Initiation of translation by cricket paralysis virus IRES requires its translocation in the ribosome.

The cricket paralysis virus internal ribosome entry site (CrPV-IRES) is a folded structure in a viral mRNA that allows initiation of translation in the absence of any host initiation factors. By using recent advances in single-particle electron cryomicroscopy, we have solved the structure of CrPV-IRES bound to the ribosome of the yeast Kluyveromyces lactis in both the canonical and rotated states at overall resolutions of 3.7 and 3.8 Å, respectively. In both states, the pseudoknot PKI of the CrPV-IRES mimics a tRNA/mRNA interaction in the decoding center of the A site of the 40S ribosomal subunit. The structure and accompanying factor-binding data show that CrPV-IRES binding mimics a pretranslocation rather than initiation state of the ribosome. Translocation of the IRES by elongation factor 2 (eEF2) is required to bring the first codon of the mRNA into the A site and to allow the start of translation.

RACK1 controls IRES-mediated translation of viruses.

Fighting viral infections is hampered by the scarcity of viral targets and their variability, resulting in development of resistance. Viruses depend on cellular molecules-which are attractive alternative targets-for their life cycle, provided that they are dispensable for normal cell functions. Using the model organism Drosophila melanogaster, we identify the ribosomal protein RACK1 as a cellular factor required for infection by internal ribosome entry site (IRES)-containing viruses. We further show that RACK1 is an essential determinant for hepatitis C virus translation and infection, indicating that its function is conserved for distantly related human and fly viruses. Inhibition of RACK1 does not affect Drosophila or human cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general translation. Our findings demonstrate a specific function for RACK1 in selective mRNA translation and uncover a target for the development of broad antiviral intervention.

The Kinase IKKß Regulates a STING- and NF-κB-Dependent Antiviral Response Pathway in Drosophila.

Antiviral immunity in Drosophila involves RNA interference and poorly characterized inducible responses. Here, we showed that two components of the IMD pathway, the kinase dIKKß and the transcription factor Relish, were required to control infection by two picorna-like viruses. We identified a set of genes induced by viral infection and regulated by dIKKß and Relish, which included an ortholog of STING. We showed that dSTING participated in the control of infection by picorna-like viruses, acting upstream of dIKKß to regulate expression of Nazo, an antiviral factor. Our data reveal an antiviral function for STING in an animal model devoid of interferons and suggest an evolutionarily ancient role for this molecule in antiviral immunity.

Regulation of ubiquitination and antiviral activity of Cactin by deubiquitinase Usp14 in Drosophila.

Cactin, a highly conserved protein, plays a crucial role in various physiological processes in eukaryotes, including innate immunity. Recently, the function of Cactin in the innate immunity of Drosophila has been explored, revealing that Cactin regulates a non-canonical signaling pathway associated with the Toll and Imd pathways via the Cactin-Deaf1 axis. In addition, Cactin exhibits specific antiviral activity against the Drosophila C virus (DCV) in Drosophila, with an unknown mechanism. During DCV infection, it has been confirmed that the protein level and antiviral activity of Cactin are regulated by ubiquitination. However, the precise ubiquitination and deubiquitination mechanisms of Cactin in Drosophila remain unexplored. In this study, we identified ubiquitin-specific protease 14 (Usp14) as a major deubiquitinase for Cactin through comprehensive deubiquitinase screening. Our results demonstrate that Usp14 interacts with the C_Cactus domain of Cactin via its USP domain. Usp14 efficiently removes K48- and K63-linked polyubiquitin chains from Cactin, thereby preventing its degradation through the ubiquitin-proteasome pathway. Usp14 significantly inhibits DCV replication in Drosophila cells by stabilizing Cactin. Moreover, Usp14-deficient fruit flies exhibit increased susceptibility to DCV infection compared to wild-type flies. Collectively, our findings reveal the regulation of ubiquitination and antiviral activity of Cactin by the deubiquitinase Usp14, providing valuable insights into the modulation of Cactin-mediated antiviral activity in Drosophila.IMPORTANCEViral infections pose a severe threat to human health, marked by high pathogenicity and mortality rates. Innate antiviral pathways, such as Toll, Imd, and JAK-STAT, are generally conserved across insects and mammals. Recently, the multi-functionality of Cactin in innate immunity has been identified in Drosophila. In addition to regulating a non-canonical signaling pathway through the Cactin-Deaf1 axis, Cactin exhibits specialized antiviral activity against the Drosophila C virus (DCV) with an unknown mechanism. A previous study emphasized the significance of the Cactin level, regulated by the ubiquitin-proteasome pathway, in modulating antiviral signaling. However, the regulatory mechanisms governing Cactin remain unexplored. In this study, we demonstrate that Usp14 stabilizes Cactin by preventing its ubiquitination and subsequent degradation. Furthermore, Usp14 plays a crucial role in regulating the antiviral function mediated by Cactin. Therefore, our findings elucidate the regulatory mechanism of Cactin in Drosophila, offering a potential target for the prevention and treatment of viral infections.

