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.

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.

Identification of long noncoding RNAs of silkworm at the early stage of Bombyx mori bidensovirus infection.

Bombyx mori bidensovirus (BmBDV) is one of the most important pathogens of silkworm. It mainly infects midgut cells of silkworm and causes losses to the sericulture industry. Long noncoding RNAs (lncRNAs) have been reported to play an important role in the regulation of antiviral immune response in silkworm. To explore whether lncRNAs are involved in BmBDV infection and immune response of silkworm, we performed a comparative transcriptome analysis to identify the lncRNAs and mRNAs between the BmBDV infected and noninfected silkworm larvae at the early stage. A total of 16,069 genes and 974 candidate lncRNAs were identified, among which 142 messenger RNA (mRNAs) and four lncRNAs were differentially expressed (DE). Target gene prediction revealed that 142 DEmRNAs were coexpressed with four DElncRNAs, suggesting that the expression of mRNA is mainly affected through trans-regulation activities. A regulatory network of DElncRNAs and DEmRNAs was constructed, showing that many genes targeted by different DElncRNAs are involved in metabolism and immunity, which implies that these genes and lncRNAs play an important role in the replication of BmBDV. Our results will help us to improve our understanding of lncRNA-mediated regulatory roles in BmBDV infection, providing a new perspective for further exploring the interaction between host and BmBDV.

Genetic diversity of RNA viruses infecting invertebrate pests of rice.

Invertebrate species are a natural reservoir of viral genetic diversity, and invertebrate pests are widely distributed in crop fields. However, information on viruses infecting invertebrate pests of crops is limited. In this report, we describe the deep metatranscriptomic sequencing of 88 invertebrate samples covering all major invertebrate pests in rice fields. We identified 296 new RNA viruses and 13 known RNA viruses. These viruses clustered within 31 families, with many highly divergent viruses constituting potentially new families and genera. Of the identified viruses, 13 RNA viruses clustered within the Fiersviridae family of bacteriophages, and 48 RNA viruses clustered within families and genera of mycoviruses. We detected known rice viruses in novel invertebrate hosts at high abundances. Furthermore, some novel RNA viruses have genome structures closely matching to known plant viruses and clustered within genera of several plant virus species. Forty-five potential insect pathogenic RNA viruses were detected in invertebrate species. Our analysis revealed that host taxonomy plays a major role and geographical location plays an important role in structuring viral diversity. Cross-species transmission of RNA viruses was detected between invertebrate hosts. Newly identified viral genomes showed extensive variation for invertebrate viral families or genera. Together, the large-scale metatranscriptomic analysis greatly expands our understanding of RNA viruses in rice invertebrate species, the results provide valuable information for developing efficient strategies to manage insect pests and virus-mediated crop diseases.

Insect-specific RNA virus affects the stylet penetration activity of brown citrus aphid (Aphis citricidus) to facilitate its transmission.

Sap-sucking insects often transmit plant viruses but also carry insect viruses, which infect insects but not plants. The impact of such insect viruses on insect host biology and ecology is largely unknown. Here, we identified a novel insect-specific virus carried by brown citrus aphid (Aphis citricidus), which we tentatively named Aphis citricidus picornavirus (AcPV). Phylogenetic analysis discovered a monophyletic cluster with AcPV and other unassigned viruses, suggesting that these viruses represent a new family in order Picornavirales. Systemic infection with AcPV triggered aphid antiviral immunity mediated by RNA interference, resulting in asymptomatic tolerance. Importantly, we found that AcPV was transmitted horizontally by secretion of the salivary gland into the feeding sites of plants. AcPV influenced aphid stylet behavior during feeding and increased the time required for intercellular penetration, thus promoting its transmission among aphids with plants as an intermediate site. The gene expression results suggested that this mechanism was linked with transcription of salivary protein genes and plant defense hormone signaling. Together, our results show that the horizontal transmission of AcPV in brown citrus aphids evolved in a manner similar to that of the circulative transmission of plant viruses by insect vectors, thus providing a new ecological perspective on the activity of insect-specific viruses found in aphids and improving the understanding of insect virus ecology.

