Novel viral RNA genomes of the vine mealybug Planococcus ficus.

The vine mealybug, Planococcus ficus (Signoret, 1875), is the most important insect pest in growing areas of the grapevine Vitis vinifera L. in several countries, including Mexico. In Mexico, Baja California (B.C.) is the region with the highest production of V. vinifera L. grapes for industrial purposes. Recently, the diversity of viruses infecting insects only (insect-specific viruses) has been broadly explored to elucidate further ecological viral-host interactions in many insect species, which in some cases has resulted in the application of virus-based biological control agents for insect pests. However, a survey of the Pl. ficus virome has not been done yet. In the present study, we pooled Pl. ficus individuals collected through different vineyards of Ensenada, B.C., Mexico and analysed them by meta-transcriptomics. Novel nearly complete genomes of five RNA viruses were retrieved. These viruses were related to the Iflaviridae and Reoviridae families, and to the Picornavirales and Tolivirales orders. A new isolate belonging to the Dicistroviridae family was also found. Phylogenetic analyses showed that these putative viral genomes group with viruses having hemipteran (including a mealybug species) or other insect hosts, or with viruses associated with insects. Our results suggest that the identified novel RNA viruses could be insect-specific viruses of Pl. ficus. This work is the first insight into the Pl. ficus virome; it guarantees further studies aimed to characterize those viruses with potential for application in biological control of this economically important insect.

Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees (Apis Mellifera).

Honey bees are of great ecological and agricultural importance around the world but are also subject to a variety of pressures that negatively affect bee health, including exposure to viral pathogens. Such viruses can cause a wide variety of devastating effects and can often be challenging to study due to multiple factors that make it difficult to separate the effects of experimental treatments from preexisting background infection. Here we present a method to mass produce large quantities of virus particles along with a high throughput bioassay to test viral infection and effects. Necessitated by the current lack of a continuous, virus-free honey bee cell line, viral particles are amplified in vivo using honey bee pupae, which are extracted from the hive in large volumes using minimally stressful methodology. These virus particles can then be used in honey bee cage bioassays to test inocula viability, as well as various other virus infection dynamics, including interactions with nutrition, pesticides, and other pathogens. A major advantage of using such particles is that it greatly reduces the chances of introducing unknown variables in subsequent experimentation when compared to current alternatives, such as infection via infected bee hemolymph or homogenate, though care should still be taken when sourcing the bees, to minimize background virus contamination. The cage assays are not a substitute for large-scale, field-realistic experiments testing virus infection effects at a colony level, but instead function as a method to establish baseline viral responses that, in combination with the semi-pure virus particles, can serve as important tools to examine various dimensions of honey bee-virus physiological interactions.

Transcriptomic responses to diet quality and viral infection in Apis mellifera.

BACKGROUND: Parts of Europe and the United States have witnessed dramatic losses in commercially managed honey bees over the past decade to what is considered an unsustainable extent. The large-scale loss of bees has considerable implications for the agricultural economy because bees are one of the leading pollinators of numerous crops. Bee declines have been associated with several interactive factors. Recent studies suggest nutritional and pathogen stress can interactively contribute to bee physiological declines, but the molecular mechanisms underlying interactive effects remain unknown. In this study, we provide insight into this question by using RNA-sequencing to examine how monofloral diets and Israeli acute paralysis virus inoculation influence gene expression patterns in bees. RESULTS: We found a considerable nutritional response, with almost 2000 transcripts changing with diet quality. The majority of these genes were over-represented for nutrient signaling (insulin resistance) and immune response (Notch signaling and JaK-STAT pathways). In our experimental conditions, the transcriptomic response to viral infection was fairly limited. We only found 43 transcripts to be differentially expressed, some with known immune functions (argonaute-2), transcriptional regulation, and muscle contraction. We created contrasts to explore whether protective mechanisms of good diet were due to direct effects on immune function (resistance) or indirect effects on energy availability (tolerance). A similar number of resistance and tolerance candidate differentially expressed genes were found, suggesting both processes may play significant roles in dietary buffering from pathogen infection. CONCLUSIONS: Through transcriptional contrasts and functional enrichment analysis, we contribute to our understanding of the mechanisms underlying feedbacks between nutrition and disease in bees. We also show that comparing results derived from combined analyses across multiple RNA-seq studies may allow researchers to identify transcriptomic patterns in bees that are concurrently less artificial and less noisy. This work underlines the merits of using data visualization techniques and multiple datasets to interpret RNA-sequencing studies.

Interacting stressors matter: diet quality and virus infection in honeybee health.

