Disclosing the virome of Aedes, Anopheles and Culex female mosquitoes, Alto Pantanal of Mato Grosso, Brazil, 2019.

Using Illumina NextSeq sequencing and bioinformatics, we identified and characterized thirty-three viral sequences of unsegmented and multipartite viral families in Aedes spp., Culex sp. and Anopheles darlingi female mosquito pools from Porto São Luiz and Pirizal, Alto Pantanal. Seventeen sequences belong to unsegmented viral families, twelve represent putative novel insect-specific viruses (ISVs) within families Chuviridae (3/33; partial genomes) and coding-complete sequences of Xinmoviridae (1/33), Rhabdoviridae (2/33) and Metaviridae (6/33); and five coding-complete sequences of already-known ISVs. Notably, two putative novel rhabdoviruses, Corixo rhabdovirus 1 and 2, were phylogenetically related to Coxipo dielmovirus, but separated from other Alpharhabdovirinae genera, sharing Anopheles spp. as host. Regarding multipartite families, sixteen segments of different putative novel viruses were identified (13 coding-complete segments) within Durnavirales (4/33), Elliovirales (1/33), Hareavirales (3/33) and Reovirales (8/33) orders. Overall, this study describes twenty-eight (28/33) putative novel ISVs and five (5/33) already described viruses using metagenomics approach.

Lack of abundant core virome in Culex mosquitoes from a temperate climate region despite a mosquito species-specific virome.

In arthropod-associated microbial communities, insect-specific viruses (ISVs) are prevalent yet understudied due to limited infectivity outside their natural hosts. However, ISVs might play a crucial role in regulating mosquito populations and influencing arthropod-borne virus transmission. Some studies have indicated a core virome in mosquitoes consisting of mostly ISVs. Employing single mosquito metagenomics, we comprehensively profiled the virome of native and invasive mosquito species in Belgium. This approach allowed for accurate host species determination, prevalence assessment of viruses and Wolbachia, and the identification of novel viruses. Contrary to our expectations, no abundant core virome was observed in Culex mosquitoes from Belgium. In that regard, we caution against rigidly defining mosquito core viromes and encourage nuanced interpretations of other studies. Nonetheless, our study identified 45 viruses of which 28 were novel, enriching our understanding of the mosquito virome and ISVs. We showed that the mosquito virome in this study is species-specific and less dependent on the location where mosquitoes from the same species reside. In addition, because Wolbachia has previously been observed to influence arbovirus transmission, we report the prevalence of Wolbachia in Belgian mosquitoes and the detection of several Wolbachia mobile genetic elements. The observed prevalence ranged from 83% to 92% in members from the Culex pipiens complex.IMPORTANCECulex pipiens mosquitoes are important vectors for arboviruses like West Nile virus and Usutu virus. Virome studies on individual Culex pipiens, and on individual mosquitoes in general, have been lacking. To mitigate this, we sequenced the virome of 190 individual Culex and 8 individual Aedes japonicus mosquitoes. We report the lack of a core virome in these mosquitoes from Belgium and caution the interpretation of other studies in this light. The discovery of new viruses in this study will aid our comprehension of insect-specific viruses and the mosquito virome in general in relation to mosquito physiology and mosquito population dynamics.

Transcriptome Analysis Reveals a Diverse Range of Novel Viruses in Australian Sugarcane Soldier Fly (Inopus flavus) Larvae.

In Australia, Soldier flies (Inopus spp.) are economically significant pests of sugarcane that currently lack a viable management strategy. Despite various research efforts, the mechanisms underlying the damage caused by soldier fly larvae remain poorly understood. Our study aims to explore whether this damage is associated with the transmission of plant viruses during larval feeding. We also explore the larval transcriptome to identify any entomopathogenic viruses with the potential to be used as biocontrol agents in future pest management programs. Seven novel virus sequences are identified and characterised using de novo assembly of RNA-Seq data obtained from salivary glands of larvae. The novel virus sequences belong to different virus families and are tentatively named SF-associated anphevirus (SFaAV), SF-associated orthomyxo-like virus (SFaOV), SF-associated narna-like virus (SFaNV), SF-associated partiti-like virus (SFaPV), SF-associated toti-like virus (SFaTV-1 and SFaTV-2) and SF-associated densovirus (SFaDV). These newly identified viruses are more likely insect-associated viruses, as phylogenetic analyses show that they cluster with other insect-specific viruses. Small RNA analysis indicates prominent peaks at both 21 nt and 26-29 nt, suggesting the activation of host siRNA and piwiRNA pathways. Our study helps to improve understanding of the virome of soldier flies and could identify insect viruses for deployment in novel pest management strategies.

