Prevalence and Transmission of Honeybee Viruses

Sample collection.Ten honeybee colonies maintained in the USDA-ARS Bee Research Laboratory apiaries, Beltsville, MD, were chosen randomly for vertical virus transmission studies. Queens were collected from each colony, isolated in a small cage individually with several attending workers, and supplied with honey. A representative brood frame containing eggs and brood of various ages was selected from each colony. Larvae were collected by uncapping brood cells and transferring them to individual microcentrifuge tubes with fine forceps. Adult bees were collected by shaking the frame and gently scraping worker bees into 50-ml conical tubes. About 50 eggs from each colony were collected with a tapered brush. Eggs were washed in 5% Clorox solution for 5 min to minimize possible surface contamination and then rinsed in DNase- and RNase-free water (Invitrogen, Carlsbad, CA) three times. Eggs were subjected to RNA extraction immediately. All samples were stored at −80°C for further analysis.

Queen feces collection.Queens were removed from each colony and initially isolated individually in small queen cages, and then each queen was transferred to a 100- by 15-mm petri dish to allow it to defecate. The clear fecal material was collected with a micropipette; about 20 to 25 μl of feces could be collected from each queen after 30 min of isolation. Collected feces were stored in −80°C freezer until used.

Tissue dissection.After feces collection, the queens were sacrificed for tissue dissection. Each live queen was fixed on the wax top of a dissecting dish with insect pins. Hemolymph was collected with a micropipette tip by making a small hole on the roof of the queen's thorax with a needle to make it bleed. About 15 to 20 μl of hemolymph was collected from each queen. Following hemolymph collection, the abdomen of the queen was opened with scissors. Tissues of spermatheca, gut, and ovaries were carefully separated and pulled out with forceps under a dissecting microscope. The head was cut off from the eviscerated body, and both the head and the remaining body of the queen were saved in different tubes. To prevent possible contamination with hemolymph, all tissues were rinsed with 1× phosphate-buffered saline once and nuclease-free water twice. All of the tissue samples were subjected to subsequent RNA extraction.

RNA extraction.For each colony, total RNA was extracted from 10 samples of larvae, 10 samples of adult bees, 1 sample of 50 eggs, 1 sample of queen feces, and 6 samples of queen tissues, including hemolymph, gut, spermatheca, ovaries, head, and eviscerated body, for a total of 28 samples per colony. Individual samples were homogenized in TRIzol reagent (RNA extraction kit; Invitrogen, Carlsbad, CA), and total RNA was extracted following the manufacturer's standard protocol. RNA samples were dissolved in diethyl pyrocarbonate-treated water in the presence of RNase inhibitor (Invitrogen, Carlsbad, CA). The concentration of total RNA was determined by measuring the absorption at 260 nm, and the purity of RNA was estimated by the absorbance ratio of 260 nm/280 nm using a spectrophotometer with a 50-μl ultramicrovolume cell holder (Ultrospec 3300 pro; Amersham Biosciences). RNA samples were stored at −80°C prior to molecular detection for viruses.

RT-PCR.All RNA samples were tested for the presence of six bee viruses, namely, acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), DWV, KBV, and sacbrood bee virus (SBV) by RT-PCR. The primers used in the study were synthesized by Invitrogen and are shown in Table 1. The GenBank accession numbers for each virus and β-actin are also included in Table 1. The Access RT-PCR system (Promega, Madison, WI) was used for RT-PCR according to the manufacturer's instructions. The reaction was performed in a total volume of 25 μl with a final concentration of 1× avian myeloblastosis virus/Tfl reaction buffer, 0.2 mM of deoxynucleoside triphosphate (dNTP), 1 μM of sense primer, 1 μM of antisense primer, 2 mM of MgSO₄, 0.1 unit of avian myeloblastosis virus reverse transcriptase, 0.1 unit of Tfl DNA polymerase, and 500 ng of total RNA. Amplification was undertaken using the PTC-100 DNA Engine (MJ Research, Waltham, MA) with the following thermal cycling profiles: one cycle at 48°C for 45 min for reverse transcription; 1 cycle of 95°C for 2 min; 40 cycles at 95°C for 30 s, 55°C for 1 min, and 68°C for 2 min; and 1 cycle of 68°C for 7 min. Negative (H₂O) and positive (previously identified positive sample) controls were included in each run of the RT-PCR. PCR products were electrophoresed in 1% agarose gel containing 0.5 μg/ml ethidium bromide and visualized by UV transillumination. A 100-bp DNA ladder (Invitrogen) was included as a standard on each gel.

