Quantitative Real-Time Reverse Transcription-PCR Analysis of Deformed Wing Virus Infection in the Honeybee ( Apis mellifera L.)

Abstract

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...

Characteristics of Honey Bee (Apis Mellifera Carnica, Pollman 1879) Queens Reared in Slovenian Commercial Breeding Stations

“…It seems that a low frequency of Nosema and virus infections exert little influence on the general health and body weight of queen honey bees (Chen et al, 2005;Roy and Kryger, 2012). However, queen rearing methods vary between apiaries, change from year to year, and could exert far greater influence over morphologically-based fitness traits of queen bees.…”

Horizontal transmission of deformed wing virus: pathological consequences in adult bees (Apis mellifera) depend on the transmission route

“…Colonies infested by V. destructor produce a considerable number of pupae damaged by both the mites and DWV transmitted by the mite. These pupae, which contain high levels of DWV (Chen et al, 2005), are removed and cannibalized in the nurse bees' hygienic efforts to combat the parasitic mite syndrome. Therefore, cannibalism of mite/DWV-damaged pupae should represent an efficient route for horizontal transmission of DWV to adult bees.…”

Varroa destructor is an effective vector of Israeli acute paralysis virus in the honeybee, Apis mellifera

“…The interpretation of qRT-PCR data was conducted using the comparative C t method (DDC t method) as described previously (Chen et al, 2005). After the validation of approximately equal amplification efficiencies of immune transcripts and the reference gene ( Supplementary Fig.…”

“…After the validation of approximately equal amplification efficiencies of immune transcripts and the reference gene ( Supplementary Fig. S2a, b, available in JGV Online), the expression of each immune transcript was determined by normalizing the C t value of the target to the reference gene b-actin (Chen et al, 2005) and relative to a calibrator, a sample representing the minimal level of the transcript. Negative controls from both healthy and weak groups (bees at day 0), showing the lowest level of gene expression and not showing significant difference between each other, were used as calibrator individually.…”