Background: Apis mellifera filamentous virus (AmFV) is a large double-stranded DNA virus of uncertain phylogenetic position that infects honey bees (Apis mellifera). Little is known about AmFV evolution or molecular aspects of infection. Accurate annotation of open-reading frames (ORFs) is challenged by weak homology to other known viruses. This study was undertaken to evaluate ORFs (including coding-frame conservation, codon bias, and purifying selection), quantify genetic variation within AmFV, identify host characteristics that covary with infection rate, and examine viral expression patterns in different tissues.
Methods: Short-read data were accessed from the Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI). Sequence reads were downloaded from accessions meeting search criteria and scanned for kmers representative of AmFV genomic sequence. Samples with kmer counts above specified thresholds were downloaded in full for mapping to reference sequences and de novo assembly.
Results: At least three distinct evolutionary lineages of AmFV exist. Clade 1 predominates in Europe but in the Americas and Africa it is replaced by the other clades as infection level increases in hosts. Only clade 3 was found at high relative abundance in hosts with African ancestry, whereas all clades achieved high relative abundance in bees of non-African ancestry. In Europe and Africa, clade 2 was generally detected only in low-level infections but was locally dominant in some North American samples. The geographic distribution of clade 3 was consistent with an introduction to the Americas with 'Africanized' honey bees in the 1950s. Localized genomic regions of very high nucleotide divergence in individual isolates suggest recombination with additional, as-yet unidentified AmFV lineages. A set of 155 high-confidence ORFs was annotated based on evolutionary conservation in six AmFV genome sequences representative of the three clades. Pairwise protein-level identity averaged 94.6% across ORFs (range 77.1-100%), which generally exhibited low evolutionary rates and moderate to strong codon bias. However, no robust example of positive diversifying selection on coding sequence was found in these alignments. Most of the genome was detected in RNA short-read alignments. Transcriptome assembly often yielded contigs in excess of 50 kb and containing ORFs in both orientations, and the termini of long transcripts were associated with tandem repeats. Lower levels of AmFV RNA were detected in brain tissue compared to abdominal tissue, and a distinct set of ORFs had minimal to no detectable expression in brain tissue. A scan of DNA accessions from the parasitic mite Varroa destructor was inconclusive with respect to replication in that species.
Discussion: Collectively, these results expand our understanding of this enigmatic virus, revealing transcriptional complexity and co-evolutionary associations with host lineage.
Keywords: Apis mellifera; Apis mellifera filamentous virus; DNA viruses; Host-pathogen coevolution; Kmer analysis.