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.

Innovative Toolbox for the Quantification of Drosophila C Virus, Drosophila A Virus, and Nora Virus.

Quantification of viral replication underlies investigations into host-virus interactions. In Drosophila melanogaster, persistent infections with Drosophila C virus, Drosophila A virus, and Nora virus are commonly observed in nature and in laboratory fly stocks. However, traditional endpoint dilution assays to quantify infectious titers are not compatible with persistently infecting isolates of these viruses that do not cause cytopathic effects in cell culture. Here we present a novel assay based on immunological detection of Drosophila C virus infection that allows quantification of infectious titers for a wider range of Drosophila C virus isolates. We also describe strand specific RT-qPCR assays for quantification of viral negative strand RNA produced during Drosophila C virus, Drosophila A virus, and Nora virus infection. Finally, we demonstrate the utility of these assays for quantification of viral replication during oral infections and persistent infections with each virus.

Wolbachia-Conferred Antiviral Protection Is Determined by Developmental Temperature.

Wolbachia is a maternally transmitted bacterium that is widespread in arthropods and filarial nematodes and confers strong antiviral protection in Drosophila melanogaster and other arthropods. Wolbachia-transinfected Aedes aegypti mosquitoes are currently being deployed to fight transmission of dengue and Zika viruses. However, the mechanism of antiviral protection and the factors influencing are still not fully understood. Here, we show that temperature modulates Wolbachia-conferred protection in Drosophila melanogaster. Temperature after infection directly impacts Drosophila C virus (DCV) replication and modulates Wolbachia protection. At higher temperatures, viruses proliferate more and are more lethal, while Wolbachia confers lower protection. Strikingly, host developmental temperature is a determinant of Wolbachia-conferred antiviral protection. While there is strong protection when flies develop from egg to adult at 25°C, the protection is highly reduced or abolished when flies develop at 18°C. However, Wolbachia-induced changes during development are not sufficient to limit virus-induced mortality, as Wolbachia is still required to be present in adults at the time of infection. This developmental effect is general, since it was present in different host genotypes, Wolbachia variants, and upon infection with different viruses. Overall, we show that Wolbachia-conferred antiviral protection is temperature dependent, being present or absent depending on the environmental conditions. This interaction likely impacts Wolbachia-host interactions in nature and, as a result, frequencies of host and symbionts in different climates. Dependence of Wolbachia-mediated pathogen blocking on developmental temperature could be used to dissect the mechanistic bases of protection and influence the deployment of Wolbachia to prevent transmission of arboviruses. IMPORTANCE Insects are often infected with beneficial intracellular bacteria. The bacterium Wolbachia is extremely common in insects and can protect them from pathogenic viruses. This effect is being used to prevent transmission of dengue and Zika viruses by Wolbachia-infected mosquitoes. To understand the biology of insects in the wild, we need to discover which factors affect Wolbachia-conferred antiviral protection. Here, we show that the temperature at which insects develop from eggs to adults can determine the presence or absence of antiviral protection. The environment, therefore, strongly influences this insect-bacterium interaction. Our work may help to provide insights into the mechanism of viral blocking by Wolbachia, deepen our understanding of the geographical distribution of host and symbiont, and incentivize further research on the temperature dependence of Wolbachia-conferred protection for control of mosquito-borne disease.

Honey bee viruses are highly prevalent but at low intensities in wild pollinators of cucurbit agroecosystems.

