Novel papillomavirus in a mallard duck with mesenchymal chondroid dermal tumors

Abstract

Papillomaviruses, which are epitheliotropic and may induce epithelial tumors, have been identified in several avian species, including ducks. An adult female mallard duck (Anas platyrhynchos) was admitted to a wildlife rehabilitation center with 2 beige, well-demarcated, firm masses: one in the subcutis under a wing, and the other on a digit of the right foot. After euthanasia, the masses were fixed in formalin for histologic examination. Both tumors had a lobular organization with cartilage cores surrounded by densely cellular interlacing bundles of spindle cells. Neoplastic chondroblasts in both masses, particularly the digital mass, contained basophilic intranuclear inclusion bodies, which consisted of assembly complexes of icosahedral virions of 44-nm diameter. Next-generation sequencing allowed whole genome assembly of a novel papillomavirus (Anas platyrhynchos papillomavirus 2) related most closely to Fulmarus glacialis papillomavirus 1 (59.49% nucleotide identity). Our case supports the observation that certain papillomaviruses can productively infect mesenchymal cells and induce neoplasia.

Papillomaviruses (PVs; Papillomaviridae) are non-enveloped, have an icosahedral capsid, and contain circular dsDNA. ²⁵ Over 130 PV species have been identified in avian species. ⁴ As many as 40% of avian species have an associated species of PV.^4,13 Most PVs have a tropism for epithelia, and can induce epithelial proliferation. ⁴ Two different viral strains, designated Fringilla papillomavirus 1 and 1a (FPV1/1a; Etapapillomavirus 1) have been isolated from epithelial neoplasms on the legs and feet of chaffinches, indicating an association between PV infection and epithelial tumors in birds. ¹³ Avian PVs are similar in size and organization to bovine papillomavirus 1 (BPV1; Deltapapillomavirus 4), with early and late genes in equivalent genomic locations. ¹⁴

The 5.7–8.6-kb PV genome contains several open-reading frames (ORFs) in the coding region. The ORFs encode 2 classes of proteins, early or nonstructural proteins (E1–E9) and late or structural proteins (L1, L2). The core proteins E1, E2, L1, and L2 are present in all PVs and are involved in viral replication and virion capsid formation. The non-coding regulatory region known as the long control region contains the origin of replication and transcription factor binding sites and is located between ORFs L1 and E6.^2,22,25 The E6, E7, and E9 ORFs are present in most avian PVs, in addition to core proteins.^4,24

Mallard ducks (Anas platyrhynchos) inhabit freshwater ponds, marshes, and shallow lakes worldwide. ⁷ Although PVs have been reported in fecal and choanal swabs from mallards, no associated lesions have been described, to our knowledge.^4,27 We report here a novel avian PV detected in 2 mesenchymal neoplasms from a mallard duck.

Materials and methods

A wild adult female mallard that had been unable to fly for several weeks was brought to the Wildlife Rehabilitation Center of Minnesota (Roseville, MN, USA). An ulcerated, 3-cm diameter, spherical mass was attached to the distal phalanx of digit 2 of the right foot (Fig. 1). In addition, an 8-cm long rectangular mass was present in the subcutis under the left wing. The duck was euthanized given a poor prognosis for recovery and release, and the masses were removed, fixed in 10% neutral-buffered formalin, and submitted to the Minnesota Veterinary Diagnostic Laboratory (MVDL; St. Paul, MN, USA) for further investigation. The formalin-fixed masses were trimmed, processed routinely, and 4-µm sections were stained with H&E. Samples from both masses were prepared for electron microscopy. The formalin-fixed tissue (1-mm³ fragments) was post-fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer and further processed as described previously. ¹

Figures 1–6.

Mesenchymal dermal tumors associated with infection by a novel papillomavirus in a mallard duck. Figure 1. The right foot of a mallard duck had an ~3-cm diameter spherical ulcerated mass between phalanges 2 and 3. Figure 2. The mass from the foot is arranged in a lobular pattern. The lobules have cartilaginous cores that are surrounded by spindle cell proliferation. H&E. Figure 3. The core of the lobules in the mass from the right foot is composed of well-differentiated cartilage. Numerous chondrocyte nuclei are expanded by basophilic inclusion bodies. Floccular eosinophilic material is scattered within the extracellular matrix. H&E. Figure 4. The spindle cell population in the periphery of the lobules in the mass from the foot is arranged in interwoven bundles. H&E. Figure 5. The spindle cell population in the axillary mass has marked anisocytosis and anisokaryosis. H&E. Figure 6. The chondrocyte population in the cores of the lobules in the axillary mass has marked anisocytosis and anisokaryosis. H&E stain.