Investigating the outcomes of virus coinfection within and across host species.

Interactions between coinfecting pathogens have the potential to alter the course of infection and can act as a source of phenotypic variation in susceptibility between hosts. This phenotypic variation may influence the evolution of host-pathogen interactions within host species and interfere with patterns in the outcomes of infection across host species. Here, we examine experimental coinfections of two Cripaviruses-Cricket Paralysis Virus (CrPV), and Drosophila C Virus (DCV)-across a panel of 25 Drosophila melanogaster inbred lines and 47 Drosophilidae host species. We find that interactions between these viruses alter viral loads across D. melanogaster genotypes, with a ~3 fold increase in the viral load of DCV and a ~2.5 fold decrease in CrPV in coinfection compared to single infection, but we find little evidence of a host genetic basis for these effects. Across host species, we find no evidence of systematic changes in susceptibility during coinfection, with no interaction between DCV and CrPV detected in the majority of host species. These results suggest that phenotypic variation in coinfection interactions within host species can occur independently of natural host genetic variation in susceptibility, and that patterns of susceptibility across host species to single infections can be robust to the added complexity of coinfection.

A novel transposable element-mediated mechanism causes antiviral resistance in Drosophila through truncating the Veneno protein.

Hosts are continually selected to evolve new defenses against an ever-changing array of pathogens. To understand this process, we examined the genetic basis of resistance to the Drosophila A virus in Drosophila melanogaster. In a natural population, we identified a polymorphic transposable element (TE) insertion that was associated with an ∼19,000-fold reduction in viral titers, allowing flies to largely escape the harmful effects of infection by this virulent pathogen. The insertion occurs in the protein-coding sequence of the gene Veneno, which encodes a Tudor domain protein. By mutating Veneno with CRISPR-Cas9 in flies and expressing it in cultured cells, we show that the ancestral allele of the gene has no effect on viral replication. Instead, the TE insertion is a gain-of-function mutation that creates a gene encoding a novel resistance factor. Viral titers remained reduced when we deleted the TE sequence from the transcript, indicating that resistance results from the TE truncating the Veneno protein. This is a novel mechanism of virus resistance and a new way by which TEs can contribute to adaptation.

Caught in the act: the invasion of a viral vector changes viral prevalence and titre in native honeybees and bumblebees.

Novel transmission routes change pathogen landscapes and may facilitate disease emergence. The varroa mite is a virus vector that switched to western honeybees at the beginning of the last century, leading to hive mortality, particularly in combination with RNA viruses. A recent invasion of varroa on the French island of Ushant introduced vector-mediated transmission to one of the last varroa-naive native honeybee populations and caused rapid changes in the honeybee viral community. These changes were characterized by a drastic increase in deformed wing virus type B prevalence and titre in honeybees, as well as knock-on effects in bumblebees, particularly in the year following the invasion. Slow bee paralysis virus also appeared in honeybees and bumblebees, with a 1 year delay, while black queen cell virus declined in honeybees. This study highlights the rapid and far-reaching effects of vector-borne transmission that can extend beyond the directly affected host species, and that the direction of the effect depends on the pathogen's virulence.

Seasonal trends of the ABPV, KBV, and IAPV complex in Italian managed honey bee (Apis mellifera L.) colonies.

Acute bee paralysis virus (ABPV), Kashmir bee virus (KBV), and Israeli acute paralysis virus (IAPV) usually persist as covert infections in honey bee colonies. They can cause rapid bee mortality in cases of severe infection, often associated with high Varroa destructor infestation, by which they are transmitted. In various countries, these viruses have been associated with colony collapse. Despite their potential danger, these viruses are often disregarded, and little information is available on their occurrence in many countries, including Italy. In 2021, 370 apiaries representing all of the Italian regions were investigated in four different months (June, September, November, and March) for the presence of ABPV, KBV, and IAPV. IAPV was not found in any of the apiaries investigated, whereas 16.45% and 0.67% of the samples tested positive for ABPV and KBV, respectively. Most ABPV cases occurred in late summer-autumn in both northern and southern regions. We observed a scattered pattern of KBV-positive colonies that did not allow any seasonal or regional trends to be discerned. Differences observed among regions and months were potentially related to the dynamics of varroa infestation, viral genetic variations, and different climatic conditions resulting in variations in bee behaviour. This study improves our understanding of the circulation of bee viruses and will contribute to better disease prevention and preservation of bee health.