Novel strains of Culex flavivirus and Hubei chryso-like virus 1 from the Anopheles mosquito in western Kenya.

Surveillance of mosquito vectors is critical for early detection, prevention and control of vector borne diseases. In this study we used advanced molecular tools, such as DNA barcoding in combination with novel sequencing technologies to discover new and already known viruses in genetically identified mosquito species. Mosquitoes were captured using BG sentinel traps in Western Kenya during May and July 2019, and homogenized individually before pooled into groups of ten mosquitoes. The pools and individual samples were then used for molecular analysis and to infect cell cultures. Of a total of fifty-four (54) 10-pools, thirteen (13) showed cytopathic effect (CPE) on VeroB4 cells, eighteen (18) showed CPE on C6/36 cells. Eight (8) 10-pools out of the 31 CPE positive pools showed CPE on both VeroB4 and C6/36 cells. When using reverse transcriptase polymerase chain reaction (RT-PCR), Sanger sequencing and Twist Comprehensive Viral Research Panel (CVRP) (Twist Biosciences), all pools were found negative by RT-PCR when using genus specific primers targeting alphaviruses, orthobunyaviruses and virus specific primers towards o'nyong-nyong virus, chikungunya virus and Sindbis virus (previously reported to circulate in the region). Interestingly, five pools were RT-PCR positive for flavivirus. Two of the RT-PCR positive pools showed CPE on both VeroB4 and C6/36 cells, two pools showed CPE on C6/36 cells alone and one pool on VeroB4 cells only. Fifty individual mosquito homogenates from the five RT-PCR positive 10-pools were analyzed further for flavivirus RNA. Of these, 19 out of the 50 individual mosquito homogenates indicated the presence of flavivirus RNA. Barcoding of the flavivirus positive mosquitoes revealed the mosquito species as Aedes aegypti (1), Mansonia uniformis (6), Anopheles spp (3), Culex pipiens (5), Culex spp (1), Coquilletidia metallica (2) and Culex quinquefasciatus (1). Of the 19 flavivirus positive individual mosquitoes, five (5) virus positive homogenates were sequenced. Genome sequences of two viruses were completed. One was identified as the single-stranded RNA Culex flavivirus and the other as the double-stranded RNA Hubei chryso-like virus 1. Both viruses were found in the same Anopheles spp. homogenate extracted from a sample that showed CPE on both VeroB4 and C6/36 cells. The detection of both viruses in a single mosquito homogenate indicated coinfection. Phylogenetic analyses suggested that the Culex flavivirus sequence detected was closely related to a Culex flavivirus isolated from Uganda in 2008. All four Hubei chryso-like virus 1 segments clusters closely to Hubei chryso-like virus 1 strains isolated in Australia, China and USA. Two novel strains of insect-specific viruses in Anopheles mosquitoes were detected and characterized.

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.

A male-killing gene encoded by a symbiotic virus of Drosophila.

In most eukaryotes, biparentally inherited nuclear genomes and maternally inherited cytoplasmic genomes have different evolutionary interests. Strongly female-biased sex ratios that are repeatedly observed in various arthropods often result from the male-specific lethality (male-killing) induced by maternally inherited symbiotic bacteria such as Spiroplasma and Wolbachia. However, despite some plausible case reports wherein viruses are raised as male-killers, it is not well understood how viruses, having much smaller genomes than bacteria, are capable of inducing male-killing. Here we show that a maternally inherited double-stranded RNA (dsRNA) virus belonging to the family Partitiviridae (designated DbMKPV1) induces male-killing in Drosophila. DbMKPV1 localizes in the cytoplasm and possesses only four genes, i.e., one gene in each of the four genomic segments (dsRNA1-dsRNA4), in contrast to ca. 1000 or more genes possessed by Spiroplasma or Wolbachia. We also show that a protein (designated PVMKp1; 330 amino acids in size), encoded by a gene on the dsRNA4 segment, is necessary and sufficient for inducing male-killing. Our results imply that male-killing genes can be easily acquired by symbiotic viruses through reassortment and that symbiotic viruses are hidden players in arthropod evolution. We anticipate that host-manipulating genes possessed by symbiotic viruses can be utilized for controlling arthropods.

