The multispecies stinkbug iflavirus Halyomorpha halys virus detected in the multispecies stinkbug egg parasitoid microwasp, Telenomus podisi (Ashmead) (Hymenoptera: Platygastridae).

Wasps are important parasitoids of stinkbugs and frequently exposed to various types of microorganisms through environmental contact and fecal-oral transmission route. Many parasitize stinkbug eggs and are commercially used in the field to control insect population. The parasitoid T. podisi is known for its high parasitism capacity and ability to target multiple species of stinkbugs. In this study we asked whether T. podisi exposed to eggs infected by a multispecies asymptomatic stinkbug virus, the Halyomorpha halys virus (HhV) would get infected. HhV is a geographically distributed multispecies iflavirus previously found to infect four stinkbug hosts, including three Brazilian species, Chinavia ubica, Euschistus heros and Diceraeus melacanthus, and T. posidi can parasitize all of them. As results, RT-PCR screening revealed positive samples for the HhV genome in two out of four tested pools of T. podisi, whereas the antigenome, indicative of replicative activity, was not detected. The wasps were raised in E. heros eggs that presented both the genome and the antigenome forms of the HhV genome. Subsequent RNA-deep sequencing of HhV positive T. podisi RNA pools yielded a complete genome of HhV with high coverage. Phylogenetic analysis positioned the isolate HhV-Tp (isolate Telenomus podisi) alongside with the stinkbug HhV. Analysis of transcriptomes from several hymenopteran species revealed HhV-Tp reads in four species. However, the transmission mechanism and the ecological significance of HhV remain elusive, warranting further studies to illuminate both the transmission process and its capacity for environmental propagation using T. podisi as a potential vector.

Begomovirus Transmission to Tomato Plants Is Not Hampered by Plant Defenses Induced by Dicyphus hesperus Knight.

Plants can respond to insect infestation and virus infection by inducing plant defenses, generally mediated by phytohormones. Moreover, plant defenses alter host quality for insect vectors with consequences for the spread of viruses. In agricultural settings, other organisms commonly interact with plants, thereby inducing plant defenses that could affect plant-virus-vector interactions. For example, plant defenses induced by omnivorous insects can modulate insect behavior. This study focused on tomato yellow leaf curl virus (TYLCV), a plant virus of the family Geminiviridae and genus Begomovirus. It is transmitted in a persistent circulative manner by the whitefly Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae), posing a global threat to tomato production. Mirids (Hemiptera: Miridae) are effective biological control agents of B. tabaci, but there is a possibility that their omnivorous nature could also interfere with the process of virus transmission. To test this hypothesis, this study first addressed to what extent the mirid bug Dicyphus hesperus Knight induces plant defenses in tomato. Subsequently, the impact of this plant-omnivore interaction on the transmission of TYLCV was evaluated. Controlled cage experiments were performed in a greenhouse setting to evaluate the impact of mirids on virus transmission and vector acquisition by B. tabaci. While we observed a reduced number of whiteflies settling on plants exposed to D. hesperus, the plant defenses induced by the mirid bug did not affect TYLCV transmission and accumulation. Additionally, whiteflies were able to acquire comparable amounts of TYLCV on mirid-exposed plants and control plants. Overall, the induction of plant defenses by D. hesperus did not influence TYLCV transmission by whiteflies on tomato.

New insights from the virome of Halyomorpha halys (Stål, 1855).

