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.

Exploring viral infections in honey bee colonies: insights from a metagenomic study in southern Brazil.

The decline in honey bee colonies in different parts of the world in recent years is due to different reasons, such as agricultural practices, climate changes, the use of chemical insecticides, and pests and diseases. Viral infections are one of the main causes leading to honey bee population declines, which have a major economic impact due to honey production and pollination. To investigate the presence of viruses in bees in southern Brazil, we used a metagenomic approach to sequence adults' samples of concentrated extracts from Apis mellifera collected in fifteen apiaries of six municipalities in the Rio Grande do Sul state, Brazil, between 2016 and 2017. High-throughput sequencing (HTS) of these samples resulted in the identification of eight previously known viruses (Apis rhabdovirus 1 (ARV-1), Acute bee paralysis virus (ABPV), Aphid lethal paralysis virus (ALPV), Black queen cell virus (BQCV), Bee Macula-like virus (BeeMLV), Deformed wing virus (DWV), Lake Sinai Virus NE (LSV), and Varroa destructor virus 3 (VDV-3)) and a thogotovirus isolate. This thogotovirus shares high amino acid identities in five of the six segments with Varroa orthomyxovirus 1, VOV-1 (98.36 to 99.34% identity). In contrast, segment 4, which codes for the main glycoprotein (GP), has no identity with VOV-1, as observed for the other segments, but shares an amino acid identity of 34-38% with other glycoproteins of viruses from the Orthomyxoviridae family. In addition, the putative thogotovirus GP also shows amino acid identities ranging from 33 to 41% with the major glycoprotein (GP64) of insect viruses of the Baculoviridae family. To our knowledge, this is the second report of a thogotovirus found in bees and given this information, this thogotovirus isolate was tentatively named Apis thogotovirus 1 (ATHOV-1). The detection of multiple viruses in bees is important to better understand the complex interactions between viruses and their hosts. By understanding these interactions, better strategies for managing viral infections in bees and protecting their populations can be developed.

Virome Analysis Reveals Diverse and Divergent RNA Viruses in Wild Insect Pollinators in Beijing, China.

Insect pollinators provide major pollination services for wild plants and crops. Honeybee viruses can cause serious damage to honeybee colonies. However, viruses of other wild pollinating insects have yet to be fully explored. In the present study, we used RNA sequencing to investigate the viral diversity of 50 species of wild pollinating insects. A total of 3 pathogenic honeybee viruses, 8 previously reported viruses, and 26 novel viruses were identified in sequenced samples. Among these, 7 novel viruses were shown to be closely related to honeybee pathogenic viruses, and 4 were determined to have potential pathogenicity for their hosts. The viruses detected in wild insect pollinators were mainly from the order Picornavirales and the families Orthomyxoviridae, Sinhaliviridae, Rhabdoviridae, and Flaviviridae. Our study expanded the species range of known insect pollinator viruses, contributing to future efforts to protect economic honeybees and wild pollinating insects.

Enoxacin Shows Broad-Spectrum Antiviral Activity against Diverse Viruses by Enhancing Antiviral RNA Interference in Insects.

