Replication of honey bee-associated RNA viruses across multiple bee species in apple orchards of Georgia, Germany and Kyrgyzstan.

The essential ecosystem service of pollination is provided largely by insects, which are considered threatened by diverse biotic and abiotic global change pressures. RNA viruses are one such pressure, and have risen in prominence as a major threat for honey bees (Apis mellifera) and global apiculture, as well as a risk factor for other bee species through pathogen spill-over between managed honey bees and sympatric wild pollinator communities. Yet despite their potential role in global bee decline, the prevalence of honey bee-associated RNA viruses in wild bees is poorly known from both geographic and taxonomic perspectives. We screened members of pollinator communities (honey bees, bumble bees and other wild bees belonging to four families) collected from apple orchards in Georgia, Germany and Kyrgyzstan for six common honey bee-associated RNA virus complexes encompassing nine virus targets. The Deformed wing virus complex (DWV genotypes A and B) had the highest prevalence across all localities and host species and was the only virus complex found in wild bee species belonging to all four studied families. Based on amplification of negative-strand viral RNA, we found evidence for viral replication in wild bee species of DWV-A/DWV-B (hosts: Andrena haemorrhoa and several Bombus spp.) and Black queen cell virus (hosts: Anthophora plumipes, several Bombus spp., Osmia bicornis and Xylocopa spp.). Viral amplicon sequences revealed that DWV-A and DWV-B are regionally distinct but identical in two or more bee species at any one site, suggesting virus is shared amongst sympatric bee taxa. This study demonstrates that honey bee associated RNA viruses are geographically and taxonomically widespread, likely infective in wild bee species, and shared across bee taxa.

Viruses of managed alfalfa leafcutting bees (Megachille rotundata Fabricus) and honey bees (Apis mellifera L.) in Western Canada: Incidence, impacts, and prospects of cross-species viral transmission.

We examined whether alfalfa leafcutting bees (ALCB, Megachille rotundata) experienced a higher incidence of seven viruses commonly found honey bees (Apis mellifera) when placed alongside honey bees for hybrid canola seed pollination. Although two viruses - sacbrood virus (SBV) and deformed wing virus (DWV) - were detected in ALCB adults, their presence appeared independent of whether honey bees were present in the same field or not. A further survey of viruses among ALCB adults in three different alfalfa seed growing regions in Western Canada confirmed the ubiquity of sacbrood virus (SBV) as well as the infrequent presence of acute bee paralysis virus (ABPV), both of which had not been previously reported on ALCB. Moreover, SBV and ABPV were detected in the cocoon stage and only in one region. Co-infection among pools of ALCB adults with both of these viruses was more closely correlated with decreasing levels of cocoon viability than infection levels in cocoons themselves. This research suggests ongoing viral transmission between honey bees and ALCB in the same fields is likely low but that co-infection with these viruses may lower ALCB productivity.

Preliminary Survey of Pathogens in the Asian Honey Bee (Apis cerana) in Thailand.

Widespread parasites, along with emerging threats, globalization, and climate change, have greatly affected honey bees' health, leading to colony losses worldwide. In this study, we investigated the detection of biotic stressors (i.e., viruses, microsporidian, bacteria, and fungi) in Apis cerana by surveying the colonies across different regions of Thailand (Chiang Mai in the north, Nong Khai and Khon Kaen in the northeast, and Chumphon and Surat Thani in the south, in addition to the Samui and Pha-ngan islands). In this study, we detected ABPV, BQCV, LSV, and Nosema ceranae in A. cerana samples through RT-PCR. ABPV was only detected from the samples of Chiang Mai, whereas we found BQCV only in those from Chumphon. LSV was detected only in the samples from the Samui and Pha-ngan islands, where historically no managed bees are known. Nosema ceranae was found in all of the regions except for Nong Khai and Khon Kaen in northeastern Thailand. Paenibacillus larvae and Ascosphaera apis were not detected in any of the A. cerana samples in this survey. The phylogenetic tree analysis of the pathogens provided insights into the pathogens' movements and their distribution ranges across different landscapes, indicating the flow of pathogens among the honey bees. Here, we describe the presence of emerging pathogens in the Asian honey bee as a valuable step in our understanding of these pathogens in terms of the decline in eastern honey bee populations.

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.

Detection of Honeybee Viruses in Vespa orientalis.

The Oriental hornet (Vespa orientalis) is spreading across the Italian territory threatening the health and wellbeing of honeybees by feeding on adult individuals and larvae and by plundering hive resources. Considering the capacity of other hornets in harboring honeybee viruses, the aim of this study was to identify the possible role of the Oriental hornet as a vector for honeybee viruses. Adult hornets were subjected to macroscopical examination to identify the presence of lesions, and to biomolecular investigation 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), Sac Brood Virus (SBV). No macroscopical alterations were found while biomolecular results showed that DWV was the most detected virus (25/30), followed by ABPV (19/30), BQCV (13/30), KBV (1/30) and SBV (1/30). No sample was found positive for CBPV. In 20/30 samples several co-infections were identified. The most frequent (17/30) was the association between DWV and ABPV, often associated to BQCV (9/17). One sample (1/30) showed the presence of four different viruses namely DWV, ABPV, BQCV and KBV. The detected viruses are the most widespread in apiaries across the Italian territory suggesting the possible passage from honeybees to V. orientalis, by predation of infected adult honeybees and larvae, and cannibalization of their carcasses. However, to date, it is still not clear if these viruses are replicative but we can suggest a role as mechanical vector of V. orientalis in spreading these viruses.

