The insect order Blattodea (cockroaches and termites) has drawn substantial research attention for their dietary habits and lifestyle of living with or around humans. In the present study, we focused on the discovery of RNA viruses hidden in Blattodea insects using the publicly available RNA sequencing datasets. Overall, 136 distinctive RNA viruses were identified from 36 Blattodea species, of which more than 70 % were most closely related to the invertebrate-associated viral groups within Picornavirales, Sobelivirales, Bunyaviricetes, Jingchuvirales, Durnavirales, Lispiviridae, Orthomyxoviridae, Permutotetraviridae, Flaviviridae and Muvirales. Several viruses were associated with pathogens of vertebrates (Paramyxoviridae), plants (Tymovirales), protozoa (Totiviridae), fungi (Narnaviridae) and bacteria (Norzivirales). Collectively, 93 complete or near-complete viral genomes were retrieved from the datasets, and several viruses appeared to have remarkable temporal and spatial distributions. Interestingly, the newly identified Periplaneta americana dicistrovirus displayed a remarkable distinct bicistronic genome arrangement from the well-recognized dicistroviruses with the translocated structural and non-structural polyprotein encoding open reading frames over the genome. These results significantly enhance our knowledge of RNA virosphere in Blattodea insects, and the novel genome architectures in dicistroviruses and other RNA viruses may break our stereotypes in the understanding of the genomic evolution and the emergence of potential novel viral species.

European foulbrood (EFB) is a prevalent disease in the European honey bee (Apis mellifera) in the United States, which can lead to colony decline and collapse. The bacterial components of EFB are well-studied, but the diversity of viral infections within infected colonies has not been explored. In this study, we use meta-transcriptomics sequencing of 12 honey bee hives, symptomatic (+, n = 6) and asymptomatic (-, n = 6) for EFB, to investigate viral infection associated with the disease. We assembled 41 viral genomes, belonging to three families (Iflaviridae, Dicistroviridae, and Sinhaliviridae), all previously reported in honey bees, including Lake Sinai virus, deformed wing virus, sacbrood virus, Black queen cell virus, and Israeli acute paralysis virus. In colonies with severe EFB, we observed a higher occurrence of viral genomes (34 genomes) in contrast to fewer recovered from healthy colonies (seven genomes) and a complete absence of Dicistroviridae genomes.We observed specific Lake Sinai virus clades associated exclusively with EFB + or EFB - colonies, in addition to EFB-afflicted colonies that exhibited an increase in relative abundance of sacbrood viruses. Multivariate analyses highlighted that a combination of site and EFB disease status influenced RNA virome composition, while EFB status alone did not significantly impact it, presenting a challenge for comparisons between colonies kept in different yards. These findings contribute to the understanding of viral dynamics in honey bee colonies compromised by EFB and underscore the need for future investigations to consider viral composition when investigating EFB.IMPORTANCEThis study on the viromes of honey bee colonies affected by European foulbrood (EFB) sheds light on the dynamics of viral populations in bee colonies in the context of a prevalent bacterial brood disease. The identification of distinct Lake Sinai virus and sacbrood virus clades associated with colonies affected by severe EFB suggests a potential connection between viral composition and disease status, emphasizing the need for further investigation into the role of viruses during EFB infection. The observed increase in sacbrood viruses during EFB infection suggests a potential viral dysbiosis, with potential implications for honey bee brood health. These findings contribute valuable insights related to beekeeping practices, offering a foundation for future research aimed at understanding and mitigating the impact of bacterial and viral infection in commercial honey bee operations and the management of EFB.