Managed and wild bee populations are in decline around the globe due to several biotic and abiotic stressors. Pathogenic viruses associated with the Western honey bee (Apis mellifera) have been identified as key contributors to losses of managed honey bee colonies, and are known to be transmitted to wild bee populations through shared floral resources. However, little is known about the prevalence and intensity of these viruses in wild bee populations, or how bee visitation to flowers impacts viral transmission in agroecosystems. This study surveyed honey bee, bumble bee (Bombus impatiens) and wild squash bee (Eucera (Peponapis) pruinosa) populations in Cucurbita agroecosystems across Pennsylvania (USA) for the prevalence and intensity of five honey bee viruses: acute bee paralysis virus (ABPV), deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV), and slow bee paralysis virus (SBPV). We investigated the potential role of bee visitation rate to flowers on DWV intensity among species in the pollinator community, with the expectation that increased bee visitation to flowers would increase the opportunity for transmission events between host species. We found that honey bee viruses are highly prevalent but in lower titers in wild E. pruinosa and B. impatiens than in A. mellifera populations throughout Pennsylvania (USA). DWV was detected in 88% of B. impatiens, 48% of E. pruinosa, and 95% of A. mellifera. IAPV was detected in 5% of B. impatiens and 4% of E. pruinosa, compared to 9% in A. mellifera. KBV was detected in 1% of B. impatiens and 5% of E. pruinosa, compared to 32% in A. mellifera. Our results indicate that DWV titers are not correlated with bee visitation in Cucurbita fields. The potential fitness impacts of these low viral titers detected in E. pruinosa remain to be investigated.

Establishment and characterization of the Bactrocera dorsalis (Diptera: Tephritidae) embryonic cell line QAU-Bd-E-2.

In this study, we successfully established a Bactrocera dorsalis (Diptera: Tephritidae) embryonic cell line, i.e., QAU-Bd-E-2, from the insect eggs. The cells have been stably passaged for more than 60 times in TNM-FH medium with 10% fetal bovine serum (FBS). QAU-Bd-E-2 cells are adherent cells. Most of the cells were round, spindle-shaped, and rod-shaped. Round cells accounted for 82.3%, with a diameter of 13.9 ± 2.6 µm; spindle-shaped cells accounted for 9.8%, with the size of 51.2 ± 11.2 µm × 10.3 ± 3.1 µm; the rod-shaped cells accounted for 7.9%, with the size of 35.2 ± 9.4 µm × 12.0 ± 2.5 µm. The mitochondrial cytochrome oxidase I subunit (CoI) gene from QAU-Bd-E-2 cells was amplified, and the 657 bp fragment had a 100% similarity with the CoI gene of B. dorsalis, suggesting that the cell line was derived from B. dorsalis. The chromosome number of QAU-Bd-E-2 cells was mostly 12, which is the same as the B. dorsalis chromosome number. The cell density of QAU-Bd-E-2 cells reached the maximum (3.4 × 106 cells/mL) at 192 h, and the population doubling time was 31.9 h. Bactrocera dorsalis cripavirus (BdCV) could replicate in QAU-Bd-E-2 cells, suggesting that this cell line could be used for in-depth study of the relationship between virus and host.

Resurrection of a Viral Internal Ribosome Entry Site from a 700 Year Old Ancient Northwest Territories Cripavirus.

The dicistrovirus intergenic region internal ribosome entry site (IGR IRES) uses an unprecedented, streamlined mechanism whereby the IRES adopts a triple-pseudoknot (PK) structure to directly bind to the conserved core of the ribosome and drive translation from a non-AUG codon. The origin of this IRES mechanism is not known. Previously, a partial fragment of a divergent dicistrovirus RNA genome, named ancient Northwest territories cripavirus (aNCV), was extracted from 700-year-old caribou feces trapped in a subarctic ice patch. The aNCV IGR sequence adopts a secondary structure similar to contemporary IGR IRES structures, however, there are subtle differences including 105 nucleotides upstream of the IRES of unknown function. Using filter binding assays, we showed that the aNCV IRES could bind to purified ribosomes, and toeprinting analysis pinpointed the start site at a GCU alanine codon adjacent to PKI. Using a bicistronic reporter RNA, the aNCV IGR can direct translation in vitro in a PKI-dependent manner. Lastly, a chimeric infectious clone swapping in the aNCV IRES supported translation and virus infection. The characterization and resurrection of a functional IGR IRES from a divergent 700-year-old virus provides a historical framework for the importance of this viral translational mechanism.

