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.

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.

Factor-Dependent Internal Ribosome Entry Site and -1 Programmed Frameshifting Signal in the Bemisia-Associated Dicistrovirus 2.

The dicistrovirus intergenic (IGR) IRES uses the most streamlined translation initiation mechanism: the IRES recruits ribosomes directly without using protein factors and initiates translation from a non-AUG codon. Several subtypes of dicistroviruses IRES have been identified; typically, the IRESs adopt two -to three overlapping pseudoknots with key stem-loop and unpaired regions that interact with specific domains of the ribosomal 40S and 60S subunits to direct translation. We previously predicted an atypical IGR IRES structure and a potential -1 programmed frameshift (-1 FS) signal within the genome of the whitefly Bemisia-associated dicistrovirus 2 (BaDV-2). Here, using bicistronic reporters, we demonstrate that the predicted BaDV-2 -1 FS signal can drive -1 frameshifting in vitro via a slippery sequence and a downstream stem-loop structure that would direct the translation of the viral RNA-dependent RNA polymerase. Moreover, the predicted BaDV-2 IGR can support IRES translation in vitro but does so through a mechanism that is not typical of known factorless dicistrovirus IGR IRES mechanisms. Using deletion and mutational analyses, the BaDV-2 IGR IRES is mapped within a 140-nucleotide element and initiates translation from an AUG codon. Moreover, the IRES does not bind directly to purified ribosomes and is sensitive to eIF2 and eIF4A inhibitors NSC1198983 and hippuristanol, respectively, indicating an IRES-mediated factor-dependent mechanism. Biophysical characterization suggests the BaDV-2 IGR IRES contains several stem-loops; however, mutational analysis suggests a model whereby the IRES is unstructured or adopts distinct conformations for translation initiation. In summary, we have provided evidence of the first -1 FS frameshifting signal and a novel factor-dependent IRES mechanism in this dicistrovirus family, thus highlighting the diversity of viral RNA-structure strategies to direct viral protein synthesis.

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.

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.

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.

The effect of Israeli acute paralysis virus infection on honey bee brood care behavior.

To protect themselves from communicable diseases, social insects utilize social immunity-behavioral, physiological, and organizational means to combat disease transmission and severity. Within a honey bee colony, larvae are visited thousands of times by nurse bees, representing a prime environment for pathogen transmission. We investigated a potential social immune response to Israeli acute paralysis virus (IAPV) infection in brood care, testing the hypotheses that bees will respond with behaviors that result in reduced brood care, or that infection results in elevated brood care as a virus-driven mechanism to increase transmission. We tested for group-level effects by comparing three different social environments in which 0%, 50%, or 100% of nurse bees were experimentally infected with IAPV. We investigated individual-level effects by comparing exposed bees to unexposed bees within the mixed-exposure treatment group. We found no evidence for a social immune response at the group level; however, individually, exposed bees interacted with the larva more frequently than their unexposed nestmates. While this could increase virus transmission from adults to larvae, it could also represent a hygienic response to increase grooming when an infection is detected. Together, our findings underline the complexity of disease dynamics in complex social animal systems.

HIF-1-mediated regulation of LDH gene unravels key insights into MCDV-1 pathogenesis in mud crabs Scylla paramamosain.

Hypoxia-inducible factors -1 (HIF-1) is a crucial transcription factor that regulates the expression of glycolytic genes. Our previous study proved that the Mud crab dicistrovirus-1 (MCDV-1) can induce aerobic glycolysis that favors viral replication in mud crab Scylla paramamosain. However, the role of HIF-1 on key glycolytic genes during the MCDV-1 infection has not been examined. In this study, the intricate interplay between HIF-1 and the key glycolysis enzyme, lactate dehydrogenase (LDH), was investigated after MCDV-1 infection. The expression of LDH was significant increased after MCDV-1 infection. Additionally, the expression of HIF-1α was upregulated following MCDV-1 infection, potentially attributed to the downregulation of prolyl hydroxylase domains 2 expression. Subsequent examination of the SpLDH promoter identified the presence of hypoxia response elements (HREs), serving as binding sites for HIF-1α. Intriguingly, experimental evidence demonstrated that SpHIF-1α actively promotes SpLDH transcription through these HREs. To further elucidate the functional significance of SpHIF-1α, targeted silencing was employed, resulting in a substantial reduction in SpLDH expression, activity, and lactate concentrations in MCDV-1-infected mud crabs. Notably, SpHIF-1α-silenced mud crabs exhibited higher survival rates and lower viral loads in hepatopancreas tissues following MCDV-1 infection. These results highlight the critical role of SpHIF-1α in MCDV-1 pathogenesis by regulating LDH gene dynamics, providing valuable insights into the molecular mechanisms underlying the virus-host interaction.