RNA virus diversity and prevalence in field and laboratory populations of melon fly throughout its distribution.

Insects have a rich diversity of RNA viruses that can either cause acute infections or persist in host populations without visible symptoms. The melon fly, Zeugodacus cucurbitae (Tephritidae) causes substantial economic losses through infestation of diverse cucurbit and other crops. Of Indomalayan origin, it is now established in many tropical regions of the world. The virome diversity of Z. cucurbitae is largely unknown across large parts of its distribution, including the Indian subcontinent. We have analysed three transcriptomes each of one field-collected and one laboratory-reared Z. cucurbitae population from Bangalore (India) and discovered genomes of ten putative RNA viruses: two sigmaviruses, one chimbavirus, one cripavirus, one noda-like virus, one nora virus, one orbivirus, one partiti-like virus, one sobemovirus and one toti-like virus. Analysis of the only available host genome of a Hawaiian Z. cucurbitae population did not detect host genome integration of the detected viruses. While all ten viruses were found in the Bangalore field population only seven were detected in the laboratory population, indicating that these seven may cause persistent covert infections. Using virus-specific RNA-dependent RNA polymerase gene primers, we detected nine of the RNA viruses with an overall low variant diversity in some but not all individual flies from four out of five Indian regions. We then screened 39 transcriptomes of Z. cucurbitae laboratory populations from eastern Asia (Guangdong, Hainan, Taiwan) and the Pacific region (Hawaii), and detected seven of the ten virus genomes. We found additional genomes of a picorna-like virus and a negev-like virus. Hawaii as the only tested population from the fly's invasive range only had one virus. Our study provides evidence of new and high RNA virus diversity in Indian populations within the original range of Z. cucurbitae, as well as the presence of persistent covert infections in laboratory populations. It builds the basis for future research of tephritid-associated RNA viruses, including their host effects, epidemiology and application potential in biological control.

Viral Interference between the Insect-Specific Virus Brejeira and the Saint Louis Encephalitis Virus In Vitro.

The Saint Louis encephalitis virus (SLEV) is an encephalitogenic arbovirus (Flaviviridae family) that has a wide geographical distribution in the western hemisphere, especially in the Americas. The negevirus Brejeira (BREV) was isolated for the first time in Brazil in 2005. This study aimed to verify the existence of a possible interfering effect of BREV on the course of SLEV infection and vice versa. We used clone C6/36 cells. Three combinations of MOIs were used (SLEV 0.1 × BREV 1; SLEV 1 × BREV 0.1; SLEV 1 × BREV 1) in the kinetics of up to 7 days and then the techniques of indirect immunofluorescence (IFA), a plaque assay on Vero cells, and RT-PCR were performed. Our results showed that the cytopathic effect (CPE) caused by BREV was more pronounced than the CPE caused by SLEV. Results of IFA, the plaque assay, and RT-PCR showed the suppression of SLEV replication in the co-infection condition in all the MOI combinations used. The SLEV suppression was dose-dependent. Therefore, the ISV Brejeira can suppress SLEV replication in Aedes albopictus cells, but SLEV does not negatively interfere with BREV replication.

Transmission mode predicts coinfection patterns of insect-specific viruses in field populations of the Queensland fruit fly.