Biophysical Studies of the Binding of Viral RNA with the 80S Ribosome Using switchSENSE.

Translation initiation, in both eukaryotes and bacteria, requires essential elements such as mRNA, ribosome , initiator tRNA, and a set of initiation factors. For each domain of life, canonical mechanisms and signals are observed to initiate protein synthesis. However, other initiation mechanism can be used, especially in viral mRNAs. Some viruses hijack cellular machinery to translate some of their mRNAs through a noncanonical initiation pathway using internal ribosome entry site (IRES), a highly structured RNAs which can directly recruit the ribosome with a restricted set of initiation factors, and in some cases even without cap and initiator tRNA. In this chapter, we describe the use of biosensors relying on electro-switchable nanolevers using the switchSENSE® technology, to investigate kinetics of the intergenic (IGR) IRES of the cricket paralysis virus (CrPV) binding to 80S yeast ribosome . This study provides a proof of concept for the application of this method on large complexes.

Reproductive activation in honeybee (Apis mellifera) workers protects against abiotic and biotic stress.

Social insect reproductives exhibit exceptional longevity instead of the classic trade-off between somatic maintenance and reproduction. Even normally sterile workers experience a significant increase in life expectancy when they assume a reproductive role. The mechanisms that enable the positive relation between the antagonistic demands of reproduction and somatic maintenance are unclear. To isolate the effect of reproductive activation, honeybee workers were induced to activate their ovaries. These reproductively activated workers were compared to controls for survival and gene expression patterns after exposure to Israeli Acute Paralysis Virus or the oxidative stressor paraquat. Reproductive activation increased survival, indicating better immunity and oxidative stress resistance. After qPCR analysis confirmed our experimental treatments at the physiological level, whole transcriptome analysis revealed that paraquat treatment significantly changed the expression of 1277 genes in the control workers but only two genes in reproductively activated workers, indicating that reproductive activation preemptively protects against oxidative stress. Significant overlap between genes that were upregulated by reproductive activation and in response to paraquat included prominent members of signalling pathways and anti-oxidants known to affect ageing. Thus, while our results confirm a central role of vitellogenin, they also point to other mechanisms to explain the molecular basis of the lack of a cost of reproduction and the exceptional longevity of social insect reproductives. Thus, socially induced reproductive activation preemptively protects honeybee workers against stressors, explaining their longevity. This article is part of the theme issue 'Ageing and sociality: why, when and how does sociality change ageing patterns?'

Viral susceptibility across host species is largely independent of dietary protein to carbohydrate ratios.

The likelihood of a successful host shift of a parasite to a novel host species can be influenced by environmental factors that can act on both the host and parasite. Changes in nutritional resource availability have been shown to alter pathogen susceptibility and the outcome of infection in a range of systems. Here, we examined how dietary protein to carbohydrate altered susceptibility in a large cross-infection experiment. We infected 27 species of Drosophilidae with an RNA virus on three food types of differing protein to carbohydrate ratios. We then measured how viral load and mortality across species was affected by changes in diet. We found that changes in the protein:carbohydrate in the diet did not alter the outcomes of infection, with strong positive inter-species correlations in both viral load and mortality across diets, suggesting no species-by-diet interaction. Mortality and viral load were strongly positively correlated, and this association was consistent across diets. This suggests changes in diet may give consistent outcomes across host species, and may not be universally important in determining host susceptibility to pathogens.

Impact of Varroa destructor and associated pathologies on the colony collapse disorder affecting honey bees.