Honeybee population declines have been linked to multiple stressors, including reduced diet diversity and increased exposure to understudied viral pathogens. Despite interest in these factors, few experimental studies have explored the interaction between diet diversity and viral infection in honeybees. Here, we used a mixture of laboratory cage and small semi-field nucleus hive experiments to determine how these factors interact. In laboratory experiments, we found that high-quality diets (polyfloral pollen and high-quality single-source pollen) have the potential to reduce mortality in the face of infection with Israeli acute paralysis virus (IAPV). There was a significant interaction between diet and virus infection on mortality, even in the presence of high virus titres, suggesting that good diets can help bees tolerate virus infection. Further, we found that extreme stress in the form of pollen starvation in conjunction with IAPV infection increase exiting behaviour from small experimental hives. Finally, we showed that higher-quality pollen diets have significantly higher iron and calcium content, suggesting micronutrient deficiencies could be an under-explored area of bee nutrition.

Honey Bee Viruses in Wild Bees: Viral Prevalence, Loads, and Experimental Inoculation.

Evidence of inter-species pathogen transmission from managed to wild bees has sparked concern that emerging diseases could be causing or exacerbating wild bee declines. While some pathogens, like RNA viruses, have been found in pollen and wild bees, the threat these viruses pose to wild bees is largely unknown. Here, we tested 169 bees, representing 4 families and 8 genera, for five common honey bee (Apis mellifera) viruses, finding that more than 80% of wild bees harbored at least one virus. We also quantified virus titers in these bees, providing, for the first time, an assessment of viral load in a broad spectrum of wild bees. Although virus detection was very common, virus levels in the wild bees were minimal-similar to or lower than foraging honey bees and substantially lower than honey bees collected from hives. Furthermore, when we experimentally inoculated adults of two different bee species (Megachile rotundata and Colletes inaequalis) with a mixture of common viruses that is lethal to honey bees, we saw no effect on short term survival. Overall, we found that honey bee RNA viruses can be commonly detected at low levels in many wild bee species, but we found no evidence that these pathogens cause elevated short-term mortality effects. However, more work on these viruses is greatly needed to assess effects on additional bee species and life stages.

Intensively Cultivated Landscape and Varroa Mite Infestation Are Associated with Reduced Honey Bee Nutritional State.

As key pollinators, honey bees are crucial to many natural and agricultural ecosystems. An important factor in the health of honey bees is the availability of diverse floral resources. However, in many parts of the world, high-intensity agriculture could result in a reduction in honey bee forage. Previous studies have investigated how the landscape surrounding honey bee hives affects some aspects of honey bee health, but to our knowledge there have been no investigations of the effects of intensively cultivated landscapes on indicators of individual bee health such as nutritional physiology and pathogen loads. Furthermore, agricultural landscapes in different regions vary greatly in forage and land management, indicating a need for additional information on the relationship between honey bee health and landscape cultivation. Here, we add to this growing body of information by investigating differences in nutritional physiology between honey bees kept in areas of comparatively low and high cultivation in an area generally high agricultural intensity in the Midwestern United States. We focused on bees collected directly before winter, because overwintering stress poses one of the most serious problems for honey bees in temperate climates. We found that honey bees kept in areas of lower cultivation exhibited higher lipid levels than those kept in areas of high cultivation, but this effect was observed only in colonies that were free of Varroa mites. Furthermore, we found that the presence of mites was associated with lower lipid levels and higher titers of deformed wing virus (DWV), as well as a non-significant trend towards higher overwinter losses. Overall, these results show that mite infestation interacts with landscape, obscuring the effects of landscape alone and suggesting that the benefits of improved foraging landscape could be lost without adequate control of mite infestations.

In vivo and in vitro infection dynamics of honey bee viruses.

The honey bee (Apis mellifera) is commonly infected by multiple viruses. We developed an experimental system for the study of such mixed viral infections in newly emerged honey bees and in the cell line AmE-711, derived from honey bee embryos. When inoculating a mixture of iflavirids [sacbrood bee virus (SBV), deformed wing virus (DWV)] and dicistrovirids [Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV)] in both live bee and cell culture assays, IAPV replicated to higher levels than other viruses despite the fact that SBV was the major component of the inoculum mixture. When a different virus mix composed mainly of the dicistrovirid Kashmir bee virus (KBV) was tested in cell culture, the outcome was a rapid increase in KBV but not IAPV. We also sequenced the complete genome of an isolate of DWV that covertly infects the AmE-711 cell line, and found that this virus does not prevent IAPV and KBV from accumulating to high levels and causing cytopathic effects. These results indicate that different mechanisms of virus-host interaction affect virus dynamics, including complex virus-virus interactions, superinfections, specific virus saturation limits in cells and virus specialization for different cell types.

Pollen Contaminated With Field-Relevant Levels of Cyhalothrin Affects Honey Bee Survival, Nutritional Physiology, and Pollen Consumption Behavior.

