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.

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.Geminiviruses are small, single-stranded DNA viruses that cause economically important plant diseases worldwide. Recent reports have shown that geminivirus-infected plants can recover or show reduction of symptoms (1,12,45). Such processes have been correlated to RNA silencing mechanisms. RNA silencing is an ancient mechanism involved in different fundamental processes, such as gene regulation, de novo histone and DNA methylation, establishment of heterochromatin, defense against viruses, and control of transposon mobility (6,13,30,53). Silencing pathways involve the cleavage of a double-stranded, or an imperfect stem-loop, RNA molecule into short 21 to 24 nucleotides (nt) RNAs by a Dicer enzyme. These RNAs, known as short interfering RNAs (siRNAs) and microRNAs, direct the silencing process in a sequence-specific manner (6). RNA silencing can occur at transcriptional (TGS, for transcriptional gene silencing) and posttranscriptional (PTGS) levels. Arabidopsis thaliana has evolved a diversity of RNA silencing pathways, small RNA classes, and Dicer-like (DCL) genes (32, 51). microRNA, trans-acting siRNA, and natural antisense transcript siRNA pathways (involving DCL1, DCL2, and DCL4 genes) are PTGS-related processes that play a crucial role in developmental gene regulation in plants (5,52,57). On the other hand, DCL3 produces 24-nt siRNAs, which operate at a nuclear level, guiding heterochromatin formation and transcriptional repression of tr...

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.

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.

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.