Insect-specific viruses (ISVs) can affect insect health and fitness, but can also interact with other insect-associated microorganisms. Despite this, ISVs are often studied in isolation from each other, in laboratory populations. Consequently, their diversity, prevalence and associations with other viruses in field populations are less known, yet these parameters are important to understanding virus epidemiology. To help address this knowledge gap, we assessed the diversity, prevalence and coinfections of three ISVs (horizontally transmitted cripavirus, biparentally transmitted sigmavirus and maternally transmitted iflavirus) in 29 field populations of Queensland fruit fly, Australia's most significant horticultural pest, in the context of their different transmission modes. We detected new virus variant diversity. In contrast to the very high virus prevalence in laboratory populations, 46.8% of 293 field flies carried one virus and 4.8% had two viruses. Cripavirus and sigmavirus occurred in all regions, while iflavirus was restricted to subtropical and tropical regions. Cripavirus was most prevalent (37.5%), followed by sigmavirus (13.7%) and iflavirus (4.4%). Cripavirus coinfected some flies with either one of the two vertically transmitted viruses. However, sigmavirus did not coinfect individuals with iflavirus. Three different modelling approaches detected negative association patterns between sigmavirus and iflavirus, consistent with the absence of such coinfections in laboratory populations. This may be linked with their maternal transmission and the ineffective paternal transmission of sigmavirus. Furthermore, we found that, unlike sigmavirus and iflavirus, cripavirus load was higher in laboratory than field flies. Laboratory and mass-rearing conditions may increase ISV prevalence and load due to increased transmission opportunities. We conclude that a combination of field and laboratory studies is needed to uncover ISV interactions and further our understanding of ISV epidemiology.

Influence of dengue virus serotypes on the abundance of Aedes aegypti insect-specific viruses (ISVs).

A comprehensive understanding of the virome in mosquito vectors is crucial for assessing the potential transmission of viral agents, designing effective vector control strategies, and advancing our knowledge of insect-specific viruses (ISVs). In this study, we utilized Oxford Nanopore Technologies metagenomics to characterize the virome of Aedes aegypti mosquitoes collected in various regions of Colombia, a country hyperendemic for dengue virus (DENV). Analyses were conducted on groups of insects with previous natural DENV infection (DENV-1 and DENV-2 serotypes), as well as mosquito samples that tested negative for virus infection (DENV-negative). Our findings indicate that the Ae. aegypti virome exhibits a similar viral composition at the ISV family and species levels in both DENV-positive and DENV-negative samples across all study sites. However, differences were observed in the relative abundance of viral families such as Phenuiviridae, Partitiviridae, Flaviviridae, Rhabdoviridae, Picornaviridae, Bromoviridae, and Virgaviridae, depending on the serotype of DENV-1 and DENV-2. In addition, ISVs are frequently found in the core virome of Ae. aegypti, such as Phasi Charoen-like phasivirus (PCLV), which was the most prevalent and showed variable abundance in relation to the presence of specific DENV serotypes. Phylogenetic analyses of the L, M, and S segments of the PCLV genome are associated with sequences from different regions of the world but show close clustering with sequences from Brazil and Guadeloupe, indicating a shared evolutionary relationship. The profiling of the Ae. aegypti virome in Colombia presented here improves our understanding of viral diversity within mosquito vectors and provides information that opens the way to possible connections between ISVs and arboviruses. Future studies aimed at deepening our understanding of the mechanisms underlying the interactions between ISVs and DENV serotypes in Ae. aegypti could provide valuable information for the design of effective vector-borne viral disease control and prevention strategies.IMPORTANCEIn this study, we employed a metagenomic approach to characterize the virome of Aedes aegypti mosquitoes, with and without natural DENV infection, in several regions of Colombia. Our findings indicate that the mosquito virome is predominantly composed of insect-specific viruses (ISVs) and that infection with different DENV serotypes (DENV-1 and DENV-2) could lead to alterations in the relative abundance of viral families and species constituting the core virome in Aedes spp. The study also sheds light on the identification of the genome and evolutionary relationships of the Phasi Charoen-like phasivirus in Ae. aegypti in Colombia, a widespread ISV in areas with high DENV incidence.

Employing oxford nanopore technologies (ONT) for understanding the ecology and transmission dynamics of flaviviruses in mosquitoes (Diptera: Culicidae) from Eastern Colombia.

Studies focused on identifying the viral species of Flavivirus in vectors are scarce in Latin America and particularly in Colombia. Therefore, the frequency of infection of the Flavivirus genus and its feeding preferences were identified in the mosquito species circulating in the municipality of Puerto Carreño-Vichada, located in the Eastern Plains of Colombia. This was done by sequencing the viral NS5 and vertebrate 12S rRNA genes, respectively, using Oxford Nanopore Technologies (ONT). A total of 1,159 mosquitoes were captured, with the most abundant species being Aedes serratus at 73.6% (n = 853). All the mosquitoes were processed in 230 pools (2-6 individuals) and 51 individuals, where 37.01% (n = 104) were found to be infected with Flavivirus. In these samples, infection by arboviruses of epidemiological importance, such as dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV), was ruled out by PCR. However, through sequencing, infection by different insect-specific viruses (ISFVs) and a medically important virus, West Nile virus (WNV), were identified in a mosquito of the Culex browni species. Additionally, the feeding patterns showed that most species present a generalist behavior. Given the above, conducting entomovirological surveillance studies is crucial, especially in areas of low anthropogenic intervention, due to the high probability that potentially pathogenic viruses could generate spillover events under deforestation scenarios.