Quantitative trait loci mapping for survival of virus infection and virus levels in honey bees.

Israeli acute paralysis virus (IAPV) is a highly virulent, Varroa-vectored virus that is of global concern for honey bee health. Little is known about the genetic basis of honey bees to withstand infection with IAPV or other viruses. We set up and analyzed a backcross between preselected honey bee colonies of low and high IAPV susceptibility to identify quantitative trait loci (QTL) associated with IAPV susceptibility. Experimentally inoculated adult worker bees were surveyed for survival and selectively sampled for QTL analysis based on SNPs identified by whole-genome resequencing and composite interval mapping. Additionally, natural titers of other viruses were quantified in the abdomen of these workers via qPCR and also used for QTL mapping. In addition to the full dataset, we analyzed distinct subpopulations of susceptible and non-susceptible workers separately. These subpopulations are distinguished by a single, suggestive QTL on chromosome 6, but we identified numerous other QTL for different abdominal virus titers, particularly in the subpopulation that was not susceptible to IAPV. The pronounced QTL differences between the susceptible and non-susceptible subpopulations indicate either an interaction between IAPV infection and the bees' interaction with other viruses or heterogeneity among workers of a single cohort that manifests itself as IAPV susceptibility and results in distinct subgroups that differ in their interaction with other viruses. Furthermore, our results indicate that low susceptibility of honey bees to viruses can be caused by both, virus tolerance and virus resistance. QTL were partially overlapping among different viruses, indicating a mixture of shared and specific processes that control viruses. Some functional candidate genes are located in the QTL intervals, but their genomic co-localization with numerous genes of unknown function delegates any definite characterization of the underlying molecular mechanisms to future studies.

The threat of honey bee RNA viruses to yellow-legged hornets: Insights from cross-species transmission events.

Viral diseases are a significant challenge in beekeeping, and recent studies have unveiled a potential link between these diseases and the yellow-legged hornets (Vespa velutina), notorious predators of honey bees. However, it remains unclear whether virus diseases are commonly shared between honey bees and hornets or are merely sporadic cross-species transmission events. To address this knowledge gap, we conducted a study utilizing hornet-keeping practices in Yunnan, Southwest China. Our findings demonstrate that deformed wing virus (DWV-A) and Israeli acute paralysis virus (IAPV) can be transmitted from honey bees to yellow-legged hornets. We detected virus replication in various hornet stages, including pupae with IAPV infections, indicating the similarities between infected hornet and honey bee stages. Furthermore, we observed signs and infection intensities of DWV-A and IAPV comparable to those in honey bees. While different polymorphisms were found in the virus isolates from yellow-legged hornets, the sequences remain similar to honey bee counterparts. While our findings suggest that DWV-A and IAPV behave like common diseases, we observed a natural elimination of the viruses in hornet colonies, with minimal alterations in viral sequences. Consequently, these events appear to be cross-species transmission from honey bees, with yellow-legged hornets acting as potential incidental hosts. Further investigations of virus monitoring in hornets promise valuable insights into the disease ecology of bee-infecting viruses.

Jute (Corchorus olitorius L.) Nanocrystalline Cellulose Inhibits Insect Virus via Gut Microbiota and Metabolism.

Natural plant nanocrystalline cellulose (NCC), exhibiting a number of exceptional performance characteristics, is widely used in food fields. However, little is known about the relationship between NCC and the antiviral effect in animals. Here, we tested the function of NCC in antiviral methods utilizing honey bees as the model organism employing Israeli acute paralysis virus (IAPV), a typical RNA virus of honey bees. In both the lab and the field, we fed the IAPV-infected bees various doses of jute NCC (JNCC) under carefully controlled conditions. We found that JNCC can reduce IAPV proliferation and improve gut health. The metagenome profiling suggested that IAPV infection significantly decreased the abundance of gut core bacteria, while JNCC therapy considerably increased the abundance of the gut core bacteria Snodgrassella alvi and Lactobacillus Firm-4. Subsequent metabolome analysis further revealed that JNCC promoted the biosynthesis of fatty acids and unsaturated fatty acids, accelerated the purine metabolism, and then increased the expression of antimicrobial peptides (AMPs) and the genes involved in the Wnt and apoptosis signaling pathways against IAPV infection. Our results highlighted that JNCC could be considered as a prospective candidate agent against a viral infection.

