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...
Surface waters frequently have been contaminated with human enteric viruses, and it is likely that animal enteric viruses have contaminated surface waters also. Bovine enteroviruses (BEV), found in cattle worldwide, usually cause asymptomatic infections and are excreted in the feces of infected animals in large numbers. In this study, the prevalence and genotype of BEV in a closed herd of cattle were evaluated and compared with BEV found in animals in the immediate environment and in environmental specimens. BEV was found in feces from 76% of cattle, 38% of white-tailed deer, and one of three Canada geese sharing the same pastures, as well as the water obtained from animal watering tanks, from the pasture, from streams running from the pasture to an adjacent river, and from the river, which emptied into the Chesapeake Bay. Furthermore, BEV was found in oysters collected from that river downstream from the farm. These findings suggest that BEV could be used as an indicator of fecal pollution originating from animals (cattle and/or deer). Partial sequence analysis of the viral genomes indicates that different viral variants coexist in the same area. The possibility of identifying the viral strains found in the animals and in the contaminated areas by sequencing the RNA genome, could provide a tool to find the origin of the contamination and should be useful for epidemiological and viral molecular evolution studies.
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