RNA interference (RNAi) functions as the major host antiviral defense in insects, while less is understood about how to utilize antiviral RNAi in controlling viral infection in insects. Enoxacin belongs to the family of synthetic antibacterial compounds based on a fluoroquinolone skeleton that has been previously found to enhance RNAi in mammalian cells. In this study, we show that enoxacin efficiently inhibited viral replication of Drosophila C virus (DCV) and cricket paralysis virus (CrPV) in cultured Drosophila cells. Enoxacin promoted the loading of Dicer-2-processed virus-derived small interfering RNA (siRNA) into the RNA-induced silencing complex, thereby enhancing the antiviral RNAi response in infected cells. Moreover, enoxacin treatment elicited RNAi-dependent in vivo protective efficacy against DCV or CrPV challenge in adult fruit flies. In addition, enoxacin also inhibited the replication of flaviviruses, including dengue virus and Zika virus, in Aedes mosquito cells in an RNAi-dependent manner. Together, our findings demonstrate that enoxacin can enhance RNAi in insects, and enhancing RNAi by enoxacin is an effective antiviral strategy against diverse viruses in insects, which may be exploited as a broad-spectrum antiviral agent to control the vector transmission of arboviruses or viral diseases in insect farming. IMPORTANCE RNAi has been widely recognized as one of the most broadly acting and robust antiviral mechanisms in insects. However, the application of antiviral RNAi in controlling viral infections in insects is less understood. Enoxacin is a fluoroquinolone compound that was previously found to enhance RNAi in mammalian cells, while its RNAi-enhancing activity has not been assessed in insects. Here, we show that enoxacin treatment inhibited viral replication of DCV and CrPV in Drosophila cells and adult fruit flies. Enoxacin promoted the loading of Dicer-generated virus-derived siRNA into the Ago2-incorporated RNA-induced silencing complex and in turn strengthened the antiviral RNAi response in the infected cells. Moreover, enoxacin displayed effective RNAi-dependent antiviral effects against flaviviruses, such as dengue virus and Zika virus, in mosquito cells. This study is the first to demonstrate that enhancing RNAi by enoxacin elicits potent antiviral effects against diverse viruses in insects.

Characterization and Tissue Tropism of Newly Identified Iflavirus and Negeviruses in Glossina morsitans morsitans Tsetse Flies.

Tsetse flies cause major health and economic problems as they transmit trypanosomes causing sleeping sickness in humans (Human African Trypanosomosis, HAT) and nagana in animals (African Animal Trypanosomosis, AAT). A solution to control the spread of these flies and their associated diseases is the implementation of the Sterile Insect Technique (SIT). For successful application of SIT, it is important to establish and maintain healthy insect colonies and produce flies with competitive fitness. However, mass production of tsetse is threatened by covert virus infections, such as the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV). This virus infection can switch from a covert asymptomatic to an overt symptomatic state and cause the collapse of an entire fly colony. Although the effects of GpSGHV infections can be mitigated, the presence of other covert viruses threaten tsetse mass production. Here we demonstrated the presence of two single-stranded RNA viruses isolated from Glossina morsitans morsitans originating from a colony at the Seibersdorf rearing facility. The genome organization and the phylogenetic analysis based on the RNA-dependent RNA polymerase (RdRp) revealed that the two viruses belong to the genera Iflavirus and Negevirus, respectively. The names proposed for the two viruses are Glossina morsitans morsitans iflavirus (GmmIV) and Glossina morsitans morsitans negevirus (GmmNegeV). The GmmIV genome is 9685 nucleotides long with a poly(A) tail and encodes a single polyprotein processed into structural and non-structural viral proteins. The GmmNegeV genome consists of 8140 nucleotides and contains two major overlapping open reading frames (ORF1 and ORF2). ORF1 encodes the largest protein which includes a methyltransferase domain, a ribosomal RNA methyltransferase domain, a helicase domain and a RdRp domain. In this study, a selective RT-qPCR assay to detect the presence of the negative RNA strand for both GmmIV and GmmNegeV viruses proved that both viruses replicate in G. m. morsitans. We analyzed the tissue tropism of these viruses in G. m. morsitans by RNA-FISH to decipher their mode of transmission. Our results demonstrate that both viruses can be found not only in the host's brain and fat bodies but also in their reproductive organs, and in milk and salivary glands. These findings suggest a potential horizontal viral transmission during feeding and/or a vertically viral transmission from parent to offspring. Although the impact of GmmIV and GmmNegeV in tsetse rearing facilities is still unknown, none of the currently infected tsetse species show any signs of disease from these viruses.

Small RNA Response to Infection of the Insect-Specific Lammi Virus and Hanko Virus in an Aedes albopictus Cell Line.