Varroa mite is the major threat to the western honey bee, Apis mellifera, and the cause of significant economic losses in the apiculture industry. Varroa destructor feeds on brood and adult bees being responsible for vectoring virus infections and other diseases. This study analyses the role of Varroa and other associated pathogens, such as viruses or the fungus Nosema ceranae, and their relationships regarding the viability of the bee colony. It has been carried out during one beekeeping season, with the subspecies A. m. iberiensis, commonly used in the apiculture industry of Spain. Our study shows a significant relationship between the presence of Varroa destructor and viral infection by deformed wing virus and acute bee paralysis virus. Nosema ceranae behaved as an opportunistic pathogen. In addition, this study explored a potential naturally occurring subset of peptides, responsible for the humoral immunity of the bees. The expression of the antimicrobial peptides abaecin and melittin showed a significant relationship with the levels of Varroa mite and the deformed wing virus.

Mode of Transmission Determines the Virulence of Black Queen Cell Virus in Adult Honey Bees, Posing a Future Threat to Bees and Apiculture.

Honey bees (Apis mellifera) can be infected by many viruses, some of which pose a major threat to their health and well-being. A critical step in the dynamics of a viral infection is its mode of transmission. Here, we compared for the first time the effect of mode of horizontal transmission of Black queen cell virus (BQCV), a ubiquitous and highly prevalent virus of A. mellifera, on viral virulence in individual adult honey bees. Hosts were exposed to BQCV either by feeding (representing direct transmission) or by injection into hemolymph (analogous to indirect or vector-mediated transmission) through a controlled laboratory experimental design. Mortality, viral titer and expression of three key innate immune-related genes were then quantified. Injecting BQCV directly into hemolymph in the hemocoel resulted in far higher mortality as well as increased viral titer and significant change in the expression of key components of the RNAi pathway compared to feeding honey bees BQCV. Our results support the hypothesis that mode of horizontal transmission determines BQCV virulence in honey bees. BQCV is currently considered a benign viral pathogen of adult honey bees, possibly because its mode of horizontal transmission is primarily direct, per os. We anticipate adverse health effects on honey bees if BQCV transmission becomes vector-mediated.

Metabolomic Analysis of Cricket paralysisvirus Infection in Drosophila S2 Cells Reveals Divergent Effects on Central Carbon Metabolism as Compared with Silkworm Bm5 Cells.

High-throughput approaches have opened new opportunities for understanding biological processes such as persistent virus infections, which are widespread. However, the potential of persistent infections to develop towards pathogenesis remains to be investigated, particularly with respect to the role of host metabolism. To explore the interactions between cellular metabolism and persistent/pathogenic virus infection, we performed untargeted and targeted metabolomic analysis to examine the effects of Cricket paralysis virus (CrPV, Dicistroviridae) in persistently infected silkworm Bm5 cells and acutely infected Drosophila S2 cells. Our previous study (Viruses 2019, 11, 861) established that both glucose and glutamine levels significantly increased during the persistent period of CrPV infection of Bm5 cells, while they decreased steeply during the pathogenic stages. Strikingly, in this study, an almost opposite pattern in change of metabolites was observed during different stages of acute infection of S2 cells. More specifically, a significant decrease in amino acids and carbohydrates was observed prior to pathogenesis, while their abundance significantly increased again during pathogenesis. Our study illustrates the occurrence of diametrically opposite changes in central carbon mechanisms during CrPV infection of S2 and Bm5 cells that is possibly related to the type of infection (acute or persistent) that is triggered by the virus.

Host range of Triatoma virus does not extend to Aedes aegypti and Apis mellifera.

Vector control is the most effective method to prevent transmission of Chagas disease. Control is mostly made through chemical insecticides although they have negative impact on wild pollinators, such as bees. Reducing pesticide use through biological alternatives could minimize the damage to these beneficial insects. Triatoma virus (TrV) is a pathogen able to kill triatomines and thus a valid candidate to be used as biological control agent. In this study we evaluate the capacity of TrV to infect an important beneficial insect (Apis mellifera) as well as a plague insect (Aedes aegypti). Results indicate that TrV does not infect the bees or mosquitoes tested in this study. The possible specificity of TrV for kissing bugs reinforces the possible use of TrV as a biological control agent for triatomines.