Protocol for the analysis of double-stranded RNAs in virus-infected insect cells using anti-dsRNA antibodies.

Characterization of double-stranded (ds)RNAs is relevant to the understanding of viral replication and immune sensing. Here, we provide a protocol describing the use of anti-dsRNA antibodies for immunofluorescence and immunoblotting in virus-infected insect cells, which can also be applied to tissues and other organisms. We describe the procedures to prepare insect cells for viral infection, followed by RNA extraction and in vitro production of synthetic dsRNA controls. We then detail the steps for dsRNA detection by immunoblotting and immunofluorescence. For complete details on the use and execution of this protocol, please refer to de Faria et al. (2022).1.

The Characterization of Three Novel Insect-Specific Viruses Discovered in the Bean Bug, Riptortus pedestris.

Insect-specific virus (ISV) is one of the most promising agents for the biological control of insects, which is abundantly distributed in hematophagous insects. However, few ISVs have been reported in Riptortus pedestris (Fabricius), one of the major pests threatening soybeans and causing great losses in yield and quality. In this work, field Riptortus pedestris was collected from six soybean-producing regions in China, and their virome was analyzed with the metatranscriptomic approach. Altogether, seven new insect RNA viruses were identified, three of which had complete RNA-dependent RNA polymerase (RdRp) and nearly full-length genome sequences, which were named Riptortus pedestris alphadrosrha-like virus 1 (RpALv1), Riptortus pedestris alphadrosrha-like virus 2 (RpALv2) and Riptortus pedestris almendra-like virus (RiALv). The three identified novel ISVs belonged to the family Rhabdoviridae, and phylogenetic tree analysis indicated that they were clustered into new distinct clades. Interestingly, the analysis of virus-derived small-interfering RNAs (vsiRNAs) indicated that only RiALv-derived siRNAs exhibited 22 nt length preference, whereas no clear 21 or 22 nt peaks were observed for RpALv1 and RpALv2, suggesting the complexity of siRNA-based antiviral immunity in R. pedestris. In conclusion, this study contributes to a better understanding of the microenvironment in R. pedestris and provides viral information for the development of potential soybean insect-specific biocontrol agents.

Arthropod-Borne Virus Surveillance as a Tool to Study the Australian Mosquito Virome.

Mosquitoes (n = 4381 in 198 pools) were collected in March and April 2018 to survey the presence of West Nile virus Kunjin strain in mosquito populations around crocodile farms in the Darwin region of the Northern Territory (NT) of Australia. While no Kunjin virus was detected in these mosquitoes, we applied our viral replicative intermediates screening system termed monoclonal antibodies to viral RNA intermediates in cells or MAVRIC to this set of samples. This resulted in the detection of 28 pools with virus replicating in C6/36 mosquito cells and the identification of three insect viruses from three distinct virus classes. We demonstrate the persistence of the insect-specific flavivirus Palm Creek virus in Coquillettidia xanthogaster mosquitoes from Darwin over almost a decade, with limited genetic drift. We also detected a novel Hubei macula-like virus 3 strain in samples from two mosquito genera, suggesting the virus, for which the sequence was originally detected in spiders and soybean thrips, might be involved in a horizontal transmission cycle between arthropods and plants. Overall, these data demonstrate the strength of the optimized MAVRIC system and contribute to our general knowledge of the mosquito virome and insect viruses.

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.

Local-scale virome depiction in Medellín, Colombia, supports significant differences between Aedes aegypti and Aedes albopictus.