RNA interference (RNAi)-mediated antiviral immunity is believed to be the primary defense against viral infection in mosquitoes. The production of virus-specific small RNA has been demonstrated in mosquitoes and mosquito-derived cell lines for viruses in all of the major arbovirus families. However, many if not all mosquitoes are infected with a group of viruses known as insect-specific viruses (ISVs), and little is known about the mosquito immune response to this group of viruses. Therefore, in this study, we sequenced small RNA from an Aedes albopictus-derived cell line infected with either Lammi virus (LamV) or Hanko virus (HakV). These viruses belong to two distinct phylogenetic groups of insect-specific flaviviruses (ISFVs). The results revealed that both viruses elicited a strong virus-derived small interfering RNA (vsiRNA) response that increased over time and that targeted the whole viral genome, with a few predominant hotspots observed. Furthermore, only the LamV-infected cells produced virus-derived Piwi-like RNAs (vpiRNAs); however, they were mainly derived from the antisense genome and did not show the typical ping-pong signatures. HakV, which is more distantly related to the dual-host flaviviruses than LamV, may lack certain unknown sequence elements or structures required for vpiRNA production. Our findings increase the understanding of mosquito innate immunity and ISFVs' effects on their host.

Viruses of the Fall Armyworm Spodoptera frugiperda: A Review with Prospects for Biological Control.

The fall armyworm (FAW), Spodoptera frugiperda, is a native pest species in the Western hemisphere. Since it was first reported in Africa in 2016, FAW has spread throughout the African continent and is now also present in several countries in Asia as well as Australia. The invasion of FAW in these areas has led to a high yield reduction in crops, leading to huge economic losses. FAW management options in the newly invaded areas are limited and mainly rely on the use of synthetic pesticides. Since there is a risk of resistance development against pesticides in addition to the negative environmental and human health impacts, other effective, sustainable, and cost-efficient control alternatives are desired. Insect pathogenic viruses fulfil these criteria as they are usually effective and highly host-specific with no significant harmful effect on beneficial insects and non-target organisms. In this review, we discuss all viruses known from FAW and their potential to be used for biological control. We specifically focus on baculoviruses and describe the recent advancements in the use of baculoviruses for biological control in the native geographic origin of FAW, and their potential use in the newly invaded areas. Finally, we identify current knowledge gaps and suggest new avenues for productive research on the use of viruses as a biopesticide against FAW.

A Systematic Review on Viruses in Mass-Reared Edible Insect Species.

Edible insects are expected to become an important nutrient source for animals and humans in the Western world in the near future. Only a few studies on viruses in edible insects with potential for industrial rearing have been published and concern only some edible insect species. Viral pathogens that can infect insects could be non-pathogenic, or pathogenic to the insects themselves, or to humans and animals. The objective of this systematic review is to provide an overview of the viruses detected in edible insects currently considered for use in food and/or feed in the European Union or appropriate for mass rearing, and to collect information on clinical symptoms in insects and on the vector role of insects themselves. Many different virus species have been detected in edible insect species showing promise for mass production systems. These viruses could be a risk for mass insect rearing systems causing acute high mortality, a drastic decline in growth in juvenile stages and in the reproductive performance of adults. Furthermore, some viruses could pose a risk to human and animal health where insects are used for food and feed.

A Diverse Viral Community from Predatory Wasps in Their Native and Invaded Range, with a New Virus Infectious to Honey Bees.

Wasps of the genus Vespula are social insects that have become major pests and predators in their introduced range. Viruses present in these wasps have been studied in the context of spillover from honey bees, yet we lack an understanding of the endogenous virome of wasps as potential reservoirs of novel emerging infectious diseases. We describe the characterization of 68 novel and nine previously identified virus sequences found in transcriptomes of Vespula vulgaris in colonies sampled from their native range (Belgium) and an invasive range (New Zealand). Many viruses present in the samples were from the Picorna-like virus family (38%). We identified one Luteo-like virus, Vespula vulgaris Luteo-like virus 1, present in the three life stages examined in all colonies from both locations, suggesting this virus is a highly prevalent and persistent infection in wasp colonies. Additionally, we identified a novel Iflavirus with similarity to a recently identified Moku virus, a known wasp and honey bee pathogen. Experimental infection of honey bees with this novel Vespula vulgaris Moku-like virus resulted in an active infection. The high viral diversity present in these invasive wasps is a likely indication that their polyphagous diet is a rich source of viral infections.

