Identification and Genome Characterization of a Novel Nege-like Virus Isolated from Aphids (Aphis gossypii) in Yunnan Province.

Negeviruses are insect-specific enveloped RNA viruses that exhibit a wide geographic distribution. A novel nege-like virus, tentatively named Aphis gossypii nege-like virus (AGNLV, GenBank: OR880429.1), was isolated from aphids (Aphis gossypii) in Lijiang City, Yunnan, China. AGNLV has a genome sequence of 9258 nt (excluding the polyA tail) encoding three open reading frames (ORFs). ORF1 (7149 nt) encodes a viral methyltransferase, a viral RNA helicase, and an RNA-dependent RNA polymerase. ORF2 (1422 nt) encodes a DiSB-ORF2_chro domain and ORF3 encodes an SP24 domain. The genome sequence of AGNLV shares the highest nucleotide identity of 60.0% and 59.5% with Wuhan house centipede virus 1 (WHCV1) and Astegopteryx formosana nege-like virus (AFNLV), respectively. Phylogenetic analysis based on the RNA-dependent RNA polymerase shows that AGNLV is clustered with other negeviruses and nege-like viruses discovered in aphids, forming a distinct "unclassified clade". Interestingly, AGNLV only encodes three ORFs, whereas AFNLV and WHCV1 have four ORFs. Structure and transmembrane domain predictions show the presence of eight alpha helices and five transmembrane helices in the AGNLV ORF3. Translational enhancement of the AGNLV 5' UTR was similar to that of the 5' UTR of plant viruses. Our findings provide evidence of the diversity and structure of nege-like viruses and are the first record of such a virus from a member of the genus Aphis.

Known and novel viruses in Belgian honey bees: yearly differences, spatial clustering, and associations with overwintering loss.

In recent years, managed honey bee colonies have been suffering from an increasing number of biotic and abiotic stressors, resulting in numerous losses of colonies worldwide. A pan-European study, EPILOBEE, estimated the colony loss in Belgium to be 32.4% in 2012 and 14.8% in 2013. In the current study, absolute viral loads of four known honey bee viruses (DWV-A, DWV-B, AmFV, and BMLV) and three novel putative honey bee viruses (Apis orthomyxovirus 1, apthili virus, and apparli virus) were determined in 300 Flemish honey bee samples, and associations with winter survival were determined. This revealed that, in addition to the known influence of DWV-A and DWV-B on colony health, one of the newly described viruses (apthili virus) shows a strong yearly difference and is also associated with winter survival. Furthermore, all scrutinized viruses revealed significant spatial clustering patterns, implying that despite the limited surface area of Flanders, local virus transmission is paramount. The vast majority of samples were positive for at least one of the seven investigated viruses, and up to 20% of samples were positive for at least one of the three novel viruses. One of those three, Apis orthomyxovirus 1, was shown to be a genuine honey bee-infecting virus, able to infect all developmental stages of the honey bee, as well as the Varroa destructor mite. These results shed light on the most prevalent viruses in Belgium and their roles in the winter survival of honey bee colonies. IMPORTANCE: The western honey bee (Apis mellifera) is a highly effective pollinator of flowering plants, including many crops, which gives honey bees an outstanding importance both ecologically and economically. Alarmingly high annual loss rates of managed honey bee colonies are a growing concern for beekeepers and scientists and have prompted a significant research effort toward bee health. Several detrimental factors have been identified, such as varroa mite infestation and disease from various bacterial and viral agents, but annual differences are often not elucidated. In this study, we utilize the viral metagenomic survey of the EPILOBEE project, a European research program for bee health, to elaborate on the most abundant bee viruses of Flanders. We complement the existing metagenomic data with absolute viral loads and their spatial and temporal distributions. Furthermore, we identify Apis orthomyxovirus 1 as a potentially emerging pathogen, as we find evidence for its active replication honey bees.

Development of a multiplex real-time quantitative reverse-transcription polymerase chain reaction for the detection of four bee viruses.