Halyomorpha halys (Stål, 1855) (Hemiptera: Pentatomidae), the brown marmorated stink bug, is an invasive pentatomid native to East-Asia, and introduced worldwide in recent times. It is a polyphagous pest with approximately 300 host plants, which, due to its plasticity, reproductive and feeding behavior, long-distance flight, and walking as well as human-mediated dispersal ability, is able to cause significant economic and ecological damage. In several cases pest control mediated by insecticide treatments leads to unsatisfactory efficacy, mostly due to insect recovery ability. Thus, the most promising method for the long-term management of this pest has been focused with growing emphasis on classical biological control strategies. In this framework, viruses have untill now been poorly investigated in H. halys with only a single virus described from the US territory. For this reason we investigated the virome associated with a small and well described population of H. halys from Piedmont (Italy) describing for the first time 7 new viral sequences belonging to different taxonomical groups. Further studies will be necessary to assess the biological and ecological effects the viruses have on their host. Due to the agricultural importance of this insect, the biological characterization of these viruses would give important information on the possibility to exploit viral entities as biological control agents. Finally, the presence of a such relevant number of viruses from a small population suggests a wide association between the brown marmorated stink bug and viral entities. Further studies to determine the possible exploitation of viral sequences to trace different populations are ongoing.

Discovery and characterization of a novel alphavirus-like RNA virus from the red firebug Pyrrhocoris apterus L. (Heteroptera).

Recent studies have shown that insects harbor numerous viruses of various taxa and that viral infections are often latent without noticeable symptoms. The red firebug Pyrrhocoris apterus, a true flightless bug from the family Pyrrhocoridae, is widely used for physiological studies on insect metabolism, endocrinology, and digestion. While exploring the transcriptome of P. apterus salivary glands, a nearly complete genomic sequence of a novel RNA virus was reconstructed. The virus, provisionally named Pyrrhocoris apterus virus 1 (PaV1), possesses eight potential open reading frames (ORFs) encoding for an array of proteins, some of which are involved in virus replication while others ensure success of the virus in multiple ways, including evasion of the host immune response. In addition to the information obtained from sequence analyses, we documented virus transmission, virus-induced mortality, host response upon persistent PaV1 infection, virion morphology, and putative virus-induced structures in salivary gland cells in a laboratory culture of red firebug. We propose that PaV1 belongs to a novel viral species of a new, yet-to-be established family.

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.

Characterization of a novel RNA virus from Nesidiocoris tenuis related to members of the genus Iflavirus.

The complete genome of a novel virus from Nesidiocoris tenuis was determined by RNA-seq and rapid amplification of cDNA ends. This virus has a single-stranded RNA genome of 10633 nucleotides (nt) in length, not including the poly(A) tail, and contains two putative open reading frames (ORFs). ORF1 encodes a polypeptide of 1320 amino acids (aa) with a predicted molecular mass of 147.92 kDa and theoretical isoelectric point (pI) of 6.96. ORF2 encodes a polypeptide of 1728 aa with a predicted molecular mass of 197.09 kDa and pI of 6.73. Phylogenetic analysis with the deduced aa sequences of the conserved RNA dependent RNA polymerase domain as well as whole genome nt sequences indicated that the virus clusters with viruses classified within the genus Iflavirus, with a high bootstrap value in the maximum-likelihood and neighbor-joining trees. However, this virus has a distinct genome structure with two ORFs, iflaviruses normally having one, suggesting the virus might be a prototype of a new genus. We named the virus isolate Nesidiocoris tenuis virus 1 (NtV-1). The prevalence of NtV-1 infection in wild samples of N. tenuis was at a low level (7.32%, 6 positive in 82 samples), suggesting a possible harmful effect to its host.

The genome sequence of a novel RNA virus in Adelphocoris suturalis.