Honey bees are exposed to a variety of environmental factors that impact their health, including nutritional stress, pathogens, and pesticides. In particular, there has been increasing evidence that sublethal exposure to pesticides can cause subtle, yet important effects on honey bee health and behavior. Here, we add to this body of knowledge by presenting data on bee-collected pollen containing sublethal levels of cyhalothrin, a pyrethroid insecticide, which, when fed to young honey bees, resulted in significant changes in lifespan, nutritional physiology,and behavior. For the first time, we show that when young, nest-aged bees are presented with pollen containing field-relevant levels of cyhalothrin, they reduce their consumption of contaminated pollen. This indicates that, at least for some chemicals, young bees are able to detect contamination in pollen and change their behavioral response, even if the contamination levels do not prevent foraging honey bees from collecting the contaminated pollen.

Challenges associated with research on RNA viruses of insects.

Dicistroviridae and Iflaviridae (part of the group formerly identified as picorna-like viruses) are rapidly growing families within the order Picornavirales. Work on these emerging groups of arthropod viruses offers a unique and exciting opportunity for virologist, but this task comes with particular challenges. The lack of cell culture systems and infectious clones has imposed limitations on the advancement of study of these viruses. Here we discuss the goals and challenges regarding the establishment of controlled systems as well as some issues associated with insect RNA virology at the organismal level. These concerns apply to RNA viruses affecting other organisms for which basic research tools are limited. A list of pitfalls associated with RNA virus research along with recommendations is provided.

Analysis of new aphid lethal paralysis virus (ALPV) isolates suggests evolution of two ALPV species.

Aphid lethal paralysis virus (ALPV; family Dicistroviridae) was first isolated from the bird cherry-oat aphid, Rhopalosiphum padi. ALPV-like virus sequences have been reported from many insects and insect predators. We identified a new isolate of ALPV (ALPV-AP) from the pea aphid, Acyrthosiphon pisum, and a new isolate (ALPV-DvV) from western corn rootworm, Diabrotica virgifera virgifera. ALPV-AP has an ssRNA genome of 9940 nt. Based on phylogenetic analysis, ALPV-AP was closely related to ALPV-AM, an ALPV isolate from honeybees, Apis mellifera, in Spain and Brookings, SD, USA. The distinct evolutionary branches suggested the existence of two lineages of the ALPV virus. One consisted of ALPV-AP and ALPV-AM, whilst all other isolates of ALPV grouped into the other lineage. The similarity of ALPV-AP and ALPV-AM was up to 88 % at the RNA level, compared with 78-79 % between ALPV-AP and other ALPV isolates. The sequence identity of proteins between ALPV-AP and ALPV-AM was 98-99 % for both ORF1 and ORF2, whilst only 85-87 % for ORF1 and 91-92 % for ORF2 between ALPV-AP and other ALPV isolates. Sequencing of RACE (rapid amplification of cDNA ends) products and cDNA clones of the virus genome revealed sequence variation in the 5' UTRs and in ORF1, indicating that ALPV may be under strong selection pressure, which could have important biological implications for ALPV host range and infectivity. Our results indicated that ALPV-like viruses infect insects in the order Coleoptera, in addition to the orders Hemiptera and Hymenoptera, and we propose that ALPV isolates be classified as two separate viral species.

Substitution of the premembrane and envelope protein genes of Modoc virus with the homologous sequences of West Nile virus generates a chimeric virus that replicates in vertebrate but not mosquito cells.

BACKGROUND: Most known flaviviruses, including West Nile virus (WNV), are maintained in natural transmission cycles between hematophagous arthropods and vertebrate hosts. Other flaviviruses such as Modoc virus (MODV) and Culex flavivirus (CxFV) have host ranges restricted to vertebrates and insects, respectively. The genetic elements that modulate the differential host ranges and transmission cycles of these viruses have not been identified. METHODS: Fusion polymerase chain reaction (PCR) was used to replace the capsid (C), premembrane (prM) and envelope (E) genes and the prM-E genes of a full-length MODV infectious cDNA clone with the corresponding regions of WNV and CxFV. Fusion products were directly transfected into baby hamster kidney-derived cells that stably express T7 RNA polymerase. At 4 days post-transfection, aliquots of each supernatant were inoculated onto vertebrate (BHK-21 and Vero) and mosquito (C6/36) cells which were then assayed for evidence of viral infection by reverse transcription-PCR, Western blot and plaque assay. RESULTS: Chimeric virus was recovered in cells transfected with the fusion product containing the prM-E genes of WNV. The virus could infect vertebrate but not mosquito cells. The in vitro replication kinetics and yields of the chimeric virus were similar to MODV but the chimeric virus produced larger plaques. Chimeric virus was not recovered in cells transfected with any of the other fusion products. CONCLUSIONS: Our data indicate that genetic elements outside of the prM-E gene region of MODV condition its vertebrate-specific phenotype.