Viruses in the families Dicistroviridae and Iflaviridae are among the main threats to western honey bees (Apis mellifera) and native bee species. Polymerase chain reaction (PCR) is the gold standard for pathogen detection in bees. However, high throughput screening for bee virus infections in singleplex PCR reactions is cumbersome and limited by the high quantities of sample RNA required. Thus, the development of a sensitive and specific multiplex PCR detection method for screening for multiple viruses simultaneously is necessary. Here, we report the development of a one-step multiplex reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay to detect four viruses commonly encountered in pollinator species. The optimized multiplex RT-qPCR protocol described in this study allows simultaneous detection of two dicistroviruses (Israeli acute paralysis virus and Black queen cell virus) and two iflaviruses (Sacbrood virus and Deformed wing virus) with high efficiency and specificity comparable to singleplex detection assays. This assay provides a broad range of detection and quantification, and the results of virus quantification in this study are similar to those performed in other studies using singleplex detection assays. This method will be particularly useful for data generation from small-bodied insect species that yield low amounts of RNA.

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.

Eggs sampling as an effective tool for identifying the incidence of viruses in honey bees involved in artificial queen rearing.

The Carniolan honey bee (Apis mellifera carnica) plays an essential role in crop pollination, environment diversity, and the production of honey bee products. However, the health of individual honey bees and their colonies is under pressure due to multiple stressors, including viruses as a significant threat to bees. Monitoring various virus infections could be a crucial selection tool during queen rearing. In the present study, samples from all developmental stages (eggs, larvae, pupae, and queens) were screened for the incidence of seven viruses during queen rearing in Slovenia. The screening of a total of 108 samples from five queen breeders was performed by the RT-qPCR assays. The results showed that the highest incidence was observed for black queen cell virus (BQCV), Lake Sinai virus 3 (LSV3), deformed wing virus B (DWV-B), and sacbrood virus (SBV). The highest viral load was detected in queens (6.07 log10 copies/queen) and larvae (5.50 log10 copies/larva) for BQCV, followed by SBV in larvae (5.47 log10 copies/larva). When comparing all the honey bee developmental stages, the eggs exhibited general screening for virus incidence and load in queen mother colonies. The results suggest that analyzing eggs is a good indicator of resilience to virus infection during queen development.

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.

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.

Detection of Insect-Specific Flaviviruses in Mosquitoes (Diptera: Culicidae) in Northeastern Regions of South Africa.

Mosquitoes in the Aedes and Culex genera are considered the main vectors of pathogenic flaviviruses worldwide. Entomological surveillance using universal flavivirus sets of primers in mosquitoes can detect not only pathogenic viruses but also insect-specific ones. It is hypothesized that insect-specific flaviviruses, which naturally infect these mosquitoes, may influence their vector competence for zoonotic arboviruses. Here, entomological surveillance was performed between January 2014 and May 2018 in five different provinces in the northeastern parts of South Africa, with the aim of identifying circulating flaviviruses. Mosquitoes were sampled using different carbon dioxide trap types. Overall, 64,603 adult mosquitoes were collected, which were screened by RT-PCR and sequencing. In total, 17 pools were found positive for insect-specific Flaviviruses in the mosquito genera Aedes (12/17, 70.59%) and Anopheles (5/17, 29.41%). No insect-specific viruses were detected in Culex species. Cell-fusing agent viruses were detected in Aedes aegypti and Aedes caballus. A range of anopheline mosquitoes, including Anopheles coustani, An. squamosus and An. maculipalpis, were positive for Culex flavivirus-like and Anopheles flaviviruses. These results confirm the presence of insect-specific flaviviruses in mosquito populations in South Africa, expands their geographical range and indicates potential mosquito species as vector species.

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 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.

Isolation of a novel insect-specific flavivirus with immunomodulatory effects in vertebrate systems.