The complete genome of a novel virus found in Adelphocoris suturalis was determined by RNA-seq and named Adelphocoris suturalis-associated virus 1 (ASV1). ASV1 has a single-stranded RNA genome of 10,845 nucleotides in length and contains five putative open reading frames (ORFs). ORF1 encodes a polypeptide of 2592 amino acids (aa) and contains four conserved domains: a viral RNA methyltransferase domain, an FtsJ-like methyltransferase domain, a viral RNA helicase domain and an RNA-dependent RNA polymerase domain. ORF2, ORF3, ORF4 and ORF5 encode polypeptides of 190, 461, 103 and 159 aa, respectively, of which only ORF5 contains a conserved domain, the Tobacco mosaic virus-coat superfamily. Phylogenetic analysis with the deduced amino acid sequences indicated that ASV1 clusters with the Drosophila-related Boutonnet virus. The similar genomic structure and high bootstrap value identified in the maximum-likelihood tree suggest that ASV1 (possibly alongside Boutonnet virus) could be considered the prototype of a new taxon of unclassified insect viruses. The prevalence of ASV1 infection in wild populations of A. suturalis was at a low level (6.60%, 14 positives from 212 samples).

A novel single-stranded RNA virus in Nesidiocoris tenuis.

The complete genome sequence of a novel single-stranded RNA virus in Nesidiocoris tenuis was determined by RNA-seq and rapid amplification of cDNA ends (RACE) methodologies and was named N. tenuis virus 1. The genomic RNA was 3970 nucleotides (nt) in length and contained two putative open reading frames (ORFs). ORF1 encoded a polypeptide with 283 amino acids containing a viral (superfamily 1) RNA helicase (Hel) domain, and ORF2 encoded a polypeptide with 294 amino acids containing an RNA-dependent RNA polymerase (RdRP) domain. Phylogenetic analysis using the deduced amino acid sequences indicated that the N. tenuis virus 1 clustered with Blackford virus; however, the low bootstrap values and unique genomic structure suggested that the virus is a prototype of a new type of unclassified viruses. The prevalence of N. tenuis virus 1 infection in field populations of N. tenuis differed between three locations, with 28.32% of the 113 sampled individuals testing positive for the virus.

A novel picorna-like virus, Riptortus pedestris virus-1 (RiPV-1), found in the bean bug, R. pedestris, after fungal infection.

A viral genome was assembled de novo from next-generation sequencing (NGS) data from bean bugs, Riptortus pedestris, infected with an entomopathogenic fungus, Beauveria bassiana (Bb), and was further confirmed via the RACE method. This is a novel insect positive-sense single-stranded RNA virus, which we named Riptortus pedestris virus-1 (RiPV-1) (GenBank accession no. KU958718). The genome of RiPV-1 consists of 10,554 nucleotides (nt), excluding the poly(A) tail, which contains a single large open reading frame (ORF) of 10,371 nt encoding a polyprotein (3456 aa) and flanked by 71 and 112 nt at the 5' and 3' untranslated regions (UTR), respectively. RiPV-1 genome organization from the 5' end contains a consensus organization of picorna-like RNA virus helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp), in addition to two putative structural proteins located at the 3' region and a poly(A) tail at the 3' end. The viral particles were approximately 30nm in diameter with some dispersal distinctive surface projections. Based on the phylogenetic analysis of the RdRp sequences, RiPV-1 was clustered in the unassigned insect RNA viruses with two other viruses, APV and KFV. These three viruses were suggested to constitute a new group of insect RNA viruses. RiPV-1 could be found in all stages of lab-reared bean bugs and was detected abundantly in the thorax, abdomen, midgut and fat body, but not in the reproductive organs and muscle. Interestingly, RiPV-1 replication was increased dramatically in bean bugs 2-6days after fungal infection. In conclusion, a novel insect RNA virus was found by NGS data assembly. This virus can provide further insight into the interaction between virus, fungus and the host.

A peculiar new virus-spermatozoon association in the bug Raphigaster nebulosa (Poda) (Heteroptera-Insecta).

The sperm of the heteropteran bug Raphigaster nebulosa (Poda) are of two types, differing in length and size of their flagella. The thicker sperm are shorter than the thinner ones and have large mitochondrial derivatives. The presence of virus particles associated with the plasma membrane of thinner sperm is described for the first time; thicker sperm are immune to virus infection. The fact that virus particles are present on thinner sperm only initiates considerations on the transmission of virus.