Agua Salud Alphavirus Infection, Dissemination and Transmission in Aedes aegypti Mosquitoes.

Mosquitoes are competent vectors for many important arthropod-borne viruses (arboviruses). In addition to arboviruses, insect-specific viruses (ISV) have also been discovered in mosquitoes. ISVs are viruses that replicate in insect hosts but are unable to infect and replicate in vertebrates. They have been shown to interfere with arbovirus replication in some cases. Despite the increase in studies on ISV-arbovirus interactions, ISV interactions with their hosts and how they are maintained in nature are still not well understood. In the present study, we investigated the infection and dissemination of the Agua Salud alphavirus (ASALV) in the important mosquito vector Aedes aegypti through different infection routes (per oral infection, intrathoracic injection) and its transmission. We show here that ASALV infects the female Ae. aegypti and replicates when mosquitoes are infected intrathoracically or orally. ASALV disseminated to different tissues, including the midgut, salivary glands and ovaries. However, we observed a higher virus load in the brain than in the salivary glands and carcasses, suggesting a tropism towards brain tissues. Our results show that ASALV is transmitted horizontally during adult and larval stages, although we did not observe vertical transmission. Understanding ISV infection and dissemination dynamics in Ae. aegypti and their transmission routes could help the use of ISVs as an arbovirus control strategy in the future.

Composition and global distribution of the mosquito virome - A comprehensive database of insect-specific viruses.

Mosquitoes are vectors for emerging and re-emerging infectious viral diseases of humans, livestock and other animals. In addition to these arthropod-borne (arbo)viruses, mosquitoes are host to an array of insect-specific viruses, collectively referred to as the mosquito virome. Mapping the mosquito virome and understanding if and how its composition modulates arbovirus transmission is critical to understand arboviral disease emergence and outbreak dynamics. In recent years, next-generation sequencing as well as PCR and culture-based methods have been extensively used to identify mosquito-associated viruses, providing insights into virus ecology and evolution. Until now, the large amount of mosquito virome data, specifically those acquired by metagenomic sequencing, has not been comprehensively integrated. We have constructed a searchable database of insect-specific viruses associated with vector mosquitoes from 175 studies, published between October 2000 and February 2022. We identify the most frequently detected and widespread viruses of the Culex, Aedes and Anopheles mosquito genera and report their global distribution. In addition, we highlight the challenges of extracting and integrating published virome data and we propose that a standardized reporting format will facilitate data interpretation and re-use by other scientists. We expect our comprehensive database, summarizing mosquito virome data collected over 20 years, to be a useful resource for future studies.

Identifying the Gut Virome of Diaphorina citri from Florida Groves.

Asian citrus psyllid (Diaphorina citri) transmits the bacterial pathogen Candidatus Liberibacter asiaticus (CLas), the putative causative agent of citrus Huanglongbing disease (HLB). Insect-specific viruses can act against insects as their natural enemies, and recently, several D. citri-associated viruses were discovered. The insect gut plays an important role as not only a pool for diverse microbes but also as a physical barrier to prevent the spread of pathogens such as CLas. However, there is little evidence of the presence of D. citri-associated viruses in the gut and of the interaction between them and CLas. Here, we dissected psyllid guts collected from five growing regions in Florida, and the gut virome was analyzed by high throughput sequencing. Four insect viruses, including D. citri-associated C virus (DcACV), D. citri densovirus (DcDV), D. citri reovirus (DcRV), and D. citri flavi-like virus (DcFLV), were identified, and their presence in the gut, including an additional D. citri cimodo-like virus (DcCLV), were confirmed with PCR-based assays. Microscopic analysis showed that DcFLV infection leads to morphological abnormalities in the nuclear structure in the infected psyllid gut cells. The complex and diverse composition of microbiota in the psyllid gut suggests a possible interaction and dynamics between CLas and the D. citri-associated viruses. Our study identified various D. citri-associated viruses that localized in the psyllid gut and provided more information that helps to evaluate the potential vectors for manipulating CLas in the psyllid gut.

