A real-time PCR method for quantification of the total and major variant strains of the deformed wing virus

Abstract

European honey bees (Apis mellifera) are critically important to global food production by virtue of their pollination services but are severely threatened by deformed wing virus (DWV) especially in the presence of the external parasite Varroa destructor. DWV exists as many viral strains with the two major variants (DWV-A and DWV-B) varying in virulence. A single plasmid standard was constructed containing three sections for the specific determination of DWV-A (VP2 capsid region), DWV-B (IRES) and a conserved region suitable for total DWV (helicase region). The assays were confirmed as specific and discriminatory with limits of detections of 25, 25 and 50 genome equivalents for DWV-A, DWV-B and total-DWV, respectively. The methods were successfully tested on Apis mellifera and V. destructor samples with varying DWV profiles. The new method determined a more accurate total DWV titre in samples with substantial DWV-B than the method currently described in the COLOSS Beebook. The proposed assays could be utilized for the screening of large quantities of bee material for both a total DWV load overview along with more detailed investigations into DWV-A and DWV-B profiles.

Fig 1. Location of quantification viral primers.

1) DWV-B primer location, 2) DWV-A primer location, 3) Pan-DWV primer location, shown on all seven viral sequences used. Bold and Underlined sequences identify which sequence was used for primer design, numbers correspond to location on genome sequences (Forward primers on the left, Reverse on the right), blue highlighted (*) areas indicate consensus and red areas indicate mismatch locations, diagonal double line break indicates removed sequence section between primer locations.

Fig 2. Standard curves and linear uncertainty plots for Pan-DWV, DWV-A and DWV-B assays.

Standard curves and linearity uncertainty (U_LINi) plots for each primer pair using the single constructed plasmid external standard containing fragments for A.) Pan-DWV, B.) DWV-A and C.) DWV-B. Data for the standard curve represent mean ± SE performed in quadruple. Linear regression performance was determined by calculating the mean bias for each load, with bars representing the linearity uncertainty (U_LINi).

Fig 3. DWV-A, DWV-B and Pan-DWV in A. mellifera from heavily Varroa-infested hives.

The DWV-A, DWV-B and Pan-DWV titres in five individual A. mellifera from the University of Aberdeen apiary employing the single plasmid construct as the external standard. Data presented are DWV genome equivalents per individual (total extracted RNA) mean ± SE performed in triplicate, n = 5.

Fig 4. DWV-A, DWV-B and Pan-DWV in Varroa destructor.

The average DWV-A, DWV-B and Pan-DWV titres in V. destructor from the University of Aberdeen apiary employing the single plasmid construct as the external standard. Data presented are DWV genome equivalents per mite (total RNA from pooled extractions), mean ± SE performed in triplicate of three pools each containing three mites.

Fig 5. DWV-A, DWV-B and Pan-DWV levels in A. mellifera from hives with differing Varroa infestations.

DWV-A, DWV-B and Pan-DWV titres in A. mellifera from three apiaries in Scotland, A) Heavily Varroa-infested colonies at the University of Aberdeen apiary (n = 9), B) Lowly Varroa-infested colonies at an Aberdeenshire apiary (n = 8), and C) Varroa-free colonies at the Beinn Eighe National Park (n = 8). Data presented are DWV genome equivalents per individual (total extracted RNA), mean ± SE performed in triplicate.

Fig 6. DWV-A, DWV-B and Pan-DWV levels in A. mellifera in some English colonies.

DWV-A, DWV-B and Pan-DWV titres in A. mellifera from four apiaries in South East England (Essex n = 4, Bedfordshire n = 4, Mid-Suffolk n = 4, East-Suffolk n = 2) employing the single plasmid construct as the external standard. Data presented are DWV genome equivalents per individual (total extracted RNA), mean ± SE performed in triplicate.

Fig 7. Comparison of the Beebook Method and the Pan-DWV method total DWV titre determination.

Total DWV titres in individual A. mellifera from two locations with different dominant strains as determined by the Beebook Method and the Pan-DWV method. A) University of Aberdeen apiary (DWV-A dominant, n = 8) and B) South East England (E = Essex, n = 4; and M = Mid-Suffolk, n = 4) (DWV-B dominant). Data presented are DWV genome equivalents per individual (total extracted RNA), mean ± SE performed on triplicates.