Patterns in Genotype Composition of Indian Isolates of the Bombyx mori Nucleopolyhedrovirus and Bombyx mori Bidensovirus.

The mulberry silkworm, Bombyx mori (L.), is a model organism of lepidopteran insects with high economic importance. The viral diseases of the silkworm caused by Bombyx mori nucleopolyhedrovirus (BmNPV) and Bombyx mori bidensovirus (BmBDV) inflict huge economic losses and significantly impact the sericulture industry of India and other countries. To understand the distribution of Indian isolates of the BmNPV and to investigate their genetic composition, an in-depth population structure analysis was conducted using comprehensive and newly developed genomic analysis methods. The seven new Indian BmNPV isolates from Anantapur, Dehradun, Ghumarwin, Jammu, Kashmir, Mysore and Salem grouped in the BmNPV clade, and are most closely related to Autographa californica multiple nucleopolyhedrovirus and Rachiplusia ou multiple nucleopolyhedrovirus on the basis of gene sequencing and phylogenetic analyses of the partial polh, lef-8 and lef-9 gene fragments. The whole genome sequencing of three Indian BmNPV isolates from Mysore (-My), Jammu (-Ja) and Dehradun (-De) was conducted, and intra-isolate genetic variability was analyzed on the basis of variable SNP positions and the frequencies of alternative nucleotides. The results revealed that the BmNPV-De and BmNPV-Ja isolates are highly similar in their genotypic composition, whereas the population structure of BmNPV-My appeared rather pure and homogenous, with almost no or few genetic variations. The BmNPV-De and BmNPV-Ja samples further contained a significant amount of BmBDV belonging to the Bidnaviridae family. We elucidated the genotype composition within Indian BmNPV and BmBDV isolates, and the results presented have broad implications for our understanding of the genetic diversity and evolution of BmNPV and co-occurring BmBDV isolates.

The first complete genome sequence of an iflavirus from the endoparasitoid wasp Tetrastichus brontispae.

The complete genome sequence of a novel iflavirus isolated from the gregarious and koinobiont endoparasitoid Tetrastichus brontispae, tentatively named "Tetrastichus brontispae RNA virus 3" (TbRV-3), was determined by total RNA and Sanger sequencing. The complete genome is 9998 nucleotides in length, 8934 nt of which encodes a putative polyprotein of 2978 amino acids. TbRV-3 was found to have a similar genome organization and to contain conserved domains and motifs found in other iflaviruses, with some variations. Phylogenetic analysis based on deduced amino acid sequences of the RdRp domain showed that TbRV-3 clustered with Dinocampus coccinellae paralysis virus (DcPV). However, the percent amino acid sequence identity of the putative capsid proteins of TbRV-3 and DcPV determined using BLASTp was below the species demarcation threshold (90%), suggesting that TbRV-3 is a new iflavirus. This is the first virus of the family Iflaviridae to be isolated from a wasp of the family Eulophidae.

A survey of mosquito-borne and insect-specific viruses in hospitals and livestock markets in western Kenya.

