ABC Assay: Method Development and Application to Quantify the Role of Three DWV Master Variants in Overwinter Colony Losses of European Honey Bees.

Jessica Kevill,Gideon Mordecai,Andrea Highfield,Stephen Martin,Declan Schroeder

doi:10.3390/v9110314

Abstract

Deformed wing virus (DWV) is one of the most prevalent honey bee viral pathogens in the world. Typical of many RNA viruses, DWV is a quasi-species, which is comprised of a large number of different variants, currently consisting of three master variants: Type A, B, and C. Little is known about the impact of each variant or combinations of variants upon the biology of individual hosts. Therefore, we have developed a new set of master variant-specific DWV primers and a set of standards that allow for the quantification of each of the master variants. Competitive reverse transcriptase polymerase chain reaction (RT-PCR) experimental design confirms that each new DWV primer set is specific to the retrospective master variant. The sensitivity of the ABC assay is dependent on whether DNA or RNA is used as the template and whether other master variants are present in the sample. Comparison of the overall proportions of each master variant within a sample of known diversity, as confirmed by next-generation sequence (NGS) data, validates the efficiency of the ABC assay. The ABC assay was used on archived material from a Devon overwintering colony loss (OCL) 2006–2007 study; further implicating DWV type A and, for the first time, possibly C in the untimely collapse of honey bee colonies. Moreover, in this study DWV type B was not associated with OCL. The use of the ABC assay will allow researchers to quickly and cost effectively pre-screen for the presence of DWV master variants in honey bees.

Highlights

RNA viruses have high mutation rates and exist as a diverse population of variants or quasi-species [1]
It is proposed that acute bee paralysis virus (ABPV), Israeli acute paralysis virus (IAPV), and Kashmir bee virus (KBV) all belong to the same cloud of ABPV variants [5,6]
The sequencing results of the three Deformed wing virus (DWV) variant plasmids confirmed that they could be used in the the PCR assays and for cRNA synthesis for the eventual use in the reverse transcriptase polymerase chain reaction (RT-PCR) assays (Table 2)

Summary

Introduction

RNA viruses have high mutation rates and exist as a diverse population of variants or quasi-species [1]. Quasi-species are a range of variants, genetically linked through mutation and organised around a master sequence or variant [2,3]; they undergo a constant process of mutation, competition, and selection [4]. This provides a cloud of variants with an evolutionary advantage, allowing them to occupy several biological niches; even a single amino acid change can completely alter the pathogenicity of a virus [4]. Of the many RNA viruses that infect the European honey bee, the acute bee paralysis virus (ABPV). Virulence is highly dependent on the mode of transmission and type of the genetic variant; for example, the differences in pathology among different strains of IAPV found globally is likely due to high levels of standing genetic variation [7]

Methods

Results

Conclusion