Eight 10-µm thick sections of formalin-fixed, paraffin-embedded material from both masses were processed for next-generation sequencing (NGS) at the Molecular Development Laboratory at the MVDL. The samples were deparaffinized, and DNA was extracted (QIAamp DNA FFPE tissue kit; Qiagen) according to the manufacturer’s protocol. Extracted DNA was processed for library preparation (Nextera Flex DNA library preparation kit; Illumina). Pooled libraries were submitted to the University of Minnesota Genomics Center for sequencing (V3 reagent kit, 75 bp, paired-end cycle, NextSeq 500 mid output mode; Illumina). The fastq files obtained were analyzed by an in-house bioinformatics pipeline for steps such as trimming and quality check with Trimmomatic, and assembled with SPAdes.^3,19 Extracted contigs were analyzed using BLASTx (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine taxonomy. Contigs belonging to PV were subjected to ORF prediction using Vgas and PuMA tools with default parameters.^17,28

We compared our sequences with PV sequences from GenBank and reference genomes available in the curated database Papillomavirus Episteme (PaVE: the papillomavirus knowledge source, 2020; https://pave.niaid.nih.gov/). ²³ The L1 taxonomy tool was used to determine the position of our sequence. All sequences were aligned using MAFFT v.7.450 with scoring matrix BLOSUM62. ⁸ Phylogenetic analysis was performed (Geneious Prime 2020.2.2). ⁹ Maximum-likelihood trees were generated in MEGA7 using complete aligned L1 protein sequences and from full genome alignment using RAxML-NG.^10,11 Maximum-likelihood trees were run with 500 bootstrap replications with a gamma distribution for rates over sites with the Whelan and Goldman (WAG) model. ²⁶

Results

The well-demarcated digital mass abutted phalanges 2 and 3 and the interphalangeal joint, but did not infiltrate these structures in the sections examined. The mass had a lobular pattern with a cartilaginous core surrounded by spindle cells, with various proportions and cellularity (Fig. 2). Basophilic inclusion bodies (IBs) frequently filled and expanded chondrocyte nuclei (Fig. 3). There was ~1 mitotic figure per 2.37 mm² in the chondrocytes. Globular eosinophilic material, up to 50-µm diameter, usually surrounded by a clear halo, was detected in chondroblasts and the extracellular matrix. Cartilaginous cores had small areas of necrosis with pallor of the extracellular matrix and absence of cells. Spindle cells were arranged in interwoven bundles (Fig. 4), and had oblong nuclei with moderate anisokaryosis, eosinophilic cytoplasm, indistinct cell borders, and ~10 mitotic figures per 2.37 mm². Scattered small lymphoplasmacytic deposits were present among the spindle cell population at the interface with the cartilaginous cores.

The axillary mass was similar to the digital mass, but the transition of the spindle cell population to the cartilaginous cores was gradual, and both populations were interspersed in some areas. Both cell types had marked anisocytosis and anisokaryosis (Figs. 5, 6). There were ~18 mitotic figures per 2.37 mm² within the spindle cell populations, and < 1 mitotic figure per 2.37 mm² in the chondroblast population. Intranuclear IBs (INIBs) were far less common in the chondroblasts of the axillary mass than in the mass on the digit.

Electron microscopic (EM) examination of the mass on the digit and the axillary mass confirmed that the INIBs consisted of replicating virus and assembly complexes, which marginalized the chromatin. Viral particles, of average size 44.0 nm (SD 3.0 nm), were either scattered randomly in the nuclei, arranged in paracrystalline arrays, or appeared as whirling filamentous structures (Figs. 7–9).

Figures 7–9.

Electron micrographs of the mass from the foot. Figure 7. A chondrocyte nucleus contains numerous scattered viral particles. Figure 8. Viral particles with an average particle size of 44 nm arranged in paracrystalline arrays within a chondrocyte nucleus. Figure 9. The viral particles are also arranged in whirling filamentous structures in a chondrocyte nucleus.

NGS identified viral DNA in both masses with the characteristics of reported animal PVs. High-quality sequence reads with a Phred score of ≥ 30 were used to assemble the genome. The whole genome of a PV of 8,350 bp was identified in the mass on the digit. A partial 154-bp genome of a PV identified in the axillary mass was identical to a portion of the full genome of the PV from the mass on the digit. The virus was recognized as novel and named Anas platyrhynchos papillomavirus 2 (AplaPV2), by the papillomavirus working group of the International Committee on Taxonomy of Viruses (ICTV). The sequence was submitted to GenBank as accession MW888448.

The GC content of the complete AplaPV2 genome is 48.6%. The genome contains 6 ORFs, which encode 4 early (E1, E2, E6, E7) and 2 late (L1, L2) proteins, and an upstream regulatory region between ORFs L1 and E6. Based on the presence of the upstream regulatory region and overlapping motifs, the genome is arranged in a circle (Fig. 10). The genome was compared to PV sequences in the PaVE database based on the L1 gene (Suppl. Fig. 1). AplaPV2 clustered with avian PVs. It was further compared to of reptile- and fish-origin PVs (Suppl. Fig. 2). The AplaPV2 sequence was compared to avian PVs based on L1 amino acid (Fig. 11) and whole genome sequences (Suppl. Fig. 3). AplaPV2 is most similar to Fulmarus glacialis papillomavirus 1 (FgPV1), with 59.49% nucleotide identity. AplaPV2 forms a different clade and is divergent from previously reported PVs infecting Anseriformes (Fig. 11).

Figure 10.

Diagram of the genome organization of Anas platyrhynchos papillomavirus 2 (AplaPV2). The genome is characterized by 4 early proteins (E1, E2, E6, E7), 2 late proteins (L1, L2), and an upstream regulatory region with E1 and E2 binding motifs.

Figure 11.

Phylogenetic tree of avian papillomaviruses constructed based on complete L1 protein. Tree was constructed in MEGA 7.0 using WAG+G model with the maximum-likelihood method and 500 bootstrap replicates. Our sequence is highlighted in bold.

Discussion

The masses described here in a mallard duck were spindle cell sarcomas with chondroid differentiation associated with