Densitometry of the RT-PCR products.PCR products of the queen tissues were analyzed by densitometry to provide a semiquantification of virus titers in different tissues. To normalize the results, the quality of all samples was checked by RT-PCR amplification of an internal control, β-actin. The intensities of the PCR bands on the gel were quantified by densitometry (LabWorks 4.0, Image Acquisition and Analysis Software; UVP). The densitometry result of each sample was divided by the densitometry result of the β-actin. The results are expressed as the ratio of the band intensity of the target PCR product to that of β-actin.

Sequencing.The specificity of the RT-PCR assay was confirmed by sequence analysis. RT-PCR bands specific for ABPV, BQCV, CBPV, DWV, KBV, and SBV were excised from the low-melting-temperature agarose gel (Invitrogen, Carlsbad, CA) and purified using Wizard PCR Prep DNA purification system (Promega, Madison, WI). Purified RT-PCR fragments were individually ligated into a TOPO cloning vector (Invitrogen, Carlsbad, CA). Recombinant plasmid DNAs were purified using a Plasmid Mini Prep kit (Bio-Rad, Hercules, CA). The nucleotide sequences of the cloned RT-PCR fragments were determined from both forward and reverse directions. The sequence data of each virus fragment were analyzed using the BLAST server at the National Center for Biotechnology Information, NIH.

Statistical analysis.Statistical analysis was performed using Statistix7 statistical software (Analytic Software). The Fisher's least significant difference comparison of means test was used to analyze for significant differences of virus titers among different tissues of the queens. The results are expressed as the mean ± standard deviation. Differences were considered statistically significant if P was <0.05.

Detection of multiple viruses from queen feces.RT-PCR was performed to screen for presence of bee viruses from samples of queen feces. Of six viruses screened, BQCV and DWV were detected in the samples of queen feces. One hundred percent of feces samples (10/10) collected from individual queens tested positive for the presence of BQCV, and 90% of feces samples (9/10) tested positive for the presence of DWV (Table 2).

Distribution of viruses in the body of queens.The results of RT-PCR analysis on tissues of hemolymph, gut, ovaries, spermatheca, head, and eviscerated body from 10 queens for the presence of six viruses are shown in Table 1. Among the six viruses screened, ABPV was not found in any materials. BQCV, CBPV, DWV, KBV, and SBV were detected in one or more queen tissues. Except for tissues of the head, which were negative for all six viruses, the other five tissue samples were found to be virus positive. The presence of DWV was found in 100% of hemolymph samples, 80% of gut samples, 100% of ovary samples, 80% of spermatheca samples, and 100% of eviscerated body samples. The presence of BQCV was found in 100% of gut samples and 70% of ovary samples. The presence of SBV was found in 40% of ovary samples, 20% of hemolymph samples, and 60% of eviscerated body samples. The presence of CBPV was found in 30% of hemolymph and 40% of eviscerated body samples. The presence of KBV was found in 20% of the eviscerated body samples.

Semiquantification of virus titers in tissues of queens.RT-PCR using the β-actin primers resulted in the amplification of the β-actin fragment from all tissue samples tested, indicating that approximately equal amounts of initial RNAs were used for RT-PCR analysis. Semiquantification of DWV levels in different tissues of queens showed that virus titer in the guts was significantly higher than in any other tissues tested. There was no significant difference in DWV titer between tissues of spermatheca and eviscerated body samples. The lowest relative DWV titers were observed in the tissues of ovaries. The ratios of the band intensity of DWV-specific PCR product relative to that of β-actin were 2.63 ± 0.68, 0.08 ± 0.25, 2.0 ± 0.45, and 2.16 ± 0.49 for gut, ovaries, spermatheca, and eviscerated body, respectively (Fig. 1A).

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FIG. 1.

