Functional Specialization of the Small Interfering RNA Pathway in Response to Virus Infection

The Asian citrus psyllid, Diaphorina citri, is the natural vector of the causal agent of Huanglongbing (HLB), or citrus greening disease. Together; HLB and D. citri represent a major threat to world citrus production. As there is no cure for HLB, insect vector management is considered one strategy to help control the disease, and D. citri viruses might be useful. In this study, we used a metagenomic approach to analyze viral sequences associated with the global population of D. citri. By sequencing small RNAs and the transcriptome coupled with bioinformatics analysis, we showed that the virus-like sequences of D. citri are diverse. We identified novel viral sequences belonging to the picornavirus superfamily, the Reoviridae, Parvoviridae, and Bunyaviridae families, and an unclassified positive-sense single-stranded RNA virus. Moreover, a Wolbachia prophage-related sequence was identified. This is the first comprehensive survey to assess the viral community from worldwide populations of an agricultural insect pest. Our results provide valuable information on new putative viruses, some of which may have the potential to be used as biocontrol agents. IMPORTANCEInsects have the most species of all animals, and are hosts to, and vectors of, a great variety of known and unknown viruses. Some of these most likely have the potential to be important fundamental and/or practical resources. In this study, we used highthroughput next-generation sequencing (NGS) technology and bioinformatics analysis to identify putative viruses associated with Diaphorina citri, the Asian citrus psyllid. D. citri is the vector of the bacterium causing Huanglongbing (HLB), currently the most serious threat to citrus worldwide. Here, we report several novel viral sequences associated with D. citri. Viruses are the most abundant microbes on our planet (1) and are found in all types of organisms. Insects are the largest and most diverse taxonomic class among animals, representing perhaps half of known animals, with more than one million species recognized worldwide (2). Insects are known to be hosts to viruses belonging to various viral taxa, including the Baculoviridae, Parvoviridae, Flaviviridae, Ascoviridae, Togaviridae, Bunyaviridae, and Rhabdoviridae (3). However, the number of currently described viral species infecting insects is relatively low compared to the number of viruses that have been discovered among prokaryotes, plants, and vertebrates. Furthermore, most of the insect viruses described to date have been discovered because of their pathogenic effects on their insect hosts or because they are pathogens of humans, other vertebrates, or economically important plants. Traditional viral detection methods that require prior knowledge of genome sequences may not be suitable for the discovery of new viruses and, in particular, viruses with a high level of genetic diversity. However, in the past decade, the development of high-throughput next-generation sequencing (NGS) technologies and bioinformatics applications has provided ne...

Section: Discussionmentioning

confidence: 53%

Diverse Array of New Viral Sequences Identified in Worldwide Populations of the Asian Citrus Psyllid (Diaphorina citri) Using Viral Metagenomics

Nouri

Salem

Nigg

et al. 2016

“…The four sequence collections were analyzed with WebLogo 3 program (http://weblogo .threeplusone.com/create.cgi) (24). The statistical significance (P Ͻ 0.05) of enriched or depleted bases of the created logos was evaluated with one-proportion Z-tests as previously described (25). For the delineation of the degenerate motif of DVNV DC at the cut site (ˆ) of vsRNAs, only the positions with statistically significantly enriched or depleted bases in the logos of all four sequence collections were selected.…”

Section: Methodsmentioning

confidence: 99%

Viral Small-RNA Analysis of Bombyx mori Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection

