Manejo de emergencias

During analysis of the BeauR-L-CTGAACAA IR mRNA the mRNA corresponding to the M gene was also sequenced as a control. This was to confirm that the M mRNA TRS was derived from the complete M TRS-B as per the current understanding of sg mRNA transcription, and that the TG mutation was not incorporated into the mRNA TRS in a similar mechanism to the IR mRNA. However, this was not found to be the case with the first M mRNA clone sequenced from BeauR-L-CTGAACAA unexpectedly showing the presence of the TG mutation at position 3 of the mRNA TRS (Fig 7.12A). A second clone of the M mRNA was subsequently sequenced and while the consensus sequence gave the

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mRNA TRS as CTTAACAA analysis of the sequence traces showed this was an error and that the dominant nucleotide at position 3 was a G with a minor T population (Fig. 7.12B).

Fig. 7.12. Sequence traces of M gene mRNAs. CK cells were infected with BeauR- IR-CTGAACAA and 20 hpi intracellular RNA was harvested. M gene mRNAs were amplified with primers Leader1 and IR(R) and PCR cloned for sequence analysis with primers M13(F) and M13(R). (A, B) Sequence traces for two clones of M mRNA. Grey boxes outline the TRS-B with position of the TRS-L mutation indicated by black arrow.

Given this finding other sg mRNAs from a BeauR-L-CTGAACAA infection were cloned and sequenced to establish whether the TG mutation is consistently incorporated into mRNA TRSs and therefore suggestive of a greater degree of flexibility in TRS usage than previously supposed (Fig. 7.13).

A

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Fig. 7.13. Sequence traces of IBV mRNAs. CK cells were infected with (A, B) BeauR-L-CTGAACAA or (C, D, E) Beau-R and 20 hpi intracellular RNA was harvested. Messenger RNAs were amplified with primers Leader1 and (A) BG147, (B) BG149, (C) BG133, (D, E) BG142, and PCR cloned for sequence analysis with primers M13(F) and M13(R). Sequence traces are shown for (A) Gene 5 (B) N (C) Spike (D, E) Two clones of Gene 3. Grey boxes outline the TRS-B with TRS-L point mutation indicated by black arrow.

A

B

C

D

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Two clones each of Gene 5 (Fig. 7.13A) and N gene (Fig. 7.13B) mRNAs were subject to sequence analysis and in all cases showed the incorporation of the TG mutation. Given that for BeauR-L-CTGAACAA both the TRS-L and the TRS-Bs for Spike and Gene 3 are CTGAACAA it was not possible to determine the source of the G at position 3 by sequence analysis. However, it was possible to analyse the Spike and Gene 3 mRNAs from a Beau-R infection to investigate whether the mRNA TRS position 3 is a G, as derived from the TRS-B, or a T, as derived from the TRS-L. Due to limited availability of clones only one clone of the Spike mRNA was sequenced and found to contain a T at position 3 (Fig. 7.13C). Two clones of the Gene 3 mRNA were available and sequenced, with one containing a T and one containing a G at position 3 of the TRS (Fig. 7.13D, E).

This result demonstrated that even in a wild type virus infection the TRS of a mRNA can be partly derived from the TRS-L. Although limited numbers of clones were analysed the data suggests that for IBV mRNAs the complete TRS-B is not necessary to permit hybridisation to the TRS-L, and there may be some flexibility in the TRS-B requirements for generation of mRNAs.

These findings could be investigated further via a more in depth analysis of the IR mRNAs transcribed during infection with rIBV BeauR-IR-CTGAACAA. The original IR mRNA clone sequenced from this virus showed the incorporation of the entire TRS-B in the mRNA TRS (see Fig. 7.8). By carrying out sequence analysis of a further four clones of IR mRNA from each replicate of BeauR-IR-CTGAACAA it was possible to determine whether the mRNA TRS is consistently derived from the TRS-B alone, as demonstrated by the first clone, or if the IR mRNA TRS is often

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derived from both the TRS-B and TRS-L as seen with the wild type IR mRNA. The sequence alignments of the IR mRNA showed that 4/4 clones for R1 and 2/4 clones for R2 contained a T nucleotide at position 3 of the TRS confirming that in many cases the mRNA TRS is still partly derived from the TRS-L (Fig. 7.14)

T/G

▼

R1 IR-mRNA (1) :CTAGATTTTTAACTTAACAAAGCGGAAATAAG-N93ATG:172

R1 IR-mRNA (2) :CTAGATTTTTAACTTAACAAAGCGGAAATAAG-N93ATG:172

R1 IR-mRNA (3) :CTAGATTTTTAACTTAACAAAGCGGAAATAAG-N93ATG:172

R1 IR-mRNA (4) :CTAGATTTTTAACTTAACAAAGCGGAAATAAG-N93ATG:172

R2 IR-mRNA (1) :CTAGATTTTTAACTGAACAAAGCGGAAATAAG-N93ATG:172

R2 IR-mRNA (2) :CTAGATTTTTAACTTAACAAAGCGGAAATAAG-N93ATG:172

R2 IR-mRNA (3) :CTAGATTTTTAACTTAACAAAGCGGAAATAAG-N93ATG:172

R2 IR-mRNA (4) :CTAGATTTTTAACTGAACAAAGCGGAAATAAG-N93ATG:172

BeauR-IR-CTGAACAA:AGAAATATACTGCTGAACAAAGCGGAAATAAG-N93ATG:25185

Beau-R :AGAAATATACTGGTGACCAAAGCGGAAATAAG-N93ATG:25185

Fig. 7.14. Sequence alignments of BeauR-IR-CTGAACAA IR mRNA. Confluent monolayers of CK cells were infected with replicate 1 (R1) or replicate 2 (R2) of BeauR-IR-CTGAACAA and intracellular RNA extracted 20 hpi. IR mRNAs were amplified by PCR with primers Leader1 and IR(R) and PCR cloned for sequence analysis. Sequence alignments are shown for four clones of each replicate against BeauR-IR-CTGAACAA and Beau-R genomic sequence. Leader sequence highlighted in blue and TRS highlighted in yellow. (▼) indicates position of the variable nucleotide. Relative positions of the mRNA and genome are given on the right along with the distance to the IR ATG.

Overall the results in this chapter have demonstrated that the IBV IR mRNA is unique among the known IBV sg mRNAs in that the associated TRS-B is a non- canonical shortened TRS-B of CAA, not CTTAACAA, and that the IR mRNA TRS is mostly derived from the TRS-L and not the TRS-B as would be expected. Furthermore, by introducing point mutations into the IR TRS-B to extend it from CAA to CTGAACAA, thus better resembling the consensus TRS, it was shown that the IR mRNA expression level could be increased 6-fold over wild type and

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demonstrated the importance of the TRS-L/TRS-B interaction in regulating mRNA expression levels. Sequence analysis of all IBV sg mRNAs showed that the TRS of individual mRNAs can be derived, in part, from the TRS-L, suggesting a greater degree of flexibility during transcription of IBV sg mRNAs with regards to the origin of individual mRNA TRSs.