Aedes aegypti and Culex pipiens complex mosquitoes are prolific vectors of arboviruses that are a global threat to human and animal health. Increased globalization and ease of travel have facilitated the worldwide dissemination of these mosquitoes and the viruses they transmit. To assess disease risk, we determined the frequency of arboviruses in western Kenyan counties bordering an area of high arboviral activity. In addition to pathogenic viruses, insect-specific flaviviruses (ISFs), some of which are thought to impair the transmission of specific pathogenic arboviruses, were also evaluated. We trapped mosquitoes in the short and long rainy seasons in 2018 and 2019 at livestock markets and hospitals. Mosquitoes were screened for dengue, chikungunya and other human pathogenic arboviruses, ISFs, and their blood-meal sources as determined by high-resolution melting analysis of (RT-)PCR products. Of 6,848 mosquitoes collected, 89% were trapped during the long rainy season, with A. aegypti (59%) and Cx. pipiens sensu lato (40%) being the most abundant. Most blood-fed mosquitoes were Cx. pipiens s.l. with blood-meals from humans, chicken, and sparrow (Passer sp.). We did not detect dengue or chikungunya viruses. However, one Culex poicilipes female was positive for Sindbis virus, 30 pools of Ae. aegypti had cell fusing agent virus (CFAV; infection rate (IR) = 1.27%, 95% CI = 0.87%-1.78%); 11 pools of Ae. aegypti had Aedes flavivirus (AeFV; IR = 0.43%, 95% CI = 0.23%-0.74%); and seven pools of Cx. pipiens s.l. (IR = 0.23%, 95% CI = 0.1%-0.45%) and one pool of Culex annulioris had Culex flavivirus. Sindbis virus, which causes febrile illness in humans, can complicate the diagnosis and prognosis of patients with fever. The presence of Sindbis virus in a single mosquito from a population of mosquitoes with ISFs calls for further investigation into the role ISFs may play in blocking transmission of other arboviruses in this region.

The virome of vector mosquitoes.

Mosquitoes are the major vectors for arthropod-borne viruses (arboviruses) of medical importance. Aedes aegypti and A. albopictus are the most prolific and widespread mosquito vectors being responsible for global transmission of dengue, Zika and Chikungunya viruses. Characterizing the collection of viruses circulating in mosquitoes, the virome, has long been of special interest. In addition to arboviruses, mosquitoes carry insect-specific viruses (ISVs) that do not directly infect vertebrates. Mounting evidence indicates that ISVs interact with arboviruses and may affect mosquito vector competence. Here, we review our current knowledge about the virome of vector mosquitoes and discuss the challenges for the field that may lead to novel strategies to prevent outbreaks of arboviruses.

Structural diversity and phylogenetic distribution of valyl tRNA-like structures in viruses.

Viruses commonly use specifically folded RNA elements that interact with both host and viral proteins to perform functions important for diverse viral processes. Examples are found at the 3' termini of certain positive-sense ssRNA virus genomes where they partially mimic tRNAs, including being aminoacylated by host cell enzymes. Valine-accepting tRNA-like structures (TLSVal) are an example that share some clear homology with canonical tRNAs but have several important structural differences. Although many examples of TLSVal have been identified, we lacked a full understanding of their structural diversity and phylogenetic distribution. To address this, we undertook an in-depth bioinformatic and biochemical investigation of these RNAs, guided by recent high-resolution structures of a TLSVal We cataloged many new examples in plant-infecting viruses but also in unrelated insect-specific viruses. Using biochemical and structural approaches, we verified the secondary structure of representative TLSVal substrates and tested their ability to be valylated, confirming previous observations of structural heterogeneity within this class. In a few cases, large stem-loop structures are inserted within variable regions located in an area of the TLS distal to known host cell factor binding sites. In addition, we identified one virus whose TLS has switched its anticodon away from valine, causing a loss of valylation activity; the implications of this remain unclear. These results refine our understanding of the structural and functional mechanistic details of tRNA mimicry and how this may be used in viral infection.

Determination of Genetically Identical Strains of Four Honeybee Viruses in Bumblebee Positive Samples.

In recent years, there has been growing evidence that certain types of honeybee viruses could be transmitted between different pollinators. Within a voluntary monitoring programme, 180 honeybee samples (Apis mellifera carnica) were collected from affected apiaries between 2007 and 2018. Also from August 2017 to August 2018, a total 148 samples of healthy bumblebees (Bombus lapidarius, B. pascuorum, B. terrestris, B. lucorum, B. hortorum, B. sylvarum, B. humilis) were collected at four different locations in Slovenia, and all samples were tested by using RT-PCR methods for six honeybee viruses. Direct sequencing of a total 158 positive samples (acute bee paralysis virus (ABPV n = 33), black queen cell virus (BQCV n = 75), sacbrood bee virus (SBV n = 25) and Lake Sinai virus (LSV n = 25)) was performed from obtained RT-PCR products. The genetic comparison of identified positive samples of bumblebees and detected honeybee field strains of ABPV, BQCV, SBV, and LSV demonstrated 98.74% to 100% nucleotide identity between both species. This study not only provides evidence that honeybees and bumblebees are infected with genetically identical or closely related viral strains of four endemically present honeybee viruses but also detected a high diversity of circulating strains in bumblebees, similar as was observed among honeybees. Important new genetic data for endemic strains circulating in honeybees and bumblebees in Slovenia are presented.