Aedes spp. comprise the primary group of mosquitoes that transmit arboviruses such as dengue, Zika, and chikungunya viruses to humans, and thus these insects pose a significant burden on public health worldwide. Advancements in next-generation sequencing and metagenomics have expanded our knowledge on the richness of RNA viruses harbored by arthropods such as Ae. aegypti and Ae. albopictus. Increasing evidence suggests that vector competence can be modified by the microbiome (comprising both bacteriome and virome) of mosquitoes present in endemic zones. Using an RNA-seq-based metataxonomic approach, this study determined the virome structure, Wolbachia presence and mitochondrial diversity of field-caught Ae. aegypti and Ae. albopictus mosquitoes in Medellín, Colombia, a municipality with a high incidence of mosquito-transmitted arboviruses. The two species are sympatric, but their core viromes differed considerably in richness, diversity, and abundance; although the community of viral species identified was large and complex, the viromes were dominated by few virus species. BLAST searches of assembled contigs suggested that at least 17 virus species (16 of which are insect-specific viruses [ISVs]) infect the Ae. aegypti population. Dengue virus 3 was detected in one sample and it was the only pathogenic virus detected. In Ae. albopictus, up to 11 ISVs and one plant virus were detected. Therefore, the virome composition appears to be species-specific. The bacterial endosymbiont Wolbachia was identified in all Ae. albopictus samples and in some Ae. aegypti samples collected after 2017. The presence of Wolbachia sp. in Ae. aegypti was not related to significant changes in the richness, diversity, or abundance of this mosquito's virome, although it was related to an increase in the abundance of Aedes aegypti To virus 2 (Metaviridae). The mitochondrial diversity of these mosquitoes suggested that the Ae. aegypti population underwent a change that started in the second half of 2017, which coincides with the release of Wolbachia-infected mosquitoes in Medellín, indicating that the population of wMel-infected mosquitoes released has introduced new alleles into the wild Ae. aegypti population of Medellín. However, additional studies are required on the dispersal speed and intergenerational stability of wMel in Medellín and nearby areas as well as on the introgression of genetic variants in the native mosquito population.

Resistance mechanism of Nid-1, a dominant non-susceptibility gene, against Bombyx mori densovirus 1 infection.

Bombyx mori densovirus 1 (BmDV1) is a pathogen that causes flacherie disease in mulberry silkworms (B. mori). The absolute resistance (non-susceptibility) to BmDV1 of certain silkworm strains is determined independently by two genes, nsd-1 and Nid-1. Previously, we investigated the expression of viral transcript in virus-inoculated silkworms carrying different nsd-1 and Nid-1 genotypes, and observed that nsd-1 and Nid-1 expression blocked the early and late steps of BmDV1 infection, respectively. In addition, we found that nsd-1 encoded a Bombyx-specific mucin-like membrane protein only present on the surface of the midgut, where BmDV1 could infect. In this study, we dissected the resistance mechanism by Nid-1 against BmDV1 infection by investigating the sequential changes in the accumulation of viral DNA, transcripts, and proteins derived from BmDV1 in susceptible strain (pxj) and Nid-1-carrying resistant strain (No. 908) after inoculation with BmDV1. Genomic PCR results showed that the BmDV1 DNA was detected immediately after the infection in both strains but rapidly decreased in the Nid-1-carrying strain No. 908 compared with the susceptible strain pxj. RT-PCR results also showed that the BmDV1 transcripts of Nid-1-carrying strain No. 908 were rapidly decreased after the infection. Moreover, BmDV1-derived proteins were not detected in No. 908 throughout the infection. These results suggest that Nid-1 expression might inhibit the accumulation of viral DNA and transcripts. As Nid-1 has not been molecularly characterized, its identification will contribute to the elucidation of the interactions between the silkworm and BmDV1.

In Vitro and In Vivo Characterization of a New Strain of Mosquito Flavivirus Derived from Culicoides.

