Insects are commonly infected with multiple viruses including those that cause sublethal, asymptomatic, and latent infections. Traditional methods for virus isolation typically lack the sensitivity required for detection of such viruses that are present at low abundance. In this respect, next generation sequencing technologies have revolutionized methods for the discovery and identification of new viruses from insects. Here we review both traditional and modern methods for virus discovery, and outline analysis of transcriptome and small RNA data for identification of viral sequences. We will introduce methods for de novo assembly of viral sequences, identification of potential viral sequences from BLAST data, and bioinformatics for generating full-length or near full-length viral genome sequences. We will also discuss implications of the ubiquity of viruses in insects and in insect cell lines. All of the methods described in this article can also apply to the discovery of viruses in other organisms.

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BackgroundThe soybean aphid has significantly impacted soybean production in the U.S. Transcriptomic analyses were conducted for further insight into leads for potential novel management strategies.Methodology/Principal FindingsTranscriptomic data were generated from whole aphids and from 2,000 aphid guts using an Illumina GAII sequencer. The sequence data were assembled de novo using the Velvet assembler. In addition to providing a general overview, we demonstrate (i) the use of the Multiple-k/Multiple-C method for de novo assembly of short read sequences, followed by BLAST annotation of contigs for increased transcript identification: From 400,000 contigs analyzed, 16,257 non-redundant BLAST hits were identified; (ii) analysis of species distributions of top non-redundant hits: 80% of BLAST hits (minimum e-value of 1.0-E3) were to the pea aphid or other aphid species, representing about half of the pea aphid genes; (iii) comparison of relative depth of sequence coverage to relative transcript abundance for genes with high (membrane alanyl aminopeptidase N) or low transcript abundance; (iv) analysis of the Buchnera transcriptome: Transcripts from 57.6% of the genes from Buchnera aphidicola were identified; (v) identification of Arsenophonus and Wolbachia as potential secondary endosymbionts; (vi) alignment of full length sequences from RNA-seq data for the putative salivary gland protein C002, the silencing of which has potential for aphid management, and the putative Bacillus thuringiensis Cry toxin receptors, aminopeptidase N and alkaline phosphatase.Conclusions/SignificanceThis study provides the most comprehensive data set to date for soybean aphid gene expression: This work also illustrates the utility of short-read transcriptome sequencing and the Multiple-k/Multiple-C method followed by BLAST annotation for rapid identification of target genes for organisms for which reference genome sequences are not available, and extends the utility to include the transcriptomes of endosymbionts.

Aphid lethal paralysis virus (ALPV; family Dicistroviridae) was first isolated from the bird cherry-oat aphid, Rhopalosiphum padi. ALPV-like virus sequences have been reported from many insects and insect predators. We identified a new isolate of ALPV (ALPV-AP) from the pea aphid, Acyrthosiphon pisum, and a new isolate (ALPV-DvV) from western corn rootworm, Diabrotica virgifera virgifera. ALPV-AP has an ssRNA genome of 9940 nt. Based on phylogenetic analysis, ALPV-AP was closely related to ALPV-AM, an ALPV isolate from honeybees, Apis mellifera, in Spain and Brookings, SD, USA. The distinct evolutionary branches suggested the existence of two lineages of the ALPV virus. One consisted of ALPV-AP and ALPV-AM, whilst all other isolates of ALPV grouped into the other lineage. The similarity of ALPV-AP and ALPV-AM was up to 88 % at the RNA level, compared with 78-79 % between ALPV-AP and other ALPV isolates. The sequence identity of proteins between ALPV-AP and ALPV-AM was 98-99 % for both ORF1 and ORF2, whilst only 85-87 % for ORF1 and 91-92 % for ORF2 between ALPV-AP and other ALPV isolates. Sequencing of RACE (rapid amplification of cDNA ends) products and cDNA clones of the virus genome revealed sequence variation in the 59 UTRs and in ORF1, indicating that ALPV may be under strong selection pressure, which could have important biological implications for ALPV host range and infectivity. Our results indicated that ALPV-like viruses infect insects in the order Coleoptera, in addition to the orders Hemiptera and Hymenoptera, and we propose that ALPV isolates be classified as two separate viral species.

The invasive soybean aphid, Aphis glycines, is a major pest in soybeans, resulting in substantial economic loss. We analyzed the A. glycines transcriptome to identify sequences derived from viruses of A. glycines. We identified sequences derived from a novel virus named Aphis glycines virus 2 (ApGlV2). The assembled virus genome sequence was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing, conserved domains were characterized, and distribution, and transmission examined. This virus has a positive sense, single-stranded RNA genome of ~4850 nt that encodes three proteins. The RNA-dependent RNA polymerase (RdRp) of ApGlV2 is a permuted RdRp similar to those of some tetraviruses, while the capsid protein is structurally similar to the capsid proteins of plant sobemoviruses. ApGlV2 also encodes a larger minor capsid protein, which is translated by a readthrough mechanism. ApGlV2 appears to be widespread in A. glycines populations and to persistently infect aphids with a 100% vertical transmission rate. ApGlV2 is susceptible to the antiviral RNA interference (RNAi) pathway. This virus, with its unique genome structure with both plant- and insect-virus characteristics, is of particular interest from an evolutionary standpoint.

Aphids are important agricultural pests with about 250 species known to cause damage by feeding on plant phloem and transmitting plant viruses. Current management relies primarily on the application of chemical insecticides, which can be deleterious to the environment, and the use of aphid resistant cultivars hampered by the presence of resistant aphid biotypes. We aim to develop novel biotechnology based approaches to better manage pestiferous aphids. To this end, we (i) identified and characterized aphid viruses, and (ii) analyzed a class of small RNA, microRNAs specific to aphids. We used transcriptome and small RNA sequencing datasets to identify viruses that naturally infect populations of various aphid species. We identified and characterized a new isolate of Aphid lethal paralysis virus, named ALPV-Ap (Dicistroviridae) from the pea aphid, Acyrthosiphon pisum and a novel virus from the soybean aphid, Aphis glycines, named Aphis glycines virus (AGV; Unclassified). ALPV-Ap, which has a longer genome than other ALPV isolates, is phylogenetically closely related to ALPV isolates from honeybees rather than from aphids. ALPV-Ap localizes to the aphid midgut and is not vertically transmitted from adult to nymph. AGV has a capsid protein similar to those of plant viruses while the RNA-dependent RNA polymerase is more closely related to those of insect viruses. Remarkably, AGV is 100% vertically transmitted. Analysis of small RNA datasets from various aphid species showed the presence of over 100 microRNAs. In addition to detection of many evolutionarily conserved miRNAs, a subset of 12 aphidspecific miRNA was identified. The wealth of information obtained from sequencing datasets will allow for investigation of virus-and miRNAs-based management of aphid populations for reduced damage and increased crop yield worldwide.