Prevalence and molecular analysis of some important viruses in honey bee colonies in Türkiye: the status of multiple infections.

In this study, seven bee viruses of significant importance for bee health in Türkiye were investigated using one-step RT-PCR. For this purpose, larvae from 1183 hives and adult bees from 1196 hives were sampled from 400 apiaries in 40 provinces. The prevalence of viral infections in hives was as follows: acute bee paralysis virus (ABPV), 6.4%; black queen cell virus (BQCV), 77%; chronic bee paralysis virus (CBPV), 3.2%; deformed wing virus (DWV), 63.8%; Israel acute bee paralysis virus (IAPV), 7%; Kashmir bee virus (KBV), 2.7%; sacbrood virus (SBV), 49.7%. Moreover, 50 different combinations of viral infections were identified in the hives. While dual infections (36.1%) were the most common in hives, triple infections with BQCV, DWV, and SBV were found to have the highest prevalence (22.1%). At least one viral infection was detected in all of the apiaries tested. Phylogenetic analysis showed that the isolates from this study generally exhibited the highest similarity to previously reported Turkish isolates. When similarity ratios and the locations and types of amino acid mutations were analyzed, it was observed that the isolates from our study exhibited high similarity to isolates from various countries, including China, the United Kingdom, Syria, and Germany.

Multiple Parallel Pathways of Translation Initiation on the CrPV IRES.

The complexity of eukaryotic translation allows fine-tuned regulation of protein synthesis. Viruses use internal ribosome entry sites (IRESs) to minimize or, like the CrPV IRES, eliminate the need for initiation factors. Here, by exploiting the CrPV IRES, we observed the entire process of initiation and transition to elongation in real time. We directly tracked the CrPV IRES, 40S and 60S ribosomal subunits, and tRNA using single-molecule fluorescence spectroscopy and identified multiple parallel initiation pathways within the system. Our results distinguished two pathways of 80S:CrPV IRES complex assembly that produce elongation-competent complexes. Following 80S assembly, the requisite eEF2-mediated translocation results in an unstable intermediate that is captured by binding of the elongator tRNA. Whereas initiation can occur in the 0 and +1 frames, the arrival of the first tRNA defines the reading frame and strongly favors 0 frame initiation. Overall, even in the simplest system, an intricate reaction network regulates translation initiation.

Ontogeny of immunity and natural viral infection in Apis mellifera drones and workers.

The most common viral diseases affecting honey bees (Apis mellifera) in Israel include deformed wing viruses (DWV-A and DWV-B) and acute paralysis viruses (ABPV and IAPV). These viruses are transmitted within and between colonies, both horizontally and vertically. All members of the colony contribute to this transmission, on the other hand individual and social immunity, particularly hygienic behaviour, may affect the outcome of the process. In this study, we evaluated the ontogeny of natural infections of DWV-A, DWV-B, ABPV and IAPV, their prevalence and loads, in workers and drones from high (H) and low (L) hygienic colonies. In parallel, we evaluated the expression of two immune genes: peptidoglycan recognition protein S2(PGRP-S2) and hymenoptaecin. The prevalence of DWV-B and IAPV increased with age and was higher in workers than in drones. ABPV was not detected in drones. The expression of both immune genes was significantly affected by age and sex. Drones from H colonies had higher expression of these genes. The increased expression of immune genes with drones' age, particularly in hygienic colonies, suggest additional value of honey bee breeding for hygienic behaviour for sustainable beekeeping.

Parasite and virus dynamics in the honeybee Apis mellifera unicolor on a tropical island recently invaded by Varroa destructor.