Mutation rate in Velvet tobacco mottle virus varies between genomic region and virus variant but is not influenced by obligatory mirid transmission.

Genomic mutation in plant viruses of cultivated plants is known to be influenced by virus, host and vector, but the factors influencing mutation in viruses of native plants in natural ecosystems are rarely studied. We have tested the effect of mode of transmission on mutation in Velvet tobacco mottle virus (VTMoV), a mirid-vectored sobemovirus associated with Nicotiana velutina, an Australian native xerophyte growing in a region isolated from anthropogenic influences. Two variants of VTMoV (K1 and R17) were passaged monthly in the alternative experimental plant host, N. clevelandii, for 2 years, either by mechanical inoculation or by transmission with the mirid Cyrtopeltis nicotianae. Sequence variations were scored after 24 passages in regions of the genome containing the open reading frames (ORFs) for the P1 and coat protein (CP). The mean mutation rate was 6.83 × 10(-4) nt/site year, but a higher overall rate was observed for the K1 (satellite -) than the R17 (satellite +) variant. The P1 ORF showed a higher frequency of non-synonymous mutations than the CP. No clear association was found between either mutation site or mutation rate and the mode of transmission, indicating that obligatory mirid transmission had not exerted a specific bottle-neck effect on sequence variation during the experimental time frame. Failure to detect any sequence motifs linked to vector transmission suggests that a specific capsid-stylet interaction is not required for transmission by mirids.

Prevalence of Buggy Creek virus (Togaviridae: Alphavirus) in insect vectors increases over time in the presence of an invasive avian host.

Invasive species can disrupt natural disease dynamics by altering pathogen transmission among native hosts and vectors. The relatively recent occupancy of cliff swallow (Petrochelidon pyrrhonota) nesting colonies in western Nebraska by introduced European house sparrows (Passer domesticus) has led to yearly increases in the prevalence of an endemic arbovirus, Buggy Creek virus (BCRV), in its native swallow bug (Oeciacus vicarius) vector at sites containing both the invasive sparrow host and the native swallow host. At sites without the invasive host, no long-term changes in prevalence have occurred. The percentage of BCRV isolates exhibiting cytopathicity in Vero-cell culture assays increased significantly with year at sites with sparrows but not at swallow-only sites, suggesting that the virus is becoming more virulent to vertebrates in the presence of the invasive host. Increased BCRV prevalence in bug vectors at mixed-species colonies may reflect high virus replication rates in house sparrow hosts, resulting in frequent virus transmission between sparrows and swallow bugs. This case represents a rare empirical example of a pathogen effectively switching to an invasive host, documented in the early phases of the host's arrival in a specialized ecosystem and illustrating how an invasive species can promote long-term changes in host-parasite transmission dynamics.

VLPs of Leptopilina boulardi share biogenesis and overall stellate morphology with VLPs of the heterotoma clade.

Viruses and virus-like particles (VLPs) of insect parasitoids modify host-parasite interactions. The Drosophila wasp, Leptopilina heterotoma, produce 300 nm spiked VLPs that bind to the host's blood cells via surface projections. L. heterotoma is a generalist wasp that attacks over a dozen Drosophila species. Oviposition introduces VLPs into the hemolymph of Drosophila larvae. VLPs lyse hemocytes and obliterate immune signaling in infected larval hosts. L. boulardi, a member of a distinct Leptopilina clade, is a specialist, whose host range is limited to the melanogaster group. As a step toward understanding a potential relationship between venom contents and host range in these wasps, we used electron microscopy to characterize VLPs from the virulent L. boulardi-17 (Lb-17) strain. While the Lb-17 VLPs can neither lyse blood cells nor suppress host defense, their biogenesis is surprisingly similar to that of L. heterotoma. Like L. heterotoma VLPs, L. boulardi VLPs are stellate; but they have fewer spikes, each spike being significantly longer than the spikes in L. heterotoma VLPs. The Lb-17 VLPs possess a dimple, making them clearly distinct from L. heterotoma VLPs. We discuss the significance of these cross-clade differences in VLP morphologies in relation to their biological activities and the host range of the wasp.