ICTV Virus Taxonomy Profile: Metaviridae.

Metaviridae is a family of retrotransposons and reverse-transcribing viruses with long terminal repeats belonging to the order Ortervirales. Members of the genera Errantivirus and Metavirus include, respectively, Saccharomyces cerevisiae Ty3 virus and its Gypsy-like relatives in drosophilids. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Metaviridae, which is available at ictv.global/report/metaviridae.

Understanding the Mechanisms Underlying Host Restriction of Insect-Specific Viruses.

Arthropod-borne viruses contribute significantly to global mortality and morbidity in humans and animals. These viruses are mainly transmitted between susceptible vertebrate hosts by hematophagous arthropod vectors, especially mosquitoes. Recently, there has been substantial attention for a novel group of viruses, referred to as insect-specific viruses (ISVs) which are exclusively maintained in mosquito populations. Recent discoveries of novel insect-specific viruses over the past years generated a great interest not only in their potential use as vaccine and diagnostic platforms but also as novel biological control agents due to their ability to modulate arbovirus transmission. While arboviruses infect both vertebrate and invertebrate hosts, the replication of insect-specific viruses is restricted in vertebrates at multiple stages of virus replication. The vertebrate restriction factors include the genetic elements of ISVs (structural and non-structural genes and the untranslated terminal regions), vertebrate host factors (agonists and antagonists), and the temperature-dependent microenvironment. A better understanding of these bottlenecks is thus warranted. In this review, we explore these factors and the complex interplay between ISVs and their hosts contributing to this host restriction phenomenon.

Insect-specific viruses and arboviruses in adult male culicids from Midwestern Brazil.

Viruses were identified from male anthropophilic mosquitoes from Mato Grosso (MT) State, Midwest Brazil from February 2017 to January 2018. Mosquitoes tested included Aedes (Stegomyia) aegypti (1139 males; 84 pools), Culex quinquefasciatus (9426 males; 179 pools), Culex sp. (3 males; 3 pools) and Psorophora albigenu (1 male; 1 pool) collected from four cities of MT. Pools were subjected to viral RNA extraction followed by RT-PCRs specific for ten flaviviruses, five alphaviruses and Simbu serogroup of orthobunyaviruses. Positive pools were passaged three times in VERO cells (alphavirus and orthobunyavirus) or C6/36 cells (flavivirus), with isolates confirmed through RT-PCR and nucleotide sequencing. We detected pools positive for Ilhéus (1 pool), dengue serotype 4 (1), Mayaro (12), equine encephalitis virus (1) yellow fever (1), Oropouche (2), Zika (4) and chikungunya (12) viruses. High throughput sequencing of arbovirus positive pools identified 35 insect-specific viruses (ISVs) from the families Circoviridae (2), Parvoviridae (2), Totiviridae (1), Flaviviridae (1), Iflaviridae (2), Mesoniviridae (4), Nodaviridae (2), Luteoviridae (1), Phasmaviridae (1) Phenuiviridae (2), Rhabdoviridae (2), Orthomyxoviridae (1), Xinmoviridae (1), and unclassified Bunyavirales (1), unclassified Picornavirales (3), unclassified Riboviria (4) and taxon Negevirus (5). From these, five novel viruses were tentatively named Mojica circovirus, Kuia iflavirus, Muxirum negevirus, Lambada picorna-like virus and Tacuru picorna-like virus. Our findings underscore the diversity and wide geographical distribution of ISVs and arboviruses infecting male culicids.

