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 investigate the nature of subgenomic mRNAs in CAVV, total RNA was isolated from CAVV-infected cells and subjected to Northern blot analysis. RNA from noninfected C6/36 cells served as a control. To avoid detection of defective interfering RNAs, cells were infected at a low multiplicity of infection (MOI) with virus obtained from limiting dilution endpoints of early-passage supernatants. Northern blot probes were generated against the most 5= 107 nt and the most 3= 556 nt of the genome (Fig. 4B) . Additional probes were generated for the major predicted ORFs (Fig. 4B) . Apart from a band corresponding to the genome size, fragments of approximately 4.7, 2.7, and 1.8 kb were detected with the 5= probe. These were also represented in a blot with the 3= probe, suggesting that these RNAs are 5= and 3= coterminal with the genome (DT, Fig. 4C ). Additional bands of ca. 1.4, 1.2, and 1.0 kb were detected with the 3= probe but not with the 5= probe, compatible with an NDT mechanism.