In La Réunion, the established honeybee subspecies Apis mellifera unicolor, an endemic subspecies of African lineage, is facing considerable challenges. Since the introduction of the Varroa destructor mite in 2017 high colony losses have been recorded. We investigated the dynamics of V. destructor and two viruses, the Deformed Wing Virus (DWV), known to be transmitted by the mite, and the Chronic Bee Paralysis Virus (CBPV), in A. m. unicolor. Colonies from two apiaries located at 300 and 900 m a.s.l were monitored twice for one year without any acaricide treatment. The brood area, V. destructor infestation rates, DWV and CBPV prevalence and load were recorded monthly. A. m. unicolor maintained brood rearing throughout the year. Varroa destructor infestation resulted in high colony mortality (up to 85 %) and high phoretic mite rates (up to 52 mites per hundred bees). The establishment of DWV in colonies occurred after that of V. destructor and the mite infestation rate had a significant effect on the virus prevalence and load. CBPV appeared only transiently throughout the surveys. The data showed that, in tropical colonies with permanent brood rearing, V. destructor and DWV can reach high levels, but are still subject to seasonal variations that appear to be influenced by environmental conditions. This suggests that beekeeping practices could be adapted by favouring sites and periods for transhumance or acaricide treatment.

Forward genetics in Wolbachia: Regulation of Wolbachia proliferation by the amplification and deletion of an addictive genomic island.

Wolbachia is one of the most prevalent bacterial endosymbionts, infecting approximately 40% of terrestrial arthropod species. Wolbachia is often a reproductive parasite but can also provide fitness benefits to its host, as, for example, protection against viral pathogens. This protective effect is currently being applied to fight arboviruses transmission by releasing Wolbachia-transinfected mosquitoes. Titre regulation is a crucial aspect of Wolbachia biology. Higher titres can lead to stronger phenotypes and fidelity of transmission but can have a higher cost to the host. Since Wolbachia is maternally transmitted, its fitness depends on host fitness, and, therefore, its cost to the host may be under selection. Understanding how Wolbachia titres are regulated and other aspects of Wolbachia biology has been hampered by the lack of genetic tools. Here we developed a forward genetic screen to identify new Wolbachia over-proliferative mutant variants. We characterized in detail two new mutants, wMelPop2 and wMelOctoless, and show that the amplification or loss of the Octomom genomic region lead to over-proliferation. These results confirm previous data and expand on the complex role of this genomic region in the control of Wolbachia proliferation. Both new mutants shorten the host lifespan and increase antiviral protection. Moreover, we show that Wolbachia proliferation rate in Drosophila melanogaster depends on the interaction between Octomom copy number, the host developmental stage, and temperature. Our analysis also suggests that the life shortening and antiviral protection phenotypes of Wolbachia are dependent on different, but related, properties of the endosymbiont; the rate of proliferation and the titres near the time of infection, respectively. We also demonstrate the feasibility of a novel and unbiased experimental approach to study Wolbachia biology, which could be further adapted to characterize other genetically intractable bacterial endosymbionts.

Tetracycline-induced gut community dysbiosis and Israeli Acute Paralysis Virus infection synergistically negatively affect honeybees.

Antibiotics are frequently employed to control bacterial diseases in honeybees, but their broad-spectrum action can disrupt the delicate balance of the gut microbiome, leading to dysbiosis. This imbalance in the gut microbiota of honeybees adversely affects their physiological health and weakens their resistance to pathogens, including viruses that significantly threaten honeybee health. In this study, we investigated whether tetracycline-induced gut microbiome dysbiosis promotes the replication of Israeli acute paralysis virus (IAPV), a key virus associated with colony losses and whether IAPV infection exacerbates gut microbiome dysbiosis. Our results demonstrated that tetracycline-induced gut microbiome dysbiosis increases the susceptibility of honeybees to IAPV infection. The viral titer in worker bees with antibiotic-induced gut microbiome dysbiosis prior to IAPV inoculation was significantly higher than in those merely inoculated with IAPV. Furthermore, we observed a synergistic effect between tetracycline and IAPV on the disruption of the honeybee gut microbiome balance. The progression of IAPV replication could, in turn, exacerbate antibiotic-induced gut microbiome dysbiosis in honeybees. Our research provides novel insights into the role of the gut microbiota in host-virus interactions, emphasizing the complex interplay between antibiotic use, gut microbiome health, and viral susceptibility in honeybees. We highlight the crucial role of a balanced gut microbiota in honey bees for their immune response against pathogens and emphasize the importance of careful, safe antibiotic use in beekeeping to protect these beneficial microbes.

The Israeli acute paralysis virus IRES captures host ribosomes by mimicking a ribosomal state with hybrid tRNAs.