The worldwide dispersal of the ectoparasitic mite Varroa destructor from its Asian origins has fundamentally transformed the relationship of the honey bee (Apis mellifera) with several of its viruses, via changes in transmission and/or host immunosuppression. The extent to which honey bee-virus relationships change after Varroa invasion is poorly understood for most viruses, in part because there are few places in the world with several geographically close but completely isolated honey bee populations that either have, or have not, been exposed long-term to Varroa, allowing for separate ecological, epidemiological, and adaptive relationships to develop between honey bees and their viruses, in relation to the mite\'s presence or absence. The Azores is one such place, as it contains islands with and without the mite. Here, we combined qPCR with meta-amplicon deep sequencing to uncover the relationship between Varroa presence, and the prevalence, load, diversity, and phylogeographic structure of eight honey bee viruses screened across the archipelago. Four viruses were not detected on any island (ABPV-Acute bee paralysis virus, KBV-Kashmir bee virus, IAPV-Israeli acute bee paralysis virus, BeeMLV-Bee macula-like virus); one (SBV-Sacbrood virus) was detected only on mite-infested islands; one (CBPV-Chronic bee paralysis virus) occurred on some islands, and two (BQCV-Black queen cell virus, LSV-Lake Sinai virus,) were present on every single island. This multi-virus screening builds upon a parallel survey of Deformed wing virus (DWV) strains that uncovered a remarkably heterogeneous viral landscape featuring Varroa-infested islands dominated by DWV-A and -B, Varroa-free islands naïve to DWV, and a refuge of the rare DWV-C dominating the easternmost Varroa-free islands. While all four detected viruses investigated here were affected by Varroa for one or two parameters (usually prevalence and/or the Richness component of ASV diversity), the strongest effect was observed for the multi-strain LSV. Varroa unambiguously led to elevated prevalence, load, and diversity (Richness and Shannon Index) of LSV, with these results largely shaped by LSV-2, a major LSV strain. Unprecedented insights into the mite-virus relationship were further gained from implementing a phylogeographic approach. In addition to enabling the identification of a novel LSV strain that dominated the unique viral landscape of the easternmost islands, this approach, in combination with the recovered diversity patterns, strongly suggests that Varroa is driving the evolutionary change of LSV in the Azores. This study greatly advances the current understanding of the effect of Varroa on the epidemiology and adaptive evolution of these less-studied viruses, whose relationship with Varroa has thus far been poorly defined.

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.

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.
OBJECTIVE: 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.

Drosophila remains a pre-eminent insect model system for host-virus interaction, but the host range and fitness consequences of the drosophilid virome are poorly understood. Metagenomic studies have reported approximately 200 viruses associated with Drosophilidae, but few isolates are available to characterize the Drosophila immune response, and most characterization has relied on injection and systemic infection. Here, we use a more natural infection route to characterize the fitness effects of infection and to study a wider range of viruses. We exposed laboratory Drosophila melanogaster to 23 naturally occurring viruses from wild-collected drosophilids. We recorded transmission rates along with two components of female fitness: survival and the lifetime number of adult offspring produced. Nine different viruses transmitted during contact with laboratory D. melanogaster, although for the majority, rates of transmission were less than 20%. Five virus infections led to a significant decrease in lifespan (D. melanogaster Nora virus, D. immigrans Nora virus, Muthill virus, galbut virus and Prestney Burn virus), and three led to a reduction in the total number of offspring. Our findings demonstrate the utility of the Drosophila model for community-level studies of host-virus interactions, and suggest that viral infection could be a substantial fitness burden on wild flies.

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.

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.

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.

BACKGROUND: Vertical transmission (VT) of arboviruses (arthropod-borne viruses) can serve as an essential link in the transmission cycle during adverse environmental conditions. The extent of VT among mosquito-borne arboviruses can vary significantly among different virus families and even among different viruses within the same genus. For example, orthobunyaviruses exhibit a higher VT rate than orthoflaviviruses and alphaviruses. Mosquitoes are also the natural hosts of a large number of insect-specific viruses (ISV) that belong to several virus families, including Bunyaviridae, Flaviviridae, and Togaviridae. Cell fusing agent virus (CFAV), an insect-specific orthoflavivirus, displays higher VT rates than other dual-host orthoflaviviruses, such as Zika and dengue viruses. High VT rates require establishment of stabilized infections in the germinal tissues of female vectors. To delve deeper into understanding the mechanisms governing these differences in VT rates and the establishment of stabilized infections, the ovary infection patterns and VT of Zika virus (ZIKV) and CFAV were compared.
METHODS: Laboratory colonized Aedes aegypti females were infected with either ZIKV or CFAV by intrathoracic injection. Ovary infection patterns were monitored by in situ hybridization using virus-specific probes, and VT was determined by detecting the presence of the virus among the progeny, using a reverse-transcription quantitative polymerase chain reaction (PCR) assay.
RESULTS: Both ZIKV and CFAV infect mosquito ovaries after intrathoracic injection. Infections then become widespread following a non-infectious blood meal. VT rates of ZIKV are similar to previously reported results (3.33%). CFAV, on the contrary transmits vertically very rarely. VT was not observed in the first gonotrophic cycle following intrathoracic injection, and only rarely in the second gonotrophic cycle. VT of CFAV is mosquito population independent, since similar results were obtained with Aedes aegypti collected from two different geographic locations.
CONCLUSIONS: Although CFAV infects mosquito ovaries, the occurrence of VT remains infrequent in artificially infected Ae. aegypti, despite the observation of high VT rates in field-collected mosquitoes. These results suggest that infections of insect-specific viruses are stabilized in mosquitoes by some as yet unidentified mechanisms.