Evidence for and against deformed wing virus spillover from honey bees to bumble bees: a reverse genetic analysis

Abstract

Deformed wing virus (DWV) is a persistent pathogen of European honey bees and the major contributor to overwintering colony losses. The prevalence of DWV in honey bees has led to significant concerns about spillover of the virus to other pollinating species. Bumble bees are both a major group of wild and commercially-reared pollinators. Several studies have reported pathogen spillover of DWV from honey bees to bumble bees, but evidence of a sustained viral infection characterized by virus replication and accumulation has yet to be demonstrated. Here we investigate the infectivity and transmission of DWV in bumble bees using the buff-tailed bumble bee Bombus terrestris as a model. We apply a reverse genetics approach combined with controlled laboratory conditions to detect and monitor DWV infection. A novel reverse genetics system for three representative DWV variants, including the two master variants of DWV-type A and B-was used. Our results directly confirm DWV replication in bumble bees but also demonstrate striking resistance to infection by certain transmission routes. Bumble bees may support DWV replication but it is not clear how infection could occur under natural environmental conditions.

Figure 1

Reverse genetics (RG) system for three DWV variants. (a) Diagram showing identical regions between genomic RNA sequences of field DWV variants (DWV-A—NC_004830.2, DWV-B—GenBank AY251269.2, B/A recombinant VDV-1-DWV-No-9—GenBank HM067438.1) and three RG clones (VVV, VDD and VVD); DWV-A and DWV-B specific regions are shown in white and black respectively, regions identical to the VDV-1-DWV-No-9 recombinant are shaded in grey, location of restriction sites introduced into cDNA of each variant is indicated by red marks, DWV genomic RNA organization is presented below to help interpretation. (b) Detection of RG DWV RNA by end-point PCR in honey bee pupae injected with in vitro synthesized RNA transcripts: “VDD”, “VVV”, and “VVD”—PCR products from pupae injected with corresponding full-length RNA, “VVDtrunc”—PCR from pupae, injected with truncated VVD RNA, “+” and “−” —PCR controls, “M”—molecular weight DNA marker; restriction digest—verification of the RG origin of detected DWV by the digest of the PCR products at artificially introduced restriction sites. (c) Detection of RG DWV in bumble bee pupae injected with VVD RNA and with virus stock obtained from RNA-injected bumble bee pupae (“VVDvir”); “VVD RNAtrunc”—PCR from pupae injected with truncated RNA, “Mock”—PBS-injected pupae, “ + ”—positive PCR control for DWV, “M”—molecular weight DNA marker; RG origin of the PCR products for all DWV-positive samples was confirmed by digest with HpaI restriction enzyme, amplification of the actin mRNA product was used as an indicator of RNA integrity and loading control.

Figure 2

Inoculation of bumble bee pupae and larvae with RG-DWV. (a) qPCR analysis of DWV accumulation in bumble bee pupae injected with VVV, VDD and VVD DWV. Pupae were injected at white-eyed (P0-P1)—“we”—and brown-eyed (P7-P8)—“be”—stages and analyzed 48 h post injection. Each value corresponds to an individual sample analyzed, black lines show mean ± SD, “GE” —genome equivalents. (b) Detection of DWV RNA in bumble bee larvae from two different age groups fed with 10⁸ GE VVD DWV: qPCR analysis of DWV levels in individual larvae samples, black-circled values correspond to samples which produced a positive result in (−)RNA assay; each value corresponds to an individual sample analyzed, error bars show mean ± SD, “GE” —genome equivalents. DWV (-)RNA assay—PCR products obtained after strand-specific reverse transcription and run in 1% agarose gel, “ + ”—positive PCR control for DWV, “M”—molecular weight DNA marker, “V”—PCR from the DWV inoculum used for larvae feeding.

Figure 3

Detection of DWV in adult bumble bees. (a) Detection of DWV RNA by end-point PCR in adult bumble bees after inoculation with VVD DWV via feeding or injections (10 samples from each group shown): “Mock” – non-treated bumble bees from the same colony, “PBS”—PBS-injected group (no virus), “10⁷ and 10⁸ fed”—bumble bees fed with sucrose solution containing 10⁷ or 10⁸ DWV GE of DWV per bee, “10⁴ and 10⁸ injected”—bumble bees injected with 10⁴ or 10⁸ DWV GE of DWV respectively. (b) Detection of the DWV (-)RNA strand in adult bumble bees injected with 10⁸ DWV GE. “ + ” and “-”—positive and negative PCR controls, “M”—molecular weight DNA marker. Detection of the bumble bee actin RNA was used to assay the quality of extracted RNA. RG origin of the PCR products for all DWV-positive samples was confirmed by restriction enzyme digest.

Figure 4

Morbidity of DWV in bumble bees. (a) qPCR analysis of DWV level in developed bumble bees injected with 10⁶ GE of VVD DWV at white-eyed (P0-P1) pupa stage. Individual values for each sample are shown with dots, lines represent mean ± SD, “GE” —genome equivalents. (b) Percentage of visually normal (“norm”), discolored (“disc”) and non-viable (“nv”) bumble bees developed from pupae in VVD DWV-injected (n = 43) and PBS-injected (“Mock”, n = 37) groups. (c) Phenotype of bumble bees developed from pupae in the incubator.