A novel negevirus isolated from Aedes vexans mosquitoes in Finland.

Negeviruses are insect-specific enveloped RNA viruses that have been detected in mosquitoes and sandflies from various geographical locations. Here, we describe a new negevirus from Northern Europe, isolated from pool of Aedes vexans mosquitoes collected in Finland, designated as Mekrijärvi negevirus (MEJNV). MEJNV had a typical negevirus genome organization, is 9,740 nucleotides in length, and has a GC content of 47.53%. The MEJNV genome contains three ORFs, each containing the following identified conserved domains: ORF1 (7,068 nt) encodes a viral methyltransferase, an FtsJ-like methyltransferase, a viral RNA helicase, and an RNA-dependent RNA polymerase, ORF2 (1,242 nt) encodes a putative virion glycoprotein, and ORF3 (660 nt) encodes a putative virion membrane protein. A distinctive feature relative to other currently known negeviruses is a 7-nucleotide-long overlap between ORF1 and ORF2. MEJNV shares the highest sequence identity with Ying Kou virus from China, with 67.71% nucleotide and 75.19% and 59.00% amino acid sequence identity in ORF 1 and ORF 2, respectively. ORF3 had the highest amino acid sequence similarity to Daeseongdong virus 1 and negevirus Nona 1, both with 77.61% identity, and to Ying Kou virus, with 71.22% identity. MEJNV is currently the northernmost negevirus described. Our report supports the view that negeviruses are a globally distributed, diverse group of viruses that can be found from mosquitoes in a wide range of terrestrial biomes from tropical to boreal forests.

Metagenomic Analysis of the Virome of Mosquito Excreta.

Traditional screening for arboviruses in mosquitoes requires a priori knowledge and the utilization of appropriate assays for their detection. Mosquitoes can also provide other valuable information, including unexpected or novel arboviruses, nonarboviral pathogens ingested from hosts they feed on, and their own genetic material. Metagenomic analysis using next-generation sequencing (NGS) is a rapidly advancing technology that allows us to potentially obtain all this information from a mosquito sample without any prior knowledge of virus, host, or vector. Moreover, it has been recently demonstrated that pathogens, including arboviruses and parasites, can be detected in mosquito excreta by molecular methods. In this study, we investigated whether RNA viruses could be detected in mosquito excreta by NGS. Excreta samples were collected from Aedes vigilax and Culex annulirostris experimentally exposed to either Ross River or West Nile viruses and from field mosquitoes collected across Queensland, Australia. Total RNA was extracted from the excreta samples, reverse transcribed to cDNA, and sequenced using the Illumina NextSeq 500 platform. Bioinformatic analyses from the generated reads demonstrate that mosquito excreta provide sufficient RNA for NGS, allowing the assembly of near-full-length viral genomes. We detected Australian Anopheles totivirus, Wuhan insect virus 33, and Hubei odonate virus 5 and identified seven potentially novel viruses closely related to members of the order Picornavirales (2/7) and to previously described, but unclassified, RNA viruses (5/7). Our results suggest that metagenomic analysis of mosquito excreta has great potential for virus discovery and for unbiased arbovirus surveillance in the near future.IMPORTANCE When a mosquito feeds on a host, it ingests not only its blood meal but also an assortment of microorganisms that are present in the blood, thus acting as an environmental sampler. By using specific tests, it is possible to detect arthropod-borne viruses (arboviruses) like dengue and West Nile viruses in mosquito excreta. Here, we explored the use of next-generation sequencing (NGS) for unbiased detection of RNA viruses present in excreta from experimentally infected and field-collected mosquitoes. We have demonstrated that mosquito excreta provide a suitable template for NGS and that it is possible to recover and assemble near-full-length genomes of both arboviruses and insect-borne viruses, including potentially novel ones. These results importantly show the direct practicality of the use of mosquito excreta for NGS, which in the future could be used for virus discovery, environmental virome sampling, and arbovirus surveillance.