Identification and analysis of putative tRNA genes in baculovirus genomes.

Transfer RNAs (tRNAs) genes are both coded for and arranged along some viral genomes representing the entire virosphere and seem to play different biological functions during infection, other than transferring the correct amino acid to a growing peptide chain. Baculovirus genome description and annotation has focused mostly on protein-coding genes, microRNA, and homologous regions. Here we carried out a large-scale in silico search for putative tRNA genes in baculovirus genomes. Ninety-six of 257 baculovirus genomes analyzed was found to contain at least one putative tRNA gene. We found great diversity in primary and secondary structure, in location within the genome, in intron presence and size, and in anti-codon identity. In some cases, genes of tRNA-containing genomes were found to have a bias for the codons specified by the tRNAs present in such genomes. Moreover, analysis revealed that most of the putative tRNA genes possessed conserved motifs for tRNA type 2 promoters, including the A-box and B-box motifs with few mismatches from the eukaryotic canonical motifs. From publicly available small RNA deep sequencing datasets of baculovirus-infected insect cells, we found evidence that a putative Autographa californica multiple nucleopolyhedrovirus Gln-tRNA gene was transcribed and modified with the addition of the non-templated 3'-CCA tail found at the end of all tRNAs. Further research is needed to determine the expression and functionality of these viral tRNAs.

Characterization and genomic analyses of a novel alphabaculovirus isolated from the black armyworm, Spodoptera cosmioides (Lepidoptera: Noctuidae).

The black armyworm Spodoptera cosmioides is a pest of increasing importance in Cry1Ac-Bt toxin crops and non-Bt crops of soybean and cotton in Brazil. Here we characterized a baculovirus isolated from extracts of S. cosmioides that died with symptoms of nuclear polyhedrosis. The putative novel virus exhibited polyhedral occlusion bodies (OBs) with virions containing multiple rod-shaped nucleocapsids, characteristic of alphabaculoviruses. The virus isolate was named Spodoptera cosmioides nucleopolyhedrovirus isolate CNPSo-72 (SpcoNPV-CNPSo-72). SpcoNPV-CNPSo-72 was lethal to third-instar S. cosmioides caterpillars but not to S. frugiperda under the tested viral concentrations. Moreover, SpcoNPV-CNPSo-72 contained a circular 147,763 bp long genome and a G + C content of 44.8% with 151 annotated ORFs (10 unique for baculovirus) and five homologous regions (hrs). The 38 currently defined baculovirus core genes were found in the SpcoNPV-CNPSo-72 genome. After phylogenetic analysis, the novel virus was found to be closely related to other members of Alphabaculovirus, especially to the Spodoptera-infecting viruses, which included Spodoptera eridania nucleopolyhedrovirus isolate 251, Spodoptera litura nucleopolyhedrovirus isolate II, Spodoptera exigua multiple nucleopolyhedrovirus isolate US-1, Spodoptera eridania nucleopolyhedrovirus isolate CNPSo-165, and Spodoptera frugiperda multiple nucleopolyhedrovirus isolate 19. Surprisingly, the new baculoviral genome was found to code for a putative arginine-associated tRNA gene with a predicted intronic sequence of 105 nt. The gene was found inside the bjdp CDS. Overall, baculoviruses are pathogens that lethally infect insect larvae and their study allows a better understanding of large DNA virus evolution, which provides important insights for the development and improvement of biological control agents.

An iflavirus found in stink bugs (Hemiptera: Pentatomidae) of four different species.

An analysis of transcriptomes from the antennae of the three South American stink bugs (Euschistus heros, Chinavia ubica, and Dichelops melacanthus) revealed the presence of picorna-like virus genome-length RNAs with high sequence identity to the genome of Halyomorpha halys virus (HhV), originally discovered in the transcriptome of the brown marmorated stink bug, Halyomorpha halys. Features of the genome, phylogenetic relationships to other viruses, and the appearances of virus-like particles isolated from host stink bugs all confirm that these viruses are iflaviruses and isolates of an undescribed species. Iflavirus RNAs were present at high levels (40%-90% of transcriptome reads) in the stink bug antennal transcriptomes. In whole-insect transcriptomes of H. halys, HhV reads were >500-fold more abundant in adults than in nymphs. We identified from field population a subject of species E. heros infected by this iflavirus. The results of the analysis suggest that these iflaviruses are able to produce large quantities of their RNAs without causing any obvious pathology to their hosts.

The complete genome of Rachiplusia nu nucleopolyhedrovirus (RanuNPV) and the identification of a baculoviral CPD-photolyase homolog.

We described a novel baculovirus isolated from the polyphagous insect pest Rachiplusia nu. The virus presented pyramidal-shaped occlusion bodies (OBs) with singly-embed nucleocapsids and a dose mortality response of 6.9 × 103 OBs/ml to third-instar larvae of R. nu. The virus genome is 128,587 bp long with a G + C content of 37.9% and 134 predicted ORFs. The virus is an alphabaculovirus closely related to Trichoplusia ni single nucleopolyhedrovirus, Chrysodeixis chalcites nucleopolyhedrovirus, and Chrysodeixis includens single nucleopolyhedrovirus and may constitute a new species. Surprisingly, we found co-evolution among the related viruses and their hosts at species level. Besides, auxiliary genes with homologs in other baculoviruses were found, e.g. a CPD-photolyase. The gene seemed to be result of a single event of horizontal transfer from lepidopterans to alphabaculovirus, followed by a transference from alpha to betabaculovirus. The predicted protein appears to be an active enzyme that ensures likely DNA protection from sunlight.

The complete genome sequence of the first hesperiid-infecting alphabaculovirus isolated from the leguminous pest Urbanus proteus (Lepidoptera: Hesperiidae).

Baculoviruses are insect viruses largely used as expression vectors and biopesticides. These viruses can efficiently infect the larval stage of several agricultural pests worldwide causing a lethal disease. In this work, we found a novel baculovirus isolated from the larval stage of Urbanus proteus (L.), the bean leafroller and characterized its complete genome. This is an important pest of several leguminous plants in Brazil and belongs to the butterfly family Hesperiidae, from where no baculovirus genome sequence has been described. This new virus was shown to have the smallest genome among all alphabaculoviruses sequenced to date, with 105,555 bp and 119 putative ORFs. We found ten unique genes, seven bro, and the 38 baculovirus core genes. UrprNPV was found to be related to the Adoxophyes-infecting baculoviruses AdorNPV and AdhoNPV with high genetic distance and a long branch length. Interestingly, few individual core gene-based phylogenies were found to support the relationship of UrprNPV to both AdorNPV and AdhoNPV. Importantly, the increase in number of completely sequenced baculovirus points to a very exciting way to understand baculovirus and its evolution and could potentially help the use of baculovirus as both biopesticides and expression vectors.