Arbovirus surveillance in mosquitoes: Historical methods, emerging technologies, and challenges ahead.

Arboviruses cause millions of infections each year; however, only limited options are available for treatment and pharmacological prevention. Mosquitoes are among the most important vectors for the transmission of several pathogens to humans. Despite advances, the sampling, viral detection, and control methods for these insects remain ineffective. Challenges arise with the increase in mosquito populations due to climate change, insecticide resistance, and human interference affecting natural habitats, which contribute to the increasing difficulty in controlling the spread of arboviruses. Therefore, prioritizing arbovirus surveillance is essential for effective epidemic preparedness. In this review, we offer a concise historical account of the discovery and monitoring of arboviruses in mosquitoes, from mosquito capture to viral detection. We then analyzed the advantages and limitations of these traditional methods. Furthermore, we investigated the potential of emerging technologies to address these limitations, including the implementation of next-generation sequencing, paper-based devices, spectroscopic detectors, and synthetic biosensors. We also provide perspectives on recurring issues and areas of interest such as insect-specific viruses.

First Report of Wenzhou sobemo-like virus 4 in Aedes albopictus (Diptera: Culicidae) in Latin America.

Insect-specific viruses (ISVs) are viruses that replicate exclusively in arthropod cells. Many ISVs have been studied in mosquitoes as many of them act as vectors for human etiological agents, such as arboviruses. Aedes (Stegomyia) albopictus is an important potential vector of several arboviruses in Brazil, such as dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV). The development of next-generation sequencing metagenomics has enabled the discovery and characterization of new ISVs. Ae. albopictus eggs were collected using oviposition traps placed in two urban parks in the city of São Paulo, Brazil. The Aedes albopictus females were divided into pools and the genetic material was extracted and processed for sequencing by metagenomics. Complete genomes of ISV Wenzhou sobemo-like virus 4 (WSLV4) were obtained in three of the four pools tested. This is the first detection of ISV WSLV4 in Ae. albopictus females in Latin America. Further studies on ISVs in Ae. albopictus are needed to better understand the role of this species in the dynamics of arbovirus transmission in the Americas.

Deciphering the Tissue Tropism of the RNA Viromes Harbored by Field-Collected Anopheles sinensis and Culex quinquefasciatus.

Arboviruses and insect-specific viruses (ISVs) are two major types of viruses harbored by mosquitoes that are distinguished by the involvement of vertebrate hosts in their transmission cycles. While intensive studies have focused on the transmission, tissue tropism, and evolution of arboviruses, these characteristics are poorly investigated in ISVs, which dominate the mosquito virome. Therefore, in this study, we collected two mosquito species, Anopheles sinensis and Culex quinquefasciatus, in the field and used a metatranscriptomics approach to characterize their RNA viromes in different tissues, such as the midgut, legs, salivary gland, eggs, and the remainder of the carcass. Blood-engorged individuals of these species were captured in 3 locations, and 60 mosquitoes were pooled from each species and location. A total of 40 viral species from diverse viral taxa associated with all viral RNA genome types were identified, among which 19 were newly identified in this study. According to the current viral taxonomy, some of these viruses, such as Yancheng Anopheles associated virus 2 (Narnaviridae) and Jiangsu Anopheles-related virus (Ghabrivirales), were novel. The two investigated mosquito species generally harbored distinct viromes. Nevertheless, the viruses were generally shared among different tissue types to various degrees. Specifically, the eggs possessed a viral community with significantly lower diversity and abundance than those in other tissues, whereas the legs and salivary glands exhibited higher viral abundance. The compositions and distributions of the viromes of different mosquito tissues were demonstrated for the first time in our study, providing important insight into the virome dynamics within individual mosquitoes. IMPORTANCE ISVs are considered to be ancestral to arboviruses. Because of their medical importance, arboviruses have been well studied from the aspects of their transmission mode, evolution of dual-host tropism, and genetic dynamics within mosquito vectors. However, the mode of ISV maintenance is poorly understood, even though many novel ISVs have been identified with the emergence of sequencing technology. In our study, in addition to the identification of a diverse virus community, the tissue tropism of RNA viromes harbored by two field-collected mosquito species was demonstrated for the first time. According to the results, the virus communities of different tissues, such as the salivary glands, midguts, legs, and eggs, can help us understand the evolution, transmission routes, and maintenance modes of mosquito-specific viruses in nature.