Occurrence of honey bee-associated pathogens in Varroa-free pollinator communities.

Australia remains the last significant land mass free of Varroa, a parasitic mite which has caused dramatic honey bee (Apis mellifera) colony losses across the globe, due to its association with the pathogenic deformed wing virus (DWV). As such, Australia continues to maintain relatively healthy honey bee populations, despite recent work showing apiaries harbor a surprisingly high prevalence of microbial pathogens. We sought to determine the prevalence of these microbial pathogens in honey bees and native pollinators actively co-foraging on mass flowering crops and to understand the extent to which they may be shared between taxa. We found high prevalences of black queen cell virus (BQCV) and sacbrood virus (SBV) in the honey bees (88% and 41% respectively), and correspondingly, these were the most common honey bee pathogens detected in native pollinator taxa, albeit at much lower prevalence; the maximum prevalence for any pathogen in a native pollinator group was 24% (BQCV in Halictidae spp.). The viral pathogens Israeli acute paralysis virus and Lake Sinai viruses 1 and 2, and the fungal parasites Nosema apis and Nosema ceranae, were only rarely detected. Phylogenetic analyses of the most common pathogens revealed similar genotypes circulating between species. Our data suggest that, in Australian orchards, pathogen prevalence in honey bees is a good predictor of pathogen prevalence in native pollinators, which raises concerns about how the viral landscape may change in native taxa if, or when, Varroa arrives.

The detection of honey bee (Apis mellifera)-associated viruses in ants.

Interspecies virus transmission involving economically important pollinators, including honey bees (Apis mellifera), has recently sparked research interests regarding pollinator health. Given that ants are common pests within apiaries in the southern U.S., the goals of this study were to (1) survey ants found within or near managed honey bee colonies, (2) document what interactions are occurring between ant pests and managed honey bees, and 3) determine if any of six commonly occurring honey bee-associated viruses were present in ants collected from within or far from apiaries. Ants belonging to 14 genera were observed interacting with managed honey bee colonies in multiple ways, most commonly by robbing sugar resources from within hives. We detected at least one virus in 89% of the ant samples collected from apiary sites (n = 57) and in 15% of ant samples collected at non-apiary sites (n = 20). We found that none of these ant samples tested positive for the replication of Deformed wing virus, Black queen cell virus, or Israeli acute paralysis virus, however. Future studies looking at possible virus transmission between ants and bees could determine whether ants can be considered mechanical vectors of honey bee-associated viruses, making them a potential threat to pollinator health.

Dicistrovirus-Host Molecular Interactions.

Members of the family Dicistroviridae are small RNA viruses containing a monopartite positive-sense RNA genome. Dicistroviruses mainly infect arthropods, causing diseases that impact agriculture and the economy. In this chapter, we provide an overview of current and past research on dicistroviruses including the viral life cycle, viral translational control mechanisms, virus structure, and the use of dicistrovirus infection in Drosophila as a model to identify insect antiviral responses. We then delve into how research on dicistrovirus mechanisms has yielded insights into ribosome dynamics, RNA structure/function and insect innate immunity signaling. Finally, we highlight the diseases caused by dicistroviruses, their impacts on agriculture including the shrimp and honey bee industries, and the potential use of dicistroviruses as biopesticides. Although knowledge of the mechanisms underlying dicistrovirus virus-host interactions is limited, the establishment of the first infectious clone should accelerate the discovery of new mechanistic insights into dicistrovirus infections and pathogenesis.

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.

Flowers as viral hot spots: Honey bees (Apis mellifera) unevenly deposit viruses across plant species.