Colony collapse disorder (CCD) is a multi-faceted syndrome decimating bee populations worldwide, and a group of viruses of the widely distributed Dicistroviridae family have been identified as a causing agent of CCD. This family of viruses employs non-coding RNA sequences, called internal ribosomal entry sites (IRESs), to precisely exploit the host machinery for viral protein production. Using single-particle cryo-electron microscopy (cryo-EM), we have characterized how the IRES of Israeli acute paralysis virus (IAPV) intergenic region captures and redirects translating ribosomes toward viral RNA messages. We reconstituted two in vitro reactions targeting a pre-translocation and a post-translocation state of the IAPV-IRES in the ribosome, allowing us to identify six structures using image processing classification methods. From these, we reconstructed the trajectory of IAPV-IRES from the early small subunit recruitment to the final post-translocated state in the ribosome. An early commitment of IRES/ribosome complexes for global pre-translocation mimicry explains the high efficiency observed for this IRES. Efforts directed toward fighting CCD by targeting the IAPV-IRES using RNA-interference technology are underway, and the structural framework presented here may assist in further refining these approaches.

The Hinge Region of the Israeli Acute Paralysis Virus Internal Ribosome Entry Site Directs Ribosomal Positioning, Translational Activity, and Virus Infection.

All viruses must usurp host ribosomes for viral protein synthesis. Dicistroviruses utilize an intergenic region internal ribosome entry site (IGR IRES) to directly recruit ribosomes and mediate translation initiation from a non-AUG start codon. The IGR IRES adopts a three-pseudoknot structure that comprises a ribosome binding domain of pseudoknot II and III (PKII and PKIII), and a tRNA-like anticodon domain (PKI) connected via a short, one to three nucleotide hinge region. Recent cryo-EM structural analysis of the dicistrovirus Taura syndrome virus (TSV) IGR IRES bound to the ribosome suggests that the hinge region may facilitate translocation of the IRES from the ribosomal A to P site. In this study, we provide mechanistic and functional insights into the role of the hinge region in IGR IRES translation. Using the honeybee dicistrovirus, Israeli acute paralysis virus (IAPV), as a model, we demonstrate that mutations of the hinge region resulted in decreased IRES-dependent translation in vitro. Toeprinting primer extension analysis of mutant IRESs bound to purified ribosomes and in rabbit reticulocyte lysates showed defects in the initial ribosome positioning on the IRES. Finally, using a hybrid dicistrovirus clone, mutations in the hinge region of the IAPV IRES resulted in decreased viral yield. Our work reveals an unexpected role of the hinge region of the dicistrovirus IGR IRES coordinating the two independently folded domains of the IRES to properly position the ribosome to start translation. IMPORTANCE Viruses must use the host cell machinery to direct viral protein expression for productive infection. One such mechanism is an internal ribosome entry site that can directly recruit host cell machinery. In this study, we have identified a novel sequence in an IRES that provides insight into the mechanism of viral gene expression. Specifically, this novel sequence promotes viral IRES activity by directly guiding the host cell machinery to start gene expression at a specific site.

Multiple Viral Protein Genome-Linked Proteins Compensate for Viral Translation in a Positive-Sense Single-Stranded RNA Virus Infection.

Viral protein genome-linked (VPg) protein plays an essential role in protein-primed replication of plus-stranded RNA viruses. VPg is covalently linked to the 5' end of the viral RNA genome via a phosphodiester bond typically at a conserved amino acid. Whereas most viruses have a single VPg, some viruses have multiple VPgs that are proposed to have redundant yet undefined roles in viral replication. Here, we use cricket paralysis virus (CrPV), a dicistrovirus that has four nonidentical copies of VPg, as a model to characterize the role of VPg copies in infection. Dicistroviruses contain two main open reading frames (ORFs) that are driven by distinct internal ribosome entry sites (IRESs). We systematically generated single and combinatorial deletions and mutations of VPg1 to VPg4 within the CrPV infectious clone and monitored viral yield in Drosophila S2 cells. Deletion of one to three VPg copies progressively decreased viral yield and delayed viral replication, suggesting a threshold number of VPgs for productive infection. Mass spectrometry analysis of CrPV VPg-linked RNAs revealed viral RNA linkage to either a serine or threonine in VPg, mutations of which in all VPgs attenuated infection. Mutating serine 4 in a single VPg abolished viral infection, indicating a dominant negative effect. Using viral minigenome reporters that monitor dicistrovirus 5' untranslated (UTR) and IRES translation revealed a relationship between VPg copy number and the ratio of distinct IRES translation activities. We uncovered a novel viral strategy whereby VPg copies in dicistrovirus genomes compensate for the relative IRES translation efficiencies to promote infection. IMPORTANCE Genetic duplication is exceedingly rare in small RNA viral genomes, as there is selective pressure to prevent RNA genomes from expanding. However, some small RNA viruses encode multiple copies of a viral protein, most notably an unusual viral protein that is linked to the viral RNA genome. Here, we investigate a family of viruses that contains multiple viral protein genome-linked proteins and reveal a novel viral strategy whereby viral protein copy number counterbalances differences in viral protein synthesis mechanisms.

