Additional file 1 of Transmission of deformed wing virus between Varroa destructor foundresses, mite offspring and infested honey bees
Creators: name: Piou, Vincent
name: Schurr, Frank
name: Dubois, Eric
name: Vétillard, Angélique
Publisher: name: figshare
Year: 2022
Subjects: subject: Medicine
subject: Neuroscience
subject: Biotechnology
subject: Evolutionary Biology
subject: FOS: Biological sciences
subject: Ecology
subject: Science Policy
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: 60506 Virology

description: Additional file 1: Text S1. Genome sequence of both DWV-A and DWV-B inocula used for the in vitro experiments. The NEBNext rRNA depletion kit (New England Biolabs; Ipwich, MA, USA; E6310) and NEBNext Ultra II directional RNA kit (New England Biolabs; E7760) were used to prepare the sequencing libraries. Libraries were pooled and sequenced with Nextseq 500 (Illumina Inc., San Diego, CA, USA) using the 150-cycle high-output kit at the sequencing core facility of the Brain &amp; Spine Institute, Paris, France. Average coverage depth was used to calculate the number of read to sample in order to get an average coverage depth of 80×. Subsample reads were de novo assembled using Spades (V3.10.0; Bankevich et al. [77]) into contigs and aligned to the reference genome to generate a consensus sequence. We are grateful to Fabrice Sircoulomb (ANSES Sophia Antipolis), Pierrick Lucas and Yannick Blanchard (ANSES Ploufragan) for performing the sequence analysis. Table S1. Viral copy number quantified by RT-qPCR in inoculated emerging honey bees, viral inocula (calculated viral copy numbers according to the dilutions injected) and foundress mites used in the in vitro study. Table S2. Primers, probe and recombinant plasmids used for RT-qPCR quantification according to Schurr et al. [63]. Table S3. Factors used to convert the viral copy number per PCR into viral copy number per bee head or per mite. Figure S1. Distributions of V. destructor RNA amounts recovered after extraction, in relation to the stage of the mite considered. These variations in the total RNA amount were not taken into account because they had a limited impact on the general reproducibility of viral copy numbers quantified by RT-qPCR. Based on the ratios of RNA recovery in the 118 samples, the maximal variability of the data was estimated to be 0.62 log10 viral copy numbers per mite. The table provides an example with a mean copy number of 105 viral copies per mite and the variations were calculated according to the convertion factor from viral copies per PCR to viral copies per mite. This variation in viral copy number remained within the tolerance limits of ± 1 log10 viral copies per mite. Figure S2. Accuracy profiles of the DWV-A and DWV-B quantitation methods. The assessment of the accuracy of the viral copy numbers quantified in individual bee heads by DWV-A and DWV-B RT-qPCRs was based on the construction and interpretation of accuracy profiles [17]. Crushed bee-head samples were spiked with pC1 and pFab1 recombinant plasmids including DWV-A or DWV-B VP3 coding sequence, respectively (recombinant plasmids used also for standard curves according to Schurr et al. [60] and processed for viral quantitation. Three plasmid load levels (106, 108 and 1010 copies/bee or head) were used to determine the standard deviation of reproducibility (SDR), the tolerance interval (± 2 × SDR), and the mean bias between the theoretical value and the mean of the obtained values of six results per load level. Both RT-qPCR methods quantified the recombinant plasmids within the limits of ± 1.0 log10 copies per bee head. The accuracy of DWV-B RT-qPCR was the lowest using spiked bee-head samples with 108 plasmid copies/bee head. A and B, Quantification accuracy of DWV-A and DWV-B RT-qPCR methods, respectively. Figure S3. Raw viral copy numbers of V. destructor females from our in vitro study. Viral copy numbers were quantified in 9 mites from a naturally infested colony before inoculation. The mites were then transferred on artificially inoculated bee pupae for 4 days (post-inoc). The remaining mites were transferred onto untreated spinning larvae for 12 days until emergence of the bees (emergence). Bees and mites were maintained in an incubator (70% RH, 34.5°C). Figure S4. Boxplot showing the log10 transformed deformed wing virus-variant B (DWV-B) copy number in naturally infected bees, mite foundresses and mite offspring from the in situ study. The proportion of naturally infected individuals is shown on the x-axis. The diamonds indicate the mean value of the log10 transformed viral copy numbers.