Transmission mechanisms of six honeybee viruses, including 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 bee virus (SBV), in honey bee colonies were investigated by reverse transcription-PCR (RT-PCR) methods. The virus status of individual queens was evaluated by examining the presence of viruses in the queens' feces and tissues, including hemolymph, gut, ovaries, spermatheca, head, and eviscerated body. Except for head tissue, all five tissues as well as queen feces were found to be positive for virus infections. When queens in bee colonies were identified as positive for BQCV, DWV, CBPV, KBV, and SBV, the same viruses were detected in their offspring, including eggs, larvae, and adult workers. On the other hand, when queens were found positive for only two viruses, BQCV and DWV, only these two viruses were detected in their offspring. The presence of viruses in the tissue of ovaries and the detection of the same viruses in queens' eggs and young larvae suggest vertical transmission of viruses from queens to offspring. To our knowledge, this is the first evidence of vertical transmission of viruses in honeybee colonies.The honeybee, Apis mellifera L., plays a vital role in agriculture by assisting in the pollination of a wide variety of crops, with an annual added market value exceeding 15 billion dollars (13). However, the health and vigor of honeybee colonies are threatened by numerous parasites and pathogens, including viruses, bacteria, protozoa, and mites. Among pathogens attacking honeybees, viruses are probably the least understood because of the lack of information about the dynamics underlying viral disease outbreaks.A most crucial stage in the dynamics of virus infections is the mode of virus transmission. In general, transmission of viruses can occur through two pathways: horizontal and vertical transmission. In horizontal transmission, viruses are transmitted among individuals of the same generation, while vertical transmission occurs from adults to their offspring. Transmission can occur through multiple routes in social organisms. Over the past several years, horizontal transmission of honeybee viruses has been documented in bee colonies. experimentally demonstrated that the parasitic mite Varroa destructor obtained deformed wing virus (DWV) from infected bees and acted as a vector to transmit the virus to uninfected bees, which developed morphological deformities or died after mites fed on them for certain periods of time. Our recent study with Kashmir bee virus (KBV) established the role of V. destructor in virus transmission and provided evidence of miteto-brood transmission and mite-to-mite acquisition of viruses in bee colonies (5). Although these results demonstrated horizontal transmission of viruses, virus transmission in honeybees is still not completely understood. For instance, in our subsequent studies with DWV (4, 6), we detected virus in honeybee eggs and young lar...

Deformed wing virus (DWV) can cause wing deformity and premature death in adult honeybees, although like many other bee viruses, DWV generally persists as a latent infection with no apparent symptoms. Using reverse transcription (RT)-PCR and Southern hybridization, we detected DWV in all life stages of honeybees, including adults with and without deformed wings. We also found DWV in the parasitic mite Varroa destructor, suggesting that this mite may be involved in the transmission of DWV. However, the detection of the virus in life stages not normally associated with mite parasitism (i.e., eggs and larvae) suggests that there are other modes of transmission. The levels of DWV in different life stages of bees were investigated by using TaqMan real-time quantitative RT-PCR. The amounts of virus varied significantly in these different stages, and the highest levels occurred in pupae and in adult worker bees with deformed wings. The variability in virus titer may reflect the different abilities of bees to resist DWV infection and replication. The epidemiology of DWV is discussed, and factors such as mite infestation, malnutrition, and climate are also considered.Deformed wing virus (DWV) is a positive-strand RNA virus that was initially isolated from adult honeybees (Apis mellifera) from Japan infested with the parasitic mite Varroa destructor (5, 6). Under laboratory condition, extracts containing DWV particles were injected into young pupal bees, and the resulting newly emerged adults had deformed wings (5). The disease and mortality caused by DWV have often been reported to be associated with outbreaks of V. destructor (11,14,23,24,25), and Bowen-Walker et al. (13) demonstrated experimentally the role of this mite in transmitting DWV from infected to healthy bees. To date, infection of DWV has been reported in honeybee colonies in Europe, Africa, and Asia (5), and we recently provided evidence of its occurrence in the United States (15).Several techniques have been employed for detecting bee viruses, including indirect fluorescent-antibody analysis, agarose gel immunodiffusion, an enzyme-linked immunosorbent assay, and reverse transcription-PCR (RT-PCR) (2,3,4,10,12,17,18,20,21,26,27). While there are techniques for detecting bee viruses, no techniques have been reported for quantification of these viruses. TaqMan real-time RT-PCR is a recently developed technique that allows accurate measurement of virus concentrations and gene expression levels based on fluorescence resonance energy transfer to detect and quantify the amplification product in one step (19). Here, we report the use of TaqMan real-time quantitative RT-PCR to identify DWV in various life stages of honeybees and to determine the relative virus levels in individual adults with and without deformed wings. MATERIALS AND METHODSSample collection. Two honeybee colonies that were maintained in the USDA-ARS Bee Research Laboratory apiaries in Beltsville, Md., had adult worker bees with deformed wings, and were infested heavily with V. destructor were chos...

Israel acute paralysis virus (IAPV) is associated with colony collapse disorder of honey bees. Nonetheless, its role in the pathogenesis of the disorder and its geographic distribution are unclear. Here, we report phylogenetic analysis of IAPV obtained from bees in the United States, Canada, Australia, and Israel and the establishment of diagnostic real-time PCR assays for IAPV detection. Our data indicate the existence of at least three distinct IAPV lineages, two of them circulating in the United States. Analysis of representatives from each proposed lineage suggested the possibility of recombination events and revealed differences in coding sequences that may have implications for virulence.Over the winter of 2006 and 2007, an estimated 23% of all beekeeping operations in the United States experienced losses of hives attributed to colony collapse disorder (CCD) (30). More than 90 cultivated crops depend on the honey bee, Apis mellifera, for pollination; thus, CCD has profound implications for the food supply (23). We recently reported that the presence in hives of the dicistrovirus Israel acute paralysis virus (IAPV) was strongly correlated with the presence of CCD (8).IAPV was first described in 2004 in Israel (21), where infected bees presented with shivering wings, progressed to paralysis, and then died outside the hive. IAPV has features comparable to those of members of the family Dicistroviridae of the superfamily Picornaviridae. Unlike members of the Picornaviridae, which have a single open reading frame (ORF) encoding a single polyprotein, viruses in the family Dicistroviridae have two ORFs encoding two polyproteins. Dicistroviruses have two internal ribosomal entry sites (IRES), one found in the 5Ј untranslated region (UTR) and the other located in the intergenic region between ORF1 and ORF2. Other viruses known to infect honey bees, and related to picornaviruses, are sacbrood virus, deformed wing virus, acute bee paralysis virus (ABPV), Kashmir bee virus (KBV), and black queen cell virus (4, 7).Given the importance of honey bees as pollinators and the reported association between CCD and IAPV, we pursued phylogenetic analysis of geographically discrete IAPV isolates. We report the complete genome sequences of representatives from each of three lineages of IAPV and demonstrate through detailed analysis of four regions of the viral genome (the 5Ј UTR and 5Ј terminus of ORF1; the 3Ј terminus of ORF1, the intergenic region, and the 5Ј terminus of ORF2; ORF2; and the RNA-dependent RNA polymerase [RdRp]) the presence on three continents of at least three clusters of IAPV, two of them currently circulating in the United States. MATERIALS AND METHODSSample collection and RNA extraction. Adult bees were collected from migratory beekeeping operations in the United States with CCD. Diseased apiaries were identified based on evidence of recent collapse of colonies within the apiary and a lack of dead bees in the collapsed colonies. From each colony, 150 adult bees were collected and stored at Ϫ80°C until they w...