Semiquantification of bee viruses in tissues of queens. (A to E, top panels) RT-PCR results on tissues of queens for the presence of bee viruses. Six tissues including hemolymph, gut, ovaries, spermatheca, head, and eviscerated body (E. body) were examined for the presence of viruses. Primer pairs specific for five viruses including BQCV, CBPV, DWV, KBV, SBV, and an internal control, β-actin, were used separately to amplify RT-PCR products of 700 bp, 455 bp, 702 bp, 415 bp, 824 bp, and 357 bp, respectively. Negative (H₂O [N]) and positive (P) controls (previously identified positive sample) were included in each run of the RT-PCR. (A to E, bottom panels) Densitometric analyses. The y axis depicts the ratio of band intensity of the virus-specific RT-PCR products to that of β-actin RT-PCR products. Results are expressed as mean ± standard deviation (n = 3). Different letters indicate a significant difference among tissues (P = 0.05). (A) Semiquantification of DWV in tissues of queens. (B) Semiquantification of BQCV in tissues of queens. (C) Semiquantification of SBV in tissues of queens. (D) Semiquantification of CBPV bee viruses in tissues of queens. (E) Semiquantification of KBV bee viruses in tissues of queens.

Semiquantification of BQCV levels showed that virus titer in the guts was significantly higher than that in the ovaries and that the ratios of the band intensity of BQCV-specific PCR product relative to that of β-actin were 1.45 ± 0.98 and 0.25 ± 0.35 for gut and ovaries, respectively (Fig. 1B).

Semiquantification of SBV levels showed that virus titer in the ovaries was significantly lower than that SBV titer in the hemolymph and eviscerated body and that SBV titer in the eviscerated body was significantly lower than in hemolymph. The ratios of the band intensity of SBV-specific PCR product relative to that of β-actin were 1.03 ± 0.60, 0.33 ± 0.30, and 0.52 ± 0.69 for hemolymph, ovaries, and eviscerated body, respectively (Fig. 1C). Infection of CBPV was detected in tissues of the hemolymph and the eviscerated body. The ratios of the band intensity of CBPV PCR product relative to that of β-actin were 0.20 ± 0.38 and 0.18 ± 0.93 for hemolymph and the eviscerated body, respectively. The band intensity of CBPV PCR product varied significantly among different samples of eviscerated body, which caused a high value for the standard deviation in the ratio of the band intensity. There was no significant difference in the titer of CBPV between tissues of hemolymph and eviscerated body (Fig. 1D). The presence of KBV was found only in eviscerated body samples, and the ratio of the band intensity of KBV specific PCR product to that of β-actin was about 0.42 ± 0.69 (Fig. 1E).

Evidence of the vertical transmission of viruses from queens to their offspring.Individual queens that were detected with virus or viruses in their feces or in any one of the dissected tissues were considered to be virus positive, although all of the queens in this study showed no pathological signs of virus infections. Among 10 queens examined, 100% were RT-PCR positive for DWV and BQCV, 60% were SBV positive, 40% were CBPV positive, and 20% were KBV positive. Based on the virus status of the queens, 10 experimental colonies were divided into two groups. In group A, queens were identified to be positive for three to five viruses, while in group B, queens were infected with only two viruses, BQCV and DWV.

The possibility of vertical transmission in the colonies was investigated by examining the virus status of the queens' offspring, including eggs, larvae, and adults in both groups. As shown in Table 3, all of the queens (n = 6) in group A were positive for BQCV, DWV, and SBV; the presence of BQCV, DWV, and SBV was found in 100% of egg samples (6/6), in 27%, 92%, and 25% of larvae, and in 3%, 37%, and 10% of adult bees, respectively. When 67% of the queens (4/6) in group A were positive for CBPV, 50% of egg samples, 17% of larvae, and 17% of adult bees were infected with CBPV. When 33% of the queens in group A were positive for KBV, 33% of egg samples were infected. No KBV was detected in larvae and adult bees. In group B, all of the queens were positive for BQCV and DWV, 100% of egg samples (4/4) were identified to be infected with the same two viruses, 22% of larvae and 5% adult bees were infected with BQCV, and 65% of larvae and13% of adult bees were infected with DWV.

Sequence confirmation of RT-PCR specificity.Sequence analysis showed that sequences of selected RT-PCR fragments representing individual viruses, including BQCV, CBPV, DWV, KBV, and SBV, and β-actin matched the corresponding virus sequences published in GenBank, indicating that amplification bands in this study were virus specific.