Reporter Flaviviruses as Tools to Demonstrate Homologous and Heterologous Superinfection Exclusion.

Binjari virus (BinJV) is a lineage II or dual-host affiliated insect-specific flavivirus previously demonstrated as replication-deficient in vertebrate cells. Previous studies have shown that BinJV is tolerant to exchanging its structural proteins (prM-E) with pathogenic flaviviruses, making it a safe backbone for flavivirus vaccines. Here, we report generation by circular polymerase extension reaction of BinJV expressing zsGreen or mCherry fluorescent protein. Recovered BinJV reporter viruses grew to high titres (107-8 FFU/mL) in Aedes albopictus C6/36 cells assayed using immunoplaque assays (iPA). We also demonstrate that BinJV reporters could be semi-quantified live in vitro using a fluorescence microplate reader with an observed linear correlation between quantified fluorescence of BinJV reporter virus-infected C6/36 cells and iPA-quantitated virus titres. The utility of the BinJV reporter viruses was then examined in homologous and heterologous superinfection exclusion assays. We demonstrate that primary infection of C6/36 cells with BinJVzsGreen completely inhibits a secondary infection with homologous BinJVmCherry or heterologous ZIKVmCherry using fluorescence microscopy and virus quantitation by iPA. Finally, BinJVzsGreen infections were examined in vivo by microinjection of Aedes aegypti with BinJVzsGreen. At seven days post-infection, a strong fluorescence in the vicinity of salivary glands was detected in frozen sections. This is the first report on the construction of reporter viruses for lineage II insect-specific flaviviruses and establishes a tractable system for exploring flavivirus superinfection exclusion in vitro and in vivo.

Novel viral RNA genomes of the vine mealybug Planococcus ficus.

The vine mealybug, Planococcus ficus (Signoret, 1875), is the most important insect pest in growing areas of the grapevine Vitis vinifera L. in several countries, including Mexico. In Mexico, Baja California (B.C.) is the region with the highest production of V. vinifera L. grapes for industrial purposes. Recently, the diversity of viruses infecting insects only (insect-specific viruses) has been broadly explored to elucidate further ecological viral-host interactions in many insect species, which in some cases has resulted in the application of virus-based biological control agents for insect pests. However, a survey of the Pl. ficus virome has not been done yet. In the present study, we pooled Pl. ficus individuals collected through different vineyards of Ensenada, B.C., Mexico and analysed them by meta-transcriptomics. Novel nearly complete genomes of five RNA viruses were retrieved. These viruses were related to the Iflaviridae and Reoviridae families, and to the Picornavirales and Tolivirales orders. A new isolate belonging to the Dicistroviridae family was also found. Phylogenetic analyses showed that these putative viral genomes group with viruses having hemipteran (including a mealybug species) or other insect hosts, or with viruses associated with insects. Our results suggest that the identified novel RNA viruses could be insect-specific viruses of Pl. ficus. This work is the first insight into the Pl. ficus virome; it guarantees further studies aimed to characterize those viruses with potential for application in biological control of this economically important insect.

Evolution and diversity of inherited viruses in the Nearctic phantom midge, Chaoborus americanus.

Inherited mutualists, parasites, and commensals occupy one of the most intimate ecological niches available to invertebrate-associated microbes. How this transmission environment influences microbial evolution is increasingly understood for inherited bacterial symbionts, but in viruses, research on the prevalence of vertical transmission and its effects on viral lineages is still maturing. The evolutionary stability of this strategy remains difficult to assess, although phylogenetic evidence of frequent host shifts and selective sweeps have been interpreted as strategies favoring parasite persistence. In this study, we describe and investigate a natural insect system in which species-wide sweeps have been restricted by the isolation of host populations. Previous work identified evidence of pronounced mitochondrial genetic structure among North American populations of the phantom midge, Chaoborus americanus. Here we take advantage of the geographical isolation in this species to investigate the diversity and persistence of its inherited virome. We identify eight novel RNA viruses from six families and use small RNA sequencing in reproductive tissues to provide evidence of vertical transmission. We report region-specific virus strains that mirror the continental phylogeography of the host, demonstrating that members of the inherited virome have independently persisted in parallel host lineages since they last shared a common ancestor in the Mid-Pleistocene. We find that the small interfering RNA pathway, a frontline of antiviral defense in insects, targets members of this inherited virome. Finally, our results suggest that the Piwi-mediated RNA silencing pathway is unlikely to function as a general antiviral defense in Chaoborus, in contrast to its role in some mosquitoes. However, we also report that this pathway generates abundant piRNAs from endogenous viral elements closely related to actively infecting inherited viruses, potentially helping to explain idiosyncratic patterns of virus-specific Piwi targeting in this insect.