Influence of virus abundances in donor colonies and nurse hives on queens of Apis mellifera during the rearing process.

Background: Honeybees are one of the three most important animals for mankind. In order to be safe and increase number of bee colonies for pollination, the breeding of queens is necessary. For several decades, bees were selected on economic and behavioral aspects. With the appearance of the neozootic mite Varroa destructor beekeepers were forced to adapt their methods. Varroa destructor can act as a vector for many different bee pathogenic viruses and by this potentiates its devastating impact. Aim: Methods of rearing queens were not evaluated since the mites' appearance. Besides scientific approaches, viruses received too little attention in regard to the rearing process of honeybee queens. Herein, we present a detailed analysis of virus abundances [Aparavirus, acute bee paralysis virus (ABPV); Triatovirus, black queen cell virus (BQCV); Cripavirus, chronic bee paralysis virus (CBPV); and Iflaviruses, deformed wings virus (DWV), Sacbrood virus (SBV), VDV-1] in breeding hives, donating first instar larvae, hives that are nursing these larvae until the pupa stage, and on queens of Apis mellifera in a breeding apiary. Methods: Nurse and donor colonies of the queen-rearing process were sampled in the year 2020 and analyzed by RT qPCR. Virus quantifications were correlated with queen mortalities and seasonal effects. Results: Virus detections increased in reared queens, however, the elevated virus titers did not increase the mortality of the queens until their exclosure. Moreover, we observed a lower interrelation between virus abundance in queens and their original donor colonies, than between nurse hives and their nursed queens. Conclusion: The bee pathogenic viruses ABPV, BQCV, CBPV, DWV, SBV, and VDV-1 do not influence the mortality of bee queens during the rearing process. Whether respective virus loads result in sublethal or long-term effects remains to be elucidated.

Detection of honey bee viruses in larvae of Vespa orientalis.

The Oriental hornet (Vespa orientalis) is one of the major predators of honey bees. It has been demonstrated that adults of V. orientalis can harbor honey bee viruses, however the transmission route of infection is still not clear. The aim of this study was to study the possible presence of honey bee viruses in V. orientalis larvae and honey bees collected from the same apiary. Therefore, 29 samples of V. orientalis larvae and 2 pools of honey bee (Apis mellifera). samples were analyzed by multiplex PCR to detect the presence of six honeybee viruses: Acute Bee Paralysis Virus (ABPV), Black Queen Cell Virus (BQCV), Chronic Bee Paralysis Virus (CBPV), Deformed Wing Virus (DWV), Kashmir Bee Virus (KBV) and Sac Brood Virus (SBV). Biomolecular analysis of V. orientalis larvae revealed that DWV was present in 24/29 samples, SBV in 10/29, BQCV in 7/29 samples and ABPV in 5/29 samples, while no sample was found positive for CBPV or KBV. From biomolecular analysis of honey bee samples DWV was the most detected virus, followed by SBV, BQCV, ABPV. No honey bee sample was found positive for CBPV or KBV. Considering the overlapping of positivities between V.orientalis larvae and honey bee samples, and that V.orientalis larvae are fed insect proteins, preferably honey bees, we can suggest the acquisition of viral particles through the ingestion of infected bees. However, future studies are needed to confirm this hypothesis and rule out any other source of infection.

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.

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.

An Insect Viral Protein Disrupts Stress Granule Formation in Mammalian Cells.

Stress granules (SGs) are cytosolic RNA-protein aggregates assembled during stress-induced translation arrest. Virus infection, in general, modulates and blocks SG formation. We previously showed that the model dicistrovirus Cricket paralysis virus (CrPV) 1A protein blocks stress granule formation in insect cells, which is dependent on a specific arginine 146 residue. CrPV-1A also inhibits SG formation in mammalian cells suggesting that this insect viral protein may be acting on a fundamental process that regulates SG formation. The mechanism underlying this process is not fully understood. Here, we show that overexpression of wild-type CrPV-1A, but not the CrPV-1A(R146A) mutant protein, inhibits distinct SG assembly pathways in HeLa cells. CrPV-1A mediated SG inhibition is independent of the Argonaute-2 (Ago-2) binding domain and the E3 ubiquitin ligase recruitment domain. CrPV-1A expression leads to nuclear poly(A)+ RNA accumulation and is correlated with the localization of CrPV-1A to the nuclear periphery. Finally, we show that the overexpression of CrPV-1A blocks FUS and TDP-43 granules, which are pathological hallmarks of neurodegenerative diseases. We propose a model whereby CrPV-1A expression in mammalian cells blocks SG formation by depleting cytoplasmic mRNA scaffolds via mRNA export inhibition. CrPV-1A provides a new molecular tool to study RNA-protein aggregates and potentially uncouple SG functions.