Zografidis

Nieuwerburgh

Κολλιοπούλου

et al. 2015

The lepidopteran innate immune response against RNA viruses remains poorly understood, while in other insects several studies have highlighted an essential role for the exo-RNAi pathway in combating viral infection. Here, by using deep-sequencing technology for viral small-RNA (vsRNA) assessment, we provide evidence that exo-RNAi is operative in the silkworm Bombyx mori against both persistent and pathogenic infection of B. mori cytoplasmic polyhedrosis virus (BmCPV) which is characterized by a segmented double-stranded RNA (dsRNA) genome. Further, we show that Dicer-2 predominantly targets viral dsRNA and produces 20-nucleotide (nt) vsRNAs, whereas an additional pathway is responsive to viral mRNA derived from segment 10. Importantly, vsRNA distributions, which define specific hot and cold spot profiles for each viral segment, to a considerable degree overlap between Dicer-2-related (19 to 21 nt) and Dicer-2-unrelated vsRNAs, suggesting a common origin for these profiles. We found a degenerate motif significantly enriched at the cut sites of vsRNAs of various lengths which link an unknown RNase to the origins of vsRNAs biogenesis and distribution. Accordingly, the indicated RNase activity may be an important early factor for the host's antiviral defense in Lepidoptera. Central to the defense of insects against RNA viruses is the triggering of the so-called exogenous RNA interference pathway (exo-RNAi), by which viral double-stranded RNA (dsRNA) is cleaved intracellularly into viral small interfering RNAs (siRNAs) that in turn silence homologous transcripts (1). It is established that Dicer-2, a cytoplasmic RNase III class enzyme, recognizes and cuts successively the dsRNA template to produce small interfering RNA duplexes of ϳ21 nucleotides (nt) characterized by a signature of 2-nt 3=-hydroxyl overhangs (2). These duplexes are subsequently incorporated into the effector complex RISC (RNA-induced silencing complex) via interactions with Argonaute-2, which, upon discarding one of the two strands (passenger strand), is followed by selection and cleavage of viral sequences bearing perfect complementarity to the remaining strand (guide strand) (1, 3). Dicer-2 may act upon the genome of the virus itself-as in the case of dsRNA viruses-and/or on viral replication and transcription intermediates, although structured, single-stranded RNA (ssRNA) may also be utilized (4). Interestingly, several deepsequencing data have revealed that approximately 18-to 30-nt viral small RNAs (vsRNAs) of infected insects are not evenly distributed along the genomes but map preferentially in distinct genomic areas (hot spots) versus genomic stretches with unmapped or less-mapped vsRNAs (cold spots) (4-13). The origin of these profiles, as well as the hypothetical functional distinction between vsRNAs deriving from hot or cold spots, remains substantially elusive, although hot spots near the end of the genome have been attributed to structured regulatory viral regions (14) and an RNAi decoy-like mechanism driven by abundant vsRNAs ...

“…E xperiments in the model organism Drosophila melanogaster have shown that RNA interference (RNAi) plays a major role in antiviral immunity in insects: (i) flies with mutations for the three key genes of the small interfering RNA (siRNA) pathway, Dicer-2, Argonaute 2, and r2d2, show increased sensitivity to infection by RNA and DNA viruses (1-6); (ii) Dicer-2-dependent 21-nucleotide siRNAs of viral origin accumulate in virus-infected flies (1,3,4,(7)(8)(9); (iii) several insect viruses express viral suppressors of RNAi (5,(10)(11)(12). The importance of this pathway in the control of viral infections has been confirmed in other insects, in particular, the vector mosquito genera Aedes and Culex, which transmit important human pathogens such as dengue virus, West Nile virus, and other arthropod-borne viruses (13)(14)(15)(16)(17)(18).…”

Section: Importancementioning

confidence: 99%

Analysis of the Contribution of Hemocytes and Autophagy to Drosophila Antiviral Immunity

Lamiable

Arnold

Faria

et al. 2016

Self Cite

112

Antiviral immunity in the model organism Drosophila melanogaster involves the broadly active intrinsic mechanism of RNA interference (RNAi) and virus-specific inducible responses. Here, using a panel of six viruses, we investigated the role of hemocytes and autophagy in the control of viral infections. Injection of latex beads to saturate phagocytosis, or genetic depletion of hemocytes, resulted in decreased survival and increased viral titers following infection with Cricket paralysis virus (CrPV), Flock House virus (FHV), and vesicular stomatitis virus (VSV) but had no impact on Drosophila C virus (DCV), Sindbis virus (SINV), and Invertebrate iridescent virus 6 (IIV6) infection. In the cases of CrPV and FHV, apoptosis was induced in infected cells, which were phagocytosed by hemocytes. In contrast, VSV did not trigger any significant apoptosis but we confirmed that the autophagy gene Atg7 was required for full virus resistance, suggesting that hemocytes use autophagy to recognize the virus. However, this recognition does not depend on the Toll-7 receptor. Autophagy had no impact on DCV, CrPV, SINV, or IIV6 infection and was required for replication of the sixth virus, FHV. Even in the case of VSV, the increases in titers were modest in Atg7 mutant flies, suggesting that autophagy does not play a major role in antiviral immunity in Drosophila. Altogether, our results indicate that, while autophagy plays a minor role, phagocytosis contributes to virus-specific immune responses in insects. IMPORTANCEPhagocytosis and autophagy are two cellular processes that involve lysosomal degradation and participate in Drosophila immunity. Using a panel of RNA and DNA viruses, we have addressed the contribution of phagocytosis and autophagy in the control of viral infections in this model organism. We show that, while autophagy plays a minor role, phagocytosis contributes to virusspecific immune responses in Drosophila. This work brings to the front a novel facet of antiviral host defense in insects, which may have relevance in the control of virus transmission by vector insects or in the resistance of beneficial insects to viral pathogens.