Analysis of Citrus Tristeza Virus Incidences within Asian Citrus Psyllid (Diaphorina citri) Populations in Florida via High-Throughput Sequencing.

The destructive citrus disease, Huanglongbing (HLB) or citrus greening, continues to devastate Florida's citrus industry. A hemipteran insect, the Asian citrus psyllid (ACP), disperses Candidatus Liberibacter asiaticus, one of the putative bacterial pathogens of HLB. This study builds upon ongoing research utilizing high-throughput sequencing to analyze the virome of ACP populations collected from citrus groves throughout Florida. Following the widespread detection of sequences aligning to the genome of citrus tristeza virus (CTV) across consecutive years in the Florida ACP virome, we continued to detect a pervasive amount of CTV in Florida ACPs during subsequent years. Simultaneously, we also detected mixed infections of CTV strains in pooled ACPs from different Florida regions. Predating the HLB epidemic, CTV has been present in Florida for many years and our results confirm its widespread and diverse persistence in Florida citrus groves through a unique lens, the ACP. CTV presence in the ACP likely results from feeding on CTV-infected citrus trees in Florida citrus groves, which may help to understand an overlapping presence of CTV and HLB, both endemic citrus pathosystems in the state, and their role in future integrated pest management strategies.

Negeviruses isolated from mosquitoes in the Brazilian Amazon.

BACKGROUND: There are several groups of viruses including Insect Specific Viruses (ISV) such as the taxon Negevirus, a group of viruses phylogenetically related to plant viruses. Negeviruses replicate in mosquito cells, but not in vertebrate cells. METHODS: Pools of hematophagous arthropods were inoculated in Vero and C6/36 cells. The cells were observed to detect possible cytopathic effect. Then, indirect immunofluorescence, RT-PCR, and nucleotide sequencing were performed. RESULTS: Seven samples which presented negative results for flaviviruses, alphaviruses and bunyaviruses, but showed cytopathic effect in C6/36 cells were sequenced. We identified the occurrence of a variety of ISVs, most of them belonging to the taxon Negevirus: The Brejeira, Negev, Cordoba and Wallerfield viruses, including a new virus for science, tentatively named Feitosa virus. CONCLUSIONS: We detected negeviruses in the Amazon region, including two viruses that were isolated for the first time in Brazil: Cordoba virus and the Negev virus and, a new virus for science: the Feitosa virus.

Identification of potential new mosquito-associated viruses of adult Aedes aegypti mosquitoes from Tocantins state, Brazil.

Medically important arboviruses such as dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) are primarily transmitted by the globally distributed mosquito Aedes aegypti. Increasing evidence suggests that the transmission of some viruses can be influenced by mosquito-specific and mosquito-borne viruses. Advancements in high-throughput sequencing (HTS) and bioinformatics have expanded our knowledge on the richness of viruses harbored by mosquitoes. HTS was used to characterize the presence of virus sequences in wild-caught adult Ae. aegypti from Tocantins (TO) state, Brazil. Samples of mosquitoes were collected in four cities of Tocantins state and submitted to RNA isolation, followed by sequencing at an Illumina HiSeq platform. Our results showed initially by Krona the presence of 3% of the sequenced reads belonging to the viral database. After further analysis, the virus sequences were found to have homology to two viral families found in insects Phenuiviridae and Metaviridae. Three possible viral strains including putative new viruses were detected and named Phasi Charoen-like phasivirus isolate To-1 (PCLV To-1), Aedes aegypti To virus 1 (AAToV1), and Aedes aegypti To virus 2 (AAToV2). The results presented in this work contribute to the growing knowledge about the diversity of viruses in mosquitoes and might be useful for future studies on the interaction between insect-specific viruses and arboviruses.