The Metagenomic Analysis of Viral Diversity in Colorado Potato Beetle Public NGS Data.

The Colorado potato beetle (CPB) is one of the most serious insect pests due to its high ecological plasticity and ability to rapidly develop resistance to insecticides. The use of biological insecticides based on viruses is a promising approach to control insect pests, but the information on viruses which infect leaf feeding beetles is scarce. We performed a metagenomic analysis of 297 CPB genomic and transcriptomic samples from the public National Center for Biotechnology Information Sequence Read Archive (NCBI SRA) database. The reads that were not aligned to the reference genome were assembled with metaSPAdes, and 13314 selected contigs were analyzed with BLAST tools. The contigs and non-aligned reads were also analyzed with Kraken2 software. A total of 3137 virus-positive contigs were attributed to different viruses belonging to 6 types, 17 orders, and 32 families, matching over 97 viral species. The annotated sequences can be divided into several groups: those that are homologous to genetic sequences of insect viruses (Adintoviridae, Ascoviridae, Baculoviridae, Dicistroviridae, Chuviridae, Hytrosaviridae, Iflaviridae, Iridoviridae, Nimaviridae, Nudiviridae, Phasmaviridae, Picornaviridae, Polydnaviriformidae, Xinmoviridae etc.), plant viruses (Betaflexiviridae, Bromoviridae, Kitaviridae, Potyviridae), and endogenous retroviral elements (Retroviridae, Metaviridae). Additionally, the full-length genomes and near-full length genome sequences of several viruses were assembled. We also found sequences belonging to Bracoviriform viruses and, for the first time, experimentally validated the presence of bracoviral genetic fragments in the CPB genome. Our work represents the first attempt to discover the viral genetic material in CPB samples, and we hope that further studies will help to identify new viruses to extend the arsenal of biopesticides against CPB.

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.

Evaluation of Molecular Epidemiology of IAPV in Several Regions of Iran.

In this study, the Israeli acute paralysis virus (IAPV), a single-stranded RNA virus, was investigated in honey bee colonies, which had a history of mortality, population decline, and parasitic diseases. Samples (adult honey bees) were collected from 328 apiaries from three provinces (Tehran, Alborz, and Mazandaran) of Iran to detect IAPV. After sample preparation, RNA was extracted and cDNA was synthesized to perform the reverse transcription polymerase chain reaction (RT-PCR) method using a PCR primer pair, and a 185 bp fragment was amplified. The results showed that out of 328 samples, 103 (31.4%) samples were positive, which were from Mazandaran (14.33%), Tehran (8.84%), and Alborz (8.23%) provinces. Subsequently, some of the positive samples were sequenced and a phylogenetic tree was drawn. The phylogenetic tree showed that the virus isolates were divided into two distinct groups, including one group that had a high similarity to the European acute bee paralysis virus (ABPV) and one group that had a high similarity to the Kashmir bee virus. In addition, the sequences of the samples in three regions were separated in a node from the strains of ABPV from Eastern Europe. Since the length of the branch between the Iranian sequences and the different strains of ABPV from Eastern Europe was short, it can be assumed that the sequences from Iran have a common ancestor with the mentioned strains of ABPV from Eastern Europe.

Fertility costs of cryptic viral infections in a model social insect.

Declining insect populations emphasize the importance of understanding the drivers underlying reductions in insect fitness. Here, we investigated viruses as a threat to social insect reproduction, using honey bees as a model species. We report that in two independent surveys (N = 93 and N = 54, respectively) of honey bee (Apis mellifera) queens taken from a total of ten beekeeping operations across British Columbia, high levels of natural viral infection are associated with decreased ovary mass. Failed (poor quality) queens displayed higher levels of viral infection, reduced sperm viability, smaller ovaries, and altered ovary protein composition compared to healthy queens. We experimentally infected queens with Israeli acute paralysis virus (IAPV) and found that the ovary masses of IAPV-injected queens were significantly smaller than control queens, demonstrating a causal relationship between viral infection and ovary size. Queens injected with IAPV also had significantly lower expression of vitellogenin, the main source of nutrition deposited into developing oocytes, and higher levels of heat-shock proteins, which are part of the honey bee's antiviral response. This work together shows that viral infections occurring naturally in the field are compromising queen reproductive success.