Genomics and proteomics of Apis mellifera filamentous virus isolated from honeybees in China

Abstract

Apis mellifera filamentous virus (AmFV) is a large DNA virus that is endemic in honeybee colonies. The genome sequence of the AmFV Swiss isolate (AmFV CH-C05) has been reported, but so far very few molecular studies have been conducted on this virus. In this study, we isolated and purified AmFV (AmFV CN) from Chinese honeybee (Apis mellifera) colonies and elucidated its genomics and proteomics. Electron microscopy showed ovoid purified virions with dimensions of 300-500 ​× ​210-285 ​nm, wrapping a 3165 ​× ​40 ​nm filamentous nucleocapsid in three figure-eight loops. Unlike AmFV CH-C05, which was reported to have a circular genome, our data suggest that AmFV CN has a linear genome of approximately 493 ​kb. A total of 197 ORFs were identified, among which 36 putative genes including 18 baculoviral homologs were annotated. The overall nucleotide similarity between the CN and CH-C05 isolates was 96.9%. Several ORFs were newly annotated in AmFV CN, including homologs of per os infectivity factor 4 (PIF4) and a putative integrase. Phylogenomic analysis placed AmFVs on a separate branch within the newly proposed virus class Naldaviricetes. Proteomic analysis revealed 47 AmFV virion-associated proteins, of which 14 had over 50% sequence coverage, suggesting that they are likely to be main structural proteins. In addition, all six of the annotated PIFs (PIF-0-5) were identified by proteomics, suggesting that they may function as entry factors in AmFV infection. This study provides fundamental information regarding the molecular biology of AmFV.

Keywords: Apis mellifera filamentous Virus (AmFV); Genome sequence; Naldaviricetes; Proteomics; Structural proteins; per os infectivity factor 4 (PIF4).

Fig. 1

Transmission electron micrographs of purified AmFV. A. Whole virions. B. Representative filamentous nucleocapsid with three figure-eight loops. Scale bar, 200 ​nm.

Fig. 2

Diagram of the AmFV CN genome. The linear genome of AmFV is shown with marked length. The arrows indicate predicted ORFs and direction of transcription. ORFs predicted to be related to DNA replication/metabolism, PIFs, and BROs are shown in red, green, and brown, respectively. ORFs identified by proteomics are displayed in a blue font.

Fig. 3

Phylogenetic tree of members of Naldaviricetes derived from concatenated protein sequences of PIFs. The maximum-likelihood (ML) tree on substitution model (LG ​+ ​G ​+ ​I) is present. Numbers on the nodes indicate ML nonparametric bootstrap supports (1000 replicates). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The classifications of viruses are indicated. Virus abbreviations and sequence accession numbers are: AcMNPV, Autographa californica multiple nucleopolyhedrovirus, NC_001623; CpGV, Cydia pomonella granulovirus, NC_002816; NeleNPV, Neodiprion lecontei nucleopolyhedrovirus, NC_005906; CuniNPV, Culex nigripalpus nucleopolyhedrovirus, NC_003084; GbNV, Gryllus bimaculatus nudivirus, NC_009240; HzNV, Helicoverpa zea nudivirus-2, NC_004156; MdSGHV, Musca domestica salivary gland hypertrophy virus, NC_010671; WSSV, White spot syndrome virus, NC_003225; AmFV CH–C05, NC_027925; AmFV CN, OK392616.