Mosquito-specific flaviviruses comprise a group of insect-specific viruses with a single positive RNA, which can affect the duplication of mosquito-borne viruses and the life growth of mosquitoes, and which have the potential to be developed as a vaccine platform for mosquito-borne viruses. In this study, a strain of mosquito flavivirus (MFV) YN15-283-02 was detected in Culicoides collected from Yunnan, China. The isolation of the purified MFV YN15-283-02 from cell culture failed, and the virus was then rescued by an infectious clone. To study the biological features of MFV YN15-283-02 in vitro and in vivo, electron microscopy, phylogenetic tree, and viral growth kinetic analyses were performed in both cell lines and mosquitoes. The rescued MFV (rMFV) YN15-283-02 duplicated and reached a peak in C6/36 cells at 6 d.p.i. with approximately 2 × 106 RNA copies/µL (RNA to cell ratio of 0.1), but without displaying a cytopathic effect. In addition, the infection rate for the rMFV in Ae.aegypti show a low level in both larvae (≤15%) and adult mosquitoes (≤12%).

DWV 3C Protease Uncovers the Diverse Catalytic Triad in Insect RNA Viruses.

Deformed wing virus (DWV) is the most prevalent Iflavirus that is infecting honey bees worldwide. However, the mechanisms of its infection and replication in host cells are poorly understood. In this study, we analyzed the structure and function of DWV 3C protease (3Cpro), which is necessary for the cleavage of the polyprotein to synthesize mature viral proteins. Thus, it is one of the nonstructural viral proteins essential for the replication. We found that the 3Cpros of DWV and picornaviruses share common enzymatic properties, including sensitivity to the same inhibitors, such as rupintrivir. The predicted structure of DWV 3Cpro by AlphaFold2, the predicted rupintrivir binding domain, and the protease activities of mutant proteins revealed that it has a Cys-His-Asn catalytic triad. Moreover, 3Cpros of other Iflaviruses and Dicistrovirus appear to contain Asn, Ser, Asp, or Glu as the third residue of the catalytic triad, suggesting diversity in insect RNA viruses. Both precursor 3Cpro with RNA-dependent RNA polymerase and mature 3Cpro are present in DWV-infected cells, suggesting that they may have different enzymatic properties and functions. DWV 3Cpro is the first 3Cpro characterized among insect RNA viruses, and our study uncovered both the common and unique characteristics among 3Cpros of Picornavirales. Furthermore, it would be possible to use the specific inhibitors of DWV 3Cpro to control DWV infection in honey bees in future. IMPORTANCE The number of managed honey bee (Apis mellifera) colonies has considerably declined in many developed countries in the recent years. Deformed wing virus (DWV) vectored by the mites is the major threat to honey bee colonies and health. To give insight into the mechanism of DWV replication in the host cells, we studied the structure-function relationship of 3C protease (3Cpro), which is necessary to cleave a viral polyprotein at the specific sites to produce the mature proteins. We found that the overall structure, some inhibitors, and processing of 3Cpro are shared between Picornavirales; however, there is diversity in the catalytic triad. DWV 3Cpro is the first viral protease characterized among insect RNA viruses and reveals the evolutionary history of 3Cpro among Picornavirales. Furthermore, DWV 3Cpro inhibitors identified in our study could also be applied to control DWV in honey bees in future.

Critical View on the Importance of Host Defense Strategies on Virus Distribution of Bee Viruses: What Can We Learn from SARS-CoV-2 Variants?