Cliff swallows, swallow bugs, and West Nile virus: an unlikely transmission mechanism.

The cliff swallow (Petrochelidon pyrrhonota) could play an important role in the transmission of West Nile virus (WNV) because of its breeding ecology, reservoir competence status, and potentially high natural exposure rates. Cliff swallows nest within colonies and their nests are occupied year-round by swallow bugs (Oeciacus vicarius), hematophagus ectoparasites that feed primarily on cliff swallows. These parasites are likely exposed to WNV while feeding on infectious blood of nesting cliff swallow adults and nestlings and thus, if competent vectors, could contribute to seasonal elevations in WNV transmission. In addition, swallow bugs remain within nests year-round and therefore could provide a potential overwintering mechanism for WNV if persistently infected. To test the hypotheses that swallow bugs are competent vectors and become persistently infected with WNV, we experimentally inoculated cliff swallow nestlings, allowed swallow bugs to feed on these birds during the acute phase of infection, and then exposed naive cliff swallow nestlings to the same swallow bugs. In addition, a subset of swallow bugs that fed on infectious swallow nestlings was maintained through a simulated overwintering period. Although swallow bugs ingested infectious blood (up to 10(6.8) plaque-forming units of WNV/mL serum) and subsequently blood-fed on naive swallows, no WNV transmission was detected, and all bugs tested WNV negative after the simulated overwintering period. Although many ecologic scenarios exist beyond the present study, our results suggest that swallow bugs may be unlikely to serve as competent biological vectors for WNV during active transmission periods or to reinitiate seasonal transmission.

Binding mode of the first aminoacyl-tRNA in translation initiation mediated by Plautia stali intestine virus internal ribosome entry site.

Eukaryotic ribosomes directly bind to the intergenic region-internal ribosome entry site (IGR-IRES) of Plautia stali intestine virus (PSIV) and initiate translation without either initiation factors or initiator Met-tRNA. We have investigated the mode of binding of the first aminoacyl-tRNA in translation initiation mediated by the IGR-IRES. Binding ability of aminoacyl-tRNA to the first codon within the IGR-IRES/80 S ribosome complex was very low in the presence of eukaryotic elongation factor 1A (eEF1A) alone but markedly enhanced by the translocase eEF2. Moreover, eEF2-dependent GTPase activity of the IRES/80 S ribosome complex was 3-fold higher than that of the free 80 S ribosome. This activation was suppressed by addition of the antibiotics pactamycin and hygromycin B, which are inhibitors of translocation. The results suggest that translocation by the action of eEF2 is essential for stable tRNA binding to the first codon of the PSIV-IRES in the ribosome. Chemical probing analysis showed that IRES binding causes a conformational change in helix 18 of 18 S rRNA at the A site such that IRES destabilizes the conserved pseudoknot within the helix. This conformational change caused by the PSIV-IRES may be responsible for the activation of eEF2 action and stimulation of the first tRNA binding to the P site without initiation factors.

Multiple coding sequences for the genome-linked virus protein (VPg) in dicistroviruses.

N-terminal Edman sequencing of the genome-linked viral protein (VPg) of Plautia stali intestine virus (PSIV, Dicistroviridae) detected heterologus residues. The VPg sequence determined was found to be triplicated in the nonstructural protein precursor. Multiple VPg-like sequences were also found in 10 of the 12 dicistroviruses with a maximum of six copies in Solenopsis invicta virus-1. We postulate that redundant VPg coding sequences facilitate multiplication of dicistroviruses, because fewer cycle of translation of the nonstructural protein precursor produces larger amounts of VPg proteins in parallel with the increased production of capsid proteins by the intergenic internal ribosome entry site mediated translation.