Sequence-independent characterization of viruses based on the pattern of viral small RNAs produced by the host

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...

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.

The rapid spread of Zika virus (ZIKV) in the Americas raised many questions about the role of Culex quinquefasciatus mosquitoes in transmission, in addition to the key role played by the vector Aedes aegypti. Here we analysed the competence of Cx. quinquefasciatus (with or without Wolbachia endosymbionts) for a ZIKV isolate. We also examined the induction of RNA interference pathways after viral challenge and the production of small virus-derived RNAs. We did not observe any infection nor such small virus-derived RNAs, regardless of the presence or absence of Wolbachia. Thus, Cx. quinquefasciatus does not support ZIKV replication and Wolbachia is not involved in producing this phenotype. In short, these mosquitoes are very unlikely to play a role in transmission of ZIKV. Micronesia and then in French Polynesia in 2013-2014, and after affecting most of the South Pacific islands, the virus was eventually detected in the Americas in 2015 [1][2][3][4]. The increased number of infections in humans included cases with unusually severe symptoms such as Guillain-Barr e syndrome and developmental abnormalities in newborns that are now described as congenital Zika syndrome [5][6][7][8]. As there are currently no licensed vaccines or specific therapies, the only way to interrupt ZIKV transmission is by controlling mosquito populations [9]. An enzootic cycle limited to Africa and Asia with occasional spillover events has been described [10,11]. In the current outbreak in the Americas, ZIKV is believed to be mainly transmitted by the human-biting mosquito Aedes aegypti [12,13]. Whether a similar enzootic cycle in the Americas can be established is not known and it has been suggested that such a process would make eradication efforts 'practically impossible' [14]. Experimental infections with the epidemic ZIKV genotypes demonstrated that populations of Ae. aegypti and Ae. albopictus mosquitoes, which are associated with a risk of local transmission [15] as well other aedine species were heterogeneously and weakly competent for transmission [16][17][18][19][20][21][22][23][24][25]. Therefore, the potential role of other anthropophilic mosquitoes in the ZIKV outbreak raised a question which took time to be addressed [26]. Culex quinquefasciatus Say is an opportunistic blood feeder predominant in urban settings throughout the tropics where it is frequently the most annoying biting pest to humans [27]. This night-active mosquito is also the vector of many pathogens including arboviruses such as West Nile and St. Louis encephalitis viruses [28], which belong to the genus Flavivirus of the family Flaviviridae and are related to ZIKV. Both Cx. quinquefasciatus and Culex pipiens (from temperate regions) mosquitoes of the Cx. pipiens complex were not able to experimentally transmit ZIKV and no viral particles were detected in mosquito saliva up to 21 days after exposure to an infectious blood meal [17-20, 25, 29-36], though different results were recorded with other Cx. quinquefasciatus strains [37,38]. Even after injection o...