An Insect Nidovirus Emerging from a Primary Tropical Rainforest

Zirkel, Florian; Kurth, Andreas; Quan, Phenix-Lan; Briese, Thomas; Ellerbrok, Heinz; Pauli, Georg; Leendertz, Fabian H.; Lipkin, W. Ian; Ziebuhr, John; Drosten, Christian; Junglen, Sandra

To identify potential fusion sites indicative of DT, one-step RT-PCRs were conducted for each sgRNA detected by Northern blotting. Sense primers were placed at intervals starting from the 5= end of the genome approximately 300 nt into the genomic sequence (see Fig. S2 in the supplemental material). Antisense primers were selected according to the projected sizes of the sgRNAs. RT-PCRs targeting the 4.7-kb and 2.7-kb bands resulted in products with sense primers up to at least 126 and 152 nt, respectively, from the 5= end of the genome. The 1.8-kb band was associated with PCR products up to 202 nt from the 5= end of the genome. PCR products of various and unexpected sizes were observed for the smaller potential sgRNAs (i.e., all sgRNAs smaller than the 1.8-kb band in the Northern blot). Representative PCR products for all putative sgRNAs were cloned and sequenced. PCR products of unexpected sizes from small subgenomic mRNAs yielded sequences indicative of misprimed amplification in various positions of the genome, and it was concluded that these subgenomic mRNAs were generated by NDT, lacking a leader consistent with the Northern blotting results. The three sgRNAs that were clearly codetected with 5= and 3= probes revealed potential sites of fusions between genome leader and downstream mRNA sequences. Different sites were used for each sgRNA, as shown in Fig. 4B . Nevertheless, all sgRNAs had an A/C-rich region immediately downstream of the fusion region in common. It should be noted that Northern blot detection intensities of the 4.7-kb and 2.7-kb bands corresponded well between the 5= and 3= probes, whereas the detection intensity of the 1.8-kb band was much greater with the 3= probe than with the 5= probe (Fig. 4C) . This matched the presence of a much smaller fusion region in the RT-PCR product of the 1.8-kb sgRNA. Given the high intensity of the 1.8-kb band, we suspected that a major fraction of the total amount of this sgRNA would not contain a fused leader element; nonetheless, several variant fusion sites were detected in parallel clones for all subgenomic mRNAs. Further studies are required to characterize in more detail the various CAVV RNA species and their functional relevance in CAVV genome expression.