Validation of quantitative real-time RT-PCR assays for the detection of six honeybee viruses

Frank Schurr,Amandine Tison,Laura Militano,Nicolas Cheviron,Fabrice Sircoulomb,Marie-Pierre Rivière,Magali Ribière-Chabert,Richard Thiéry,Eric Dubois

doi:10.1016/j.jviromet.2019.04.020

Abstract

Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV), Chronic bee paralysis virus (CBPV), Deformed wing virus (DWV), Sacbrood virus (SBV) and Varroa destructor virus 1 (VDV1) are the six main honeybee viruses reported in Europe. We assessed the accuracy (trueness and precision) of reverse transcriptase quantitative TaqMan® PCR methods (RT-qPCR) for quantifying ABPV, BQCV, DWV, VDV1 and SBV loads. Once the systematic bias in quantitative results had been corrected (overestimation in ABPV and BQCV quantification and underestimation in that of SBV and VDV1), measurements were taken to determine the viral load ranges for which quantification uncertainty was below ± 1 log10 equivalent of genome copies per bee (hereafter reported as genome copies/bee). The accuracy range of RT-qPCR was found to be between 6.4 and 10.4 log10 genome copies/bee for ABPV, between 3.0 and 10.0 log10 genome copies/bee for BQCV, between 2.4 and 10.4 log10 genome copies/bee for DWV and between 3.4 and 10.4 log10 genome copies/bee for SBV. Outside these ranges, the results’ uncertainty is higher. VDV1 RT-qPCR accuracy was outside accuracy limits for all viral loads. Using these RT-qPCR methods, we quantified viral loads in naturally-infected honeybees. The viral load distribution and clinical signs reported with the honeybee samples allowed us to define a threshold that could be used to differentiate between covert and overt infections. These methods will be useful in diagnosing the main viral infections impairing honeybee health.

Highlights

As many as 15 distinct honeybee virus species-complexes have been reported to infect honeybees in nature with one or more strains or sub-species (de Miranda et al, 2015)
Losses can be greater if colonies are infested by Varroa destructor; this honeybee mite is a vector of Acute bee paralysis virus (ABPV), there is no evidence of viral replication in the mite
We describe the accuracy of five quantitative TaqMan® RT-qPCRs in quantifying ABPV, Black queen cell virus (BQCV), Deformed wing virus (DWV), Sacbrood virus (SBV) and Varroa destructor virus-1 (VDV1) in honeybees

Summary

Introduction

As many as 15 distinct honeybee virus species-complexes have been reported to infect honeybees in nature with one or more strains or sub-species (de Miranda et al, 2015). Acute bee paralysis virus (ABPV; family: Dicistroviridae) normally persists at low viral loads with no obvious symptoms (covert infection). Under certain conditions ABPV can become extremely virulent, reducing adult honeybee and brood populations without any clinical signs being observed in the field (de Miranda and Genersch, 2010). When honeybees were experimentally inoculated with ABPV, paralysis and death were observed five days later (Bailey et al, 1963). Losses can be greater if colonies are infested by Varroa destructor; this honeybee mite is a vector of ABPV, there is no evidence of viral replication in the mite (de Miranda et al, 2010). Larvae infected by Sacbrood virus (SBV; family: Iflaviridae) stop pupating and fluid accumulates between the larva’s body and unshed skin, forming a saccule. Black queen cell virus (BQCV; family: Dicistroviridae) seems to be less harmful than

Methods

Results

Conclusion