We describe the isolation and characterization of a novel insect-specific flavivirus (ISFV), tentatively named Aripo virus (ARPV), that was isolated from Psorophora albipes mosquitoes collected in Trinidad. The ARPV genome was determined and phylogenetic analyses showed that it is a dual host associated ISFV, and clusters with the main mosquito-borne flaviviruses. ARPV antigen was significantly cross-reactive with Japanese encephalitis virus serogroup antisera, with significant cross-reactivity to Ilheus and West Nile virus (WNV). Results suggest that ARPV replication is limited to mosquitoes, as it did not replicate in the sandfly, culicoides or vertebrate cell lines tested. We also demonstrated that ARPV is endocytosed into vertebrate cells and is highly immunomodulatory, producing a robust innate immune response despite its inability to replicate in vertebrate systems. We show that prior infection or coinfection with ARPV limits WNV-induced disease in mouse models, likely the result of a robust ARPV-induced type I interferon response.

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.

Special Issue "Evolution and Diversity of Insect Viruses".

Insects are crucial for ecosystem functions and services and directly influence human well-being and health [...].

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.

Post-COVID-19 Action: Guarding Africa's Crops against Viral Epidemics Requires Research Capacity Building That Unifies a Trio of Transdisciplinary Interventions.

The COVID-19 pandemic has shown that understanding the genomics of a virus, diagnostics and breaking virus transmission is essential in managing viral pandemics. The same lessons can apply for plant viruses. There are plant viruses that have severely disrupted crop production in multiple countries, as recently seen with maize lethal necrosis disease in eastern and southern Africa. High-throughput sequencing (HTS) is needed to detect new viral threats. Equally important is building local capacity to develop the tools required for rapid diagnosis of plant viruses. Most plant viruses are insect-vectored, hence, biological insights on virus transmission are vital in modelling disease spread. Research in Africa in these three areas is in its infancy and disjointed. Despite intense interest, uptake of HTS by African researchers is hampered by infrastructural gaps. The use of whole-genome information to develop field-deployable diagnostics on the continent is virtually inexistent. There is fledgling research into plant-virus-vector interactions to inform modelling of viral transmission. The gains so far have been modest but encouraging, and therefore must be consolidated. For this, I propose the creation of a new Research Centre for Africa. This bold investment is needed to secure the future of Africa's crops from insect-vectored viral diseases.

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.

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.

Longitudinal monitoring of honey bee colonies reveals dynamic nature of virus abundance and indicates a negative impact of Lake Sinai virus 2 on colony health.

Honey bees (Apis mellifera) are important pollinators of plants, including those that produce nut, fruit, and vegetable crops. Therefore, high annual losses of managed honey bee colonies in the United States and many other countries threaten global agriculture. Honey bee colony deaths have been associated with multiple abiotic and biotic factors, including pathogens, but the impact of virus infections on honey bee colony population size and survival are not well understood. To further investigate seasonal patterns of pathogen presence and abundance and the impact of viruses on honey bee colony health, commercially managed colonies involved in the 2016 California almond pollination event were monitored for one year. At each sample date, colony health and pathogen burden were assessed. Data from this 50-colony cohort study illustrate the dynamic nature of honey bee colony health and the temporal patterns of virus infection. Black queen cell virus, deformed wing virus, sacbrood virus, and the Lake Sinai viruses were the most readily detected viruses in honey bee samples obtained throughout the year. Analyses of virus prevalence and abundance revealed pathogen-specific trends including the overall increase in deformed wing virus abundance from summer to fall, while the levels of Lake Sinai virus 2 (LSV2) decreased over the same time period. Though virus prevalence and abundance varied in individual colonies, analyses of the overall trends reveal correlation with sample date. Total virus abundance increased from November 2015 (post-honey harvest) to the end of the almond pollination event in March 2016, which coincides with spring increase in colony population size. Peak total virus abundance occurred in late fall (August and October 2016), which correlated with the time period when the majority of colonies died. Honey bee colonies with larger populations harbored less LSV2 than weaker colonies with smaller populations, suggesting an inverse relationship between colony health and LSV2 abundance. Together, data from this and other longitudinal studies at the colony level are forming a better understanding of the impact of viruses on honey bee colony losses.