Characterization of a Novel RNA Virus Discovered in the Autumnal Moth Epirrita autumnata in Sweden

de Miranda, Joachim R.; Hedman, Harald; Onorati, Piero; Stephan, Jörg; Karlberg, Olof; Bylund, Helena; Terenius, Olle

The infection status of Abiskovirus was assessed from evidence for negative-strand virus RNA and/or sub-genomic RNA in virus-positive samples. Both types of molecules are only produced during active infection and are therefore indicative that the virus is truly infectious in the host. However, only positive evidence for the presence of such molecules is informative, since these molecules are temporary infection intermediates and the infection process may already have passed the stage where such molecules are produced. The evidence was provided by four pairs of strand-specific RT-qPCR assays: two in the genomic region and two in the putative sub-genomic region (Figure 1 ; Supplementary Materials Table S1 ). Negative-strand Abisko virus RNA was detected by all four Viruses 2017, 9, 214 6 of 12 negative-strand assays in one of the four virus-positive E. autumnata adult samples (78A) and by three of the assays in another adult (86A), with assay neg2(g) just missing the detection threshold ( Figure 2 ). No evidence of negative-strand RNA was recovered from the other two adult samples (31A; 61A) or the diseased larval sample (7L), even though positive-strand RNA was detected in abundance. The average ratio of positive to negative strand RNA for all pairs of assays was around 275:1 and 100:1 for samples 78A and 86A respectively, which is normal for RNA viruses [28] . There was no evidence of significant amounts of subgenomic RNA in any of the samples, since the assays located in ORF-1 (genomic RNA only) detected the same amount of RNA as those located in ORF-2 and ORF-3 (genomic plus subgenomic RNA: Figure 2 ; [29] ). If Abisko virus indeed translates its ORFs through ribosome scanning, whether with or without subgenomic mRNAs, then the sequence context surrounding the AUG codon has a major role in deciding where translation is initiated [23] . This context is particularly important for ORF-3, which is downstream from ORF-2 with which it has to compete for translation initiation. The only ORF-3 AUG codon with a superior Kozak context than the putative start codon of ORF-2 is 91 nt downstream from the end of ORF-2 and without any product. The key positions of the Kozak sequence are at −3 (A/G), −1 (C/G), and +4 (G) relative to the AUG start codon [23] . The Kozak context of the nominal start of ORF-3 (UCU 8971 AUGUUA) is about as sub-optimal as can be and inferior to either of the possible ORF-2 start codons (UAA 8266 AUGCGC; AAU 8473 AUGACU), neither of which are particular optimal themselves. Therefore, unless ORF-3 is translated through a dedicated IRES hidden in the CP gene (e.g., [27] ), it may well be produced as a read-through product of ORF-2, through a leaky UAG stop codon. Such a strategy has plenty of precedence elsewhere in the virus world, particularly for coat proteins that thus acquire extensions with potentially useful functions for cell entry, infectivity, and host-range [26] .