Bees, both wild and domesticated ones, are hosts to a plethora of viruses, with most of them infecting a wide range of bee species and genera. Although viral discovery and research on bee viruses date back over 50 years, the last decade is marked by a surge of new studies, new virus discoveries, and reports on viral transmission in and between bee species. This steep increase in research on bee viruses was mainly initiated by the global reports on honeybee colony losses and the worldwide wild bee decline, where viruses are regarded as one of the main drivers. While the knowledge gained on bee viruses has significantly progressed in a short amount of time, we believe that integration of host defense strategies and their effect on viral dynamics in the multi-host viral landscape are important aspects that are currently still missing. With the large epidemiological dataset generated over the last two years on the SARS-CoV-2 pandemic, the role of these defense mechanisms in shaping viral dynamics has become eminent. Integration of these dynamics in a multi-host system would not only greatly aid the understanding of viral dynamics as a driver of wild bee decline, but we believe bee pollinators and their viruses provide an ideal system to study the multi-host viruses and their epidemiology.

Insect-specific viruses in the Parvoviridae family: Genetic lineage characterization and spatiotemporal dynamics of the recently established Brevihamaparvovirus genus.

The analysis of the viruses allocated to the recently established Brevihamaparvovirus genus (Parvoviridae family), which includes all previously known brevidensoviruses, has not yet been carried out on an extensive basis. As a result, no detailed genetic lineage characterization has ever been performed for this group of insect-specific viruses. Using a wide range of molecular tools, we have explored this taxon by calculating Shannon entropy values, intra- and inter-taxon genetic distances, analysed sequence polymorphisms, and evaluated selective pressures acting on the viral genome. While the calculated Brevihamaparvovirus mutation rates were within the range of those of other parvoviruses, their genomes look to be under strong purifying selection, and are also characterized by low diversity and entropy. Furthermore, even though recombination events are quite common among parvoviruses, no evidence of recombination (either intra or intergenic) was found in the Brevihamaparvoviruses sequences analyzed. An extended taxonomic analysis and reevaluation of existing Brevihamaparvoviruses sequences, many still unclassified, was performed using cut-off values defining NS1 identity between viral sequences from the Parvovirus family. Two existing genetic lineages, Dipteran Brevihamaparvovirus 1 and Dipteran Brevihamaparvovirus 2, were rearranged and the creation of a new one, Dipteran Brevihamaparvovirus 3, was suggested. Finally, despite the uncertainties associated with both the time estimates of the most recent common ancestors, which could span from twenty thousand years before the current era to way earlier (in the last century), and the dispersal routes proposed for Brevihamaparvoviruses sequences by phylodynamic reconstruction, the analyses here presented could help define how future studies should be conducted as more isolates continue to be identified in the future, and contribute to eliminating possible analytical biases.

Intrinsic variation in the vertically transmitted core virome of the mosquito Aedes aegypti.

Virome studies among metazoans have revealed the ubiquity of RNA viruses in animals, contributing to a fundamental rethinking of the relationships between organisms and their microbiota. Mosquito viromes, often scrutinized due to their public health relevance, may also provide insight into broadly applicable concepts, such as a "core virome," a set of viruses consistently associated with a host species or population that may fundamentally impact its basic biology. A subset of mosquito-associated viruses (MAVs) could comprise such a core, and MAVs can be categorized as (i) arboviruses, which alternate between mosquito and vertebrate hosts, (ii) insect-specific viruses, which cannot replicate in vertebrate cells, and (iii) viruses with unknown specificity. MAVs have been widely characterized in the disease vector Aedes aegypti, and the occurrence of a core virome in this species has been proposed but remains unclear. Using a wild population previously surveyed for MAVs and a common laboratory strain, we investigated viromes in reproductive tissue via metagenomic RNA sequencing. Virome composition varied across samples, but four groups comprised >97% of virus sequences: a novel partiti-like virus (Partitiviridae), a toti-like virus (Totiviridae), unclassified Riboviria, and four orthomyxo-like viruses (Orthormyxoviridae). Whole or partial genomes for the partiti-like virus, toti-like virus, and one orthomyxo-like virus were assembled and analysed phylogenetically. Multigenerational maintenance of these MAVs was confirmed by RT-PCR, indicating vertical transmission as a mechanism for persistence. This study provides fundamental information regarding MAV ecology and variability in A. aegypti and the potential for vertically maintained core viromes at the population level.

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.