparison of the amino acid sequences. NCBI GenBank accession numbers: AJ311317.1, MK728875.1, FJ904720.1, FJ904721, FJ904722, FJ904723.1, FJ904713, EU817497.1 and MN548287.