The Epidemiological Situation of the Managed Honey Bee (Apis mellifera) Colonies in the Italian Region Emilia-Romagna.

The recent decades witnessed the collapse of honey bee colonies at a global level. The major drivers of this collapse include both individual and synergic pathogen actions, threatening the colonies' survival. The need to define the epidemiological pattern of the pathogens that are involved has led to the establishment of monitoring programs in many countries, Italy included. In this framework, the health status of managed honey bees in the Emilia-Romagna region (northern Italy) was assessed, throughout the year 2021, on workers from 31 apiaries to investigate the presence of major known and emerging honey bee pathogens. The prevalence and abundance of DWV, KBV, ABPV, CBPV, Nosema ceranae, and trypanosomatids (Lotmaria passim, Crithidia mellificae, Crithidia bombi) were assessed by molecular methods. The most prevalent pathogen was DWV, followed by CBPV and N. ceranae. Trypanosomatids were not found in any of the samples. Pathogens had different peaks in abundance over the months, showing seasonal trends that were related to the dynamics of both bee colonies and Varroa destructor infestation. For some of the pathogens, a weak but significant correlation was observed between abundance and geographical longitude. The information obtained in this study increases our understanding of the epidemiological situation of bee colonies in Emilia-Romagna and helps us to implement better disease prevention and improved territorial management of honey bee health.

Viruses in the Invasive Hornet Vespa velutina.

The Asian yellow-legged hornet Vespa velutina nigrithorax, a major predator of honeybees, is spreading in Europe in part due to a lack of efficient control methods. In this study, as a first step to identify biological control agents, we characterized viral RNA sequences present in asymptomatic or symptomatic hornets. Among 19 detected viruses, the honey bee virus Deformed wing virus-B was predominant in all the samples, particularly in muscles from the symptomatic hornet, suggesting a putative cause of the deformed wing symptom. Interestingly, two new viruses closely related to Acyrthosiphon pisumvirus and Himetobi Pvirus and viruses typically associated with honey bees, Acute bee paralysis virus and Black queen cell virus, were detected in the brain and muscles, and may correspond to the circulation and possible replication forms of these viruses in the hornet. Aphid lethal paralysis virus, Bee Macula-like virus, and Moku virus, which are known to infect honey bees, were also identified in the gut virus metagenome of hornets. Therefore, our study underlined the urgent need to study the host range of these newly discovered viruses in hornets to determine whether they represent a new threat for honey bees or a hope for the biocontrol of V. velutina.

Molecular Identification of Six Honeybee Viruses in Iranian Apiaries.

The identification of honeybee viruses is of serious importance, particularly considering the lack of information on the natural incidence of viral infections in honeybee populations worldwide. Moreover, the global spread of Varroa destructor in honeybee colonies has a significant effect on the viral infection. In the present study, 160 samples of adult bee from apparently healthy colonies but with a background of parasitic diseases, tremor, and paralysis, were collected during 2011-2012. The samples belonged to 23 different provinces of Iran. They were sent to Razi Vaccine and Serum Research Institute, Karaj, Iran, for further analysis, and examined for the presence of viruses using reverse transcription polymerase chain reaction assay. According to the results, out of 160 samples, 9 (5.8 %), 40 (25.6 %), 12 (7.8 %), 34 (21.8 %), 7 (4.5 %), and 29 (18.5%) cases were positive for acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Kashmir bee virus (KBV), and sacbrood virus (SBV). The samples collected from 18 provinces (78 %) were positive for at least one virus. Among all samples, 83 (53.2 %) specimens were infected with at least one virus. The highest prevalent virus was BQCV, followed by DWV, SBV, CBPV, ABPV, and KBV, respectively.

Artificial ribonucleases inactivate a wide range of viruses using their ribonuclease, membranolytic, and chaotropic-like activities.

Artificial ribonucleases (aRNases) are small compounds catalysing RNA cleavage. Recently we demonstrated that aRNases readily inactivate various viruses in vitro. Here, for three series of aRNases (1,4-diazabicyclo [2.2.2]octane-based and peptide-like compounds) we show that apart from ribonuclease activity the aRNases display chaotropic-like and membranolytic activities. The levels of membranolytic and chaotropic-like activities correlate well with the efficiency of various viruses inactivation (enveloped, non-enveloped, RNA-, DNA-containing). We evaluated the impact of these activities on the efficiency of virus inactivation and found: i) the synergism between membranolytic and chaotropic-like activities is sufficient for the inactivation of enveloped viruses (influenza A, encephalitis, vaccinia viruses) for 1,4-diazabicyclo [2.2.2]octane based aRNases, ii) the inactivation of non-enveloped viruses (encephalomyocarditis, acute bee paralysis viruses) is totally dependent on the synergism of chaotropic-like and ribonuclease activities, iii) ribonuclease activity plays a leading role in the inactivation of RNA viruses by aRNases Dp12F6, Dtr12 and K-D-1, iv) peptide-like aRNases (L2-3, K-2) being effective virus killers have a more specific mode of action. Obtained results clearly demonstrate that aRNases represent a new class of broad-spectrum virus-inactivating agents.