RNA viruses, once considered specific to honey bees, are suspected of spilling over from managed bees into wild pollinators; however, transmission routes are largely unknown. A widely accepted yet untested hypothesis states that flowers serve as bridges in the transmission of viruses between bees. Here, using a series of controlled experiments with captive bee colonies, we examined the role of flowers in bee virus transmission. We first examined if honey bees deposit viruses on flowers and whether bumble bees become infected after visiting contaminated flowers. We then examined whether plant species differ in their propensity to harbor viruses and if bee visitation rates increase the likelihood of virus deposition on flowers. Our experiment demonstrated, for the first time, that honey bees deposit viruses on flowers. However, the two viruses we examined, black queen cell virus (BQCV) and deformed wing virus (DWV), were not equally distributed across plant species, suggesting that differences in floral traits, virus ecology and/or foraging behavior may mediate the likelihood of deposition. Bumble bees did not become infected after visiting flowers previously visited by honey bees suggesting that transmission via flowers may be a rare occurrence and contingent on multiplicative factors and probabilities such as infectivity of virus strain across bee species, immunocompetence, virus virulence, virus load, and the probability a bumble bee will contact a virus particle on a flower. Our study is among the first to experimentally examine the role of flowers in bee virus transmission and uncovers promising avenues for future research.

Non-self mutation: double-stranded RNA elicits antiviral pathogenic response in a Drosophila model of expanded CAG repeat neurodegenerative diseases.

Inflammation is activated prior to symptoms in neurodegenerative diseases, providing a plausible pathogenic mechanism. Indeed, genetic and pharmacological ablation studies in animal models of several neurodegenerative diseases demonstrate that inflammation is required for pathology. However, while there is growing evidence that inflammation-mediated pathology may be the common mechanism underlying neurodegenerative diseases, including those due to dominantly inherited expanded repeats, the proximal causal agent is unknown. Expanded CAG.CUG repeat double-stranded RNA causes inflammation-mediated pathology when expressed in Drosophila. Repeat dsRNA is recognized by Dicer-2 as a foreign or 'non-self' molecule triggering both antiviral RNA and RNAi pathways. Neither of the RNAi pathway cofactors R2D2 nor loquacious are necessary, indicating antiviral RNA activation. RNA modification enables avoidance of recognition as 'non-self' by the innate inflammatory surveillance system. Human ADAR1 edits RNA conferring 'self' status and when co-expressed with expanded CAG.CUG dsRNA in Drosophila the pathology is lost. Cricket Paralysis Virus protein CrPV-1A is a known antagonist of Argonaute-2 in Drosophila antiviral defense. CrPV-1A co-expression also rescues pathogenesis, confirming anti-viral-RNA response. Repeat expansion mutation therefore confers 'non-self' recognition of endogenous RNA, thereby providing a proximal, autoinflammatory trigger for expanded repeat neurodegenerative diseases.

Direct transmission by injection affects competition among RNA viruses in honeybees.

The arrival of the ectoparasitic mite Varroa destructor on the western honeybee Apis mellifera saw a change in the diversity and prevalence of honeybee RNA viruses. One virus in particular, deformed wing virus (DWV) has become closely associated with V. destructor, leading many to conclude that V. destructor has affected viral virulence by changing the mode of transmission. While DWV is normally transmitted via feeding and faeces, V. destructor transmits viruses by direct injection. This change could have resulted in higher viral prevalence causing increased damage to the bees. Here we test the effect of a change in the mode of transmission on the composition and levels of honeybee RNA viruses in the absence of V. destructor. We find a rapid increase in levels of two viruses, sacbrood virus (SBV) and black queen cell virus (BQCV) after direct injection of viral extracts into honeybee pupae. In pupae injected with high levels of DWV extracted from symptomatic adult bees, DWV levels rapidly decline in the presence of SBV and BQCV. Further, we observe high mortality in honeybee pupae when injected with SBV and BQCV, whereas injecting pupae with high levels of DWV results in near 100% survival. Our results suggest a different explanation for the observed association between V. destructor and DWV. Instead of V. destructor causing an increase in DWV virulence, we hypothesize that direct virus inoculation, such as that mediated by a vector, quickly eliminates the most virulent honeybee viruses resulting in an association with less virulent viruses such as DWV.