Lymantria dispar iflavirus 1 (LdIV1), a new model to study iflaviral persistence in lepidopterans.

The cell line IPLB-LD-652Y, derived from the gypsy moth (Lymantria dispar L.), is routinely used to study interactions between viruses and insect hosts. Here we report the full genome sequence and biological characteristics of a small RNA virus, designated Lymantria dispar iflavirus 1 (LdIV1), that was discovered to persistently infect IPLB-LD-652Y. LdIV1 belongs to the genus Iflavirus. LdIV1 formed icosahedral particles of approx. 30 nm in diameter and contained a 10, 044 nt polyadenylated, positive-sense RNA genome encoding a predicted polyprotein of 2980 aa. LdIV1 was induced by a viral suppressor of RNA silencing, suggesting that acute infection is restricted by RNA interference (RNAi). We detected LdIV1 in all tested tissues of gypsy-moth larvae and adults, but the virus was absent from other L. dispar-derived cell lines. We confirmed LdIV1 infectivity in two of these cell lines (IPLB-LD-652 and IPLB-LdFB). Our results provide a novel system to explore persistent infections in lepidopterans and a new model for the study of iflaviruses, a rapidly expanding group of viruses, many of which covertly infect their hosts.

RNA silencing against geminivirus: complementary action of posttranscriptional gene silencing and transcriptional gene silencing in host recovery.

RNA silencing in plants is a natural defense system mechanism against invading nucleic acids such as viruses. Geminiviruses, a family of plant viruses characterized by a circular, single-stranded DNA genome, are thought to be both inducers and targets of RNA silencing. Some natural geminivirus-host interactions lead to symptom remission or host recovery, a process commonly associated with RNA silencing-mediated defense. Pepper golden mosaic virus (PepGMV)-infected pepper plants show a recovery phenotype, which has been associated with the presence of virus-derived small RNAs. The results presented here suggest that PepGMV is targeted by both posttranscriptional and transcriptional gene silencing mechanisms. Two types of virus-related small interfering RNAs (siRNAs) were detected: siRNAs of 21 to 22 nucleotides (nt) in size that are related to the coding regions (Rep, TrAP, REn, and movement protein genes) and a 24-nt population primarily associated to the intergenic regions. Methylation levels of the PepGMV A intergenic and coat protein (CP) coding region were measured by a bisulfite sequencing approach. An inverse correlation was observed between the methylation status of the intergenic region and the concentration of viral DNA and symptom severity. The intergenic region also showed a methylation profile conserved in all times analyzed. The CP region, on the other hand, did not show a defined profile, and its methylation density was significantly lower than the one found on the intergenic region. The participation of both PTGS and TGS mechanisms in host recovery is discussed.

Symptom Remission and Specific Resistance of Pepper Plants After Infection by Pepper golden mosaic virus.

ABSTRACT Pepper golden mosaic virus (PepGMV) is an important begomovirus infecting solanaceous crops in Mexico and Central America. Under controlled conditions for growth and inoculation with a low-pressure biolistic device, PepGMV-infected pepper plants consistently showed symptom remission or host recovery 12 to 15 days postinoculation (dpi). Inoculated plants initially developed the characteristic PepGMV symptoms; however, newer leaves presented a significant decrease or disappearance of symptoms. Younger asymptomatic, recovered leaves accumulated lower quantities of viral DNA and transcripts than the ones found in the symptomatic tissue. Nonetheless, viral DNA did not disappear during the evaluation period (up to 35 dpi), suggesting that a population of viral molecules escape from plant defensive mechanisms to maintain a subliminal, symptomless infection. Recovery was correlated with a specific resistance to PepGMV but not to Pepper huasteco yellow vein virus, a different gemi-nivirus commonly found in mixed infections with PepGMV. Virus-related small interfering RNAs were detected in practically all tissues (from symptomatic to recovered leaves) but it was not possible to establish a correlation between concentration and symptom severity. The participation of a posttranscriptional gene silencing mechanism in the recovery process and specific resistance is discussed.

Use of geminiviral vectors for functional genomics.

Virus-induced gene silencing (VIGS) can be used to study the function of a gene by downregulating its expression and analyzing the resulting phenotype. VIGS is a handy tool that is less time consuming and labor intensive than other methods for generating mutants. Geminiviruses are particularly convenient and valuable choices as VIGS vectors in functional genomics. The small size of their DNA genome, the simplicity of the methods for inoculation, their wide host range and their conserved genome organization are just a few of the advantageous characteristics that this group of viruses has to offer. Geminivirus-based vectors have proved to be very efficient in VIGS systems, and further development of these systems will most probably permit their application in studies of the functional genomics of important crops that are recalcitrant to other forms of analysis.