Sequence recombination and conservation of Varroa destructor virus-1 and deformed wing virus in field collected honey bees (Apis mellifera)

Abstract

We sequenced small (s) RNAs from field collected honeybees (Apis mellifera) and bumblebees (Bombuspascuorum) using the Illumina technology. The sRNA reads were assembled and resulting contigs were used to search for virus homologues in GenBank. Matches with Varroadestructor virus-1 (VDV1) and Deformed wing virus (DWV) genomic sequences were obtained for A. mellifera but not B. pascuorum. Further analyses suggested that the prevalent virus population was composed of VDV-1 and a chimera of 5'-DWV-VDV1-DWV-3'. The recombination junctions in the chimera genomes were confirmed by using RT-PCR, cDNA cloning and Sanger sequencing. We then focused on conserved short fragments (CSF, size > 25 nt) in the virus genomes by using GenBank sequences and the deep sequencing data obtained in this study. The majority of CSF sites confirmed conservation at both between-species (GenBank sequences) and within-population (dataset of this study) levels. However, conserved nucleotide positions in the GenBank sequences might be variable at the within-population level. High mutation rates (Pi>10%) were observed at a number of sites using the deep sequencing data, suggesting that sequence conservation might not always be maintained at the population level. Virus-host interactions and strategies for developing RNAi treatments against VDV1/DWV infections are discussed.

Figure 1. Comparison of assembled virus genomes.

(A) A Neighbour Joint tree constructed with 1000 bootstraps. NCBI reference sequences were labelled with virus names followed by the GenBank accession numbers. The Maq assembled sequences were labelled accordingly to the reference sequences used. The Sacbrood virus (SBV) reference genome was used as an outer sequence. (B) SNP profile using the 3 viral reference sequences. (C) SNP profile using the 3 Maq assembled virus genomes.

Figure 2. Virus derived small interfering (vsiRNA) mapping profiles.

(A) Unique (non-redundant) vsiRNAs mapped to either VDV-1 or DWV or KV genomes. Shared reads were excluded. (B) Positions of vsiRNAs mapped to DWV and KV genomes. Shared reads with VDV-1 were excluded. (C) Shared vsiRNAs mapped to all of the three virus genomes. Note that the Y-axis represents both plus and minus strands and the scale is different among the three panels.

Figure 3. Sanger sequencing validation of chimera virus.

(A) Unrooted Neighbour Joint tree of the 5’ recombination junction using the sequences of RT-PCR products amplified by primers F1R1. The tree was constructed with 1000 bootstraps and scores higher than 50% were displayed. Reference sequences are labelled with the NCBI accession numbers followed by virus names. The Sanger sequences obtained in this work (NCBI Accession No: KC691296-KC691301) are labelled as F1 followed by in-house sequence IDs. (B) SNP profile of the 5’ recombination junction using the Sanger sequences, showing a decrease of polymorphism after the recombination junction. (C) Unrooted Neighbour Joint tree of the 3’ recombination junction using the sequences of RT-PCR products amplified by primers F5R5. The tree was constructed with 1000 bootstraps and scores higher than 50% are displayed. Reference sequences are labelled with the NCBI accession numbers followed by virus names. The Sanger sequences obtained in this work (GenBank Accession No: KC691302-KC691308) are labelled as F5 followed by in-house sequence IDs. (D) SNP profile of the 3’ recombination junction using the Sanger sequences, showing that nucleotide SNPs increased after the recombination junction.

Figure 4. Conserved short fragments (CSF) of the virus population.

Between-species conserved regions (consecutively larger than 25 nt, without any SNP) were identified by using sequence alignment of the VDV-1/DWV/KV clade (Figure S5). Within-population SNP was calculated by using the sRNA deep sequencing dataset. Each CSF was labelled using DWV genome positions. Colour coded positions represented within-population mutation rate (Pi) as Pi = 0 (dark green), Pi < 5% (light green), 5% ≤ Pi < 10% (yellow) and Pi ≥ 10% (red). The mutation rate at each nucleotide position was reported in Table S3. Black dots label CSFs with 20 consecutive low Pi sites at the within-population level.