Recovery of the first full-length genome sequence of a parapoxvirus directly from a clinical sample

Abstract

We recovered the first full-length poxvirus genome, including the terminal hairpin region, directly from complex clinical material using a combination of second generation short read and third generation nanopore sequencing technologies. The complete viral genome sequence was directly recovered from a skin lesion of a grey seal thereby preventing sequence changes due to in vitro passaging of the virus. Subsequent analysis of the proteins encoded by this virus identified genes specific for skin adaptation and pathogenesis of parapoxviruses. These data warrant the classification of seal parapoxvirus, tentatively designated SePPV, as a new species within the genus Parapoxvirus.

Figure 1

Macroscopic (A), electronmicroscopical (B) and histological analysis (C) of a harbor seal infected with seal parapoxvirus. (A) Ulcerative nodular skin lesion were identified on both front fins and the muzzle of the animal. (B) Histological changes of hair follicle epithelium characterized by ballooning degeneration and cytoplasmic eosinophilic inclusion bodies (arrows), magnification 600x. Normal hair follicle epithelium of an unaffected grey seal is shown in the upper right corner, HE, magnification 600x. (C) Electron microscopy pictures of the cytoplasm of a hair follicle epithelial cell isolated from an infected seal skin lesion. Magnification 37,500x. Mature and immature virus like particles were densely packed. The ovoid to lancealate shape of the core virions are clearly visible. (D) In situ hybridization using a digoxigenin-labeled parapoxvirus-specific DNA probe: parapox virus specific signal is detected in hair follicle epithelial cells of the suprabasal layers (arrow), while no specific signal was detected in the basal layers. Left panel represents an overview with a 100 x magnification, right panel shows a blown up of the highlighted area, magnification: 200x.

Figure 2

Genome Characterization of the full-length genome seal parapoxvirus sequence. (A) Sequence alignment of seal parapoxvirus (KY382358) to all four reference genomes (ORFV: AY386264.1, PVNZ: NC_025963.1, BPSV: NC_005337.1, PCPV: NC_013804.1) in the genus parapoxvirus. The alignment was performed using the global alignment program AVID implemented in VISTA (tool for comparative genomics). Alignments were visualized with VISTA point (Calc Window, bp: 100; Min Cons Width, bp: 100; Cons Identity, %: 90 Minimum Y, %:50). The graph represents percent conservation between the aligned sequences at a given coordinate on the base sequence. Highly conserved regions, with a conservation higher than 90%, are shown in pink. (B) G + C genome profile of all reference genomes (AY386264.1, NC_025963.1, NC_005337.1, NC_013804.1) listed in Genbank for parapoxviruses together with the newly identified seal parapoxvirus. Each trace represents the % G + C content of the indicated viral genome. GC content is indicated by the color scheme with blue representing a GC content range from 0–33.3%, black from 33.3–66.6% and red from 66.6–100%. (C) Terminal hairpin sequences of the seal parapoxvirus genome. SePPV hairpin terminus consists of an incomplementary base-paired and AT rich sequence. Telomere resolution sequence is underlined.

Figure 3

Phylogenetic tree analysis based on 47 proteins. Protein sequences were considered being conserved, if the corresponding sequence of SePPV yielded BLASTP alignments over at least 90% of the SePPV protein sequence length with sequences of all 14 representative genomes. The following sequences were used: Red Deer Parapoxvirus (PVNZ) HL953 (NC_025963.1); ORFV (AY386264.1); PCPV (NC_013804.1); BPSV (NC_005337.1); Vaccinia Virus (NC_006998.1); Variola Virus (NC_001611.1); Myxoma Virus (NC_001132.2); swinepox virus (NC_003389.1); deerpox virus (NC_006966.1); sheep pox virus (NC_004002.1); lumpy skin disease virus (NC_003027.1); fowlpox virus (NC_002188.1); rabbit fibroma virus (NC_001266.1); molluscum contagiosum virus (NC_001731.1).