Identification of a novel dicistro-like virus associated with the roots of tomato plants.

Viruses belonging to the family Dicistroviridae have a monopartite positive-sense single-stranded RNA genome and infect a variety of arthropods. Using high-throughput sequencing, we detected a novel dicistro-like virus, tentatively named "tomato root-associated dicistro-like virus" (TRaDLV), in the roots of tomato plants showing yellow mosaic symptoms on the leaves. The diseased tomato plants were coinfected with multiple plant viruses, and TRaDLV was present in the roots but not in the leaves. The genome of TRaDLV is 8726 nucleotides in length, excluding the poly(A) tail, and contains two open reading frames (ORFs) separated by an intergenic region (IGR). The TRaDLV genome showed characteristics similar to those of dicistroviruses, including the presence of a 3C-like protease domain, repeated amino acid sequences representing multiple copies of viral genome-linked protein (VPg)-like sequences in the ORF1 polyprotein, and a series of stem-loop structures resembling an internal ribosome entry site in the IGR. Phylogenetic analysis revealed that TRaDLV clustered with unclassified dicistro-like viruses from invertebrates or identified in samples of plant-derived material. These findings indicate the existence of a novel dicistro-like virus that may associate with plant roots or a root-inhabiting organism.

Cryo-EM of ribosomal 80S complexes with termination factors reveals the translocated cricket paralysis virus IRES.

The cricket paralysis virus (CrPV) uses an internal ribosomal entry site (IRES) to hijack the ribosome. In a remarkable RNA-based mechanism involving neither initiation factor nor initiator tRNA, the CrPV IRES jumpstarts translation in the elongation phase from the ribosomal A site. Here, we present cryoelectron microscopy (cryo-EM) maps of 80S⋅CrPV-STOP ⋅ eRF1 ⋅ eRF3 ⋅ GMPPNP and 80S⋅CrPV-STOP ⋅ eRF1 complexes, revealing a previously unseen binding state of the IRES and directly rationalizing that an eEF2-dependent translocation of the IRES is required to allow the first A-site occupation. During this unusual translocation event, the IRES undergoes a pronounced conformational change to a more stretched conformation. At the same time, our structural analysis provides information about the binding modes of eRF1 ⋅ eRF3 ⋅ GMPPNP and eRF1 in a minimal system. It shows that neither eRF3 nor ABCE1 are required for the active conformation of eRF1 at the intersection between eukaryotic termination and recycling.

Transmission modes and efficiency of iflavirus and cripavirus in Queensland fruit fly, Bactrocera tryoni.

Infections of insects with insect-specific RNA viruses are common and can affect host fitness and health. Previously, persistent RNA virus infections were detected in tephritid fruit flies, including the Queensland fruit fly (Bactrocera tryoni), Australia's most significant horticultural pest. Their transmission modes and efficiency are unclear yet may influence virus epidemiology in field and laboratory populations. Using standard RT-PCR and RT-qPCR we detected iflavirus, cripavirus and sigmavirus in five laboratory populations recently established with field-collected B.tryoni. Virus absence in some individuals suggested that virus transmission is incomplete. Random virus segregation in an isofemale experiment resulted in the establishment of isofemale lines with and without iflavirus and cripavirus. In infected lines, viral loads normalised against host gene transcripts were variable, but did not differ between pupae and adults. Iflavirus and cripavirus were transmitted horizontally, with viruses detected (including at low viral loads) in many previously uninfected individuals after four days, and in most after 12 days cohabitation with infected flies. Iflavirus, but not cripavirus, was transmitted vertically, and surface-sterilised embryos contained high loads. Furthermore, high iflavirus loads in individual females resulted in high loads in their offspring. We demonstrated that viruses are highly prevalent in laboratory populations and that it is possible to establish and maintain uninfected fly lines for the assessment of virus transmission and host effects. This is important for pest management strategies such as the sterile insect technique which requires the mass-rearing of flies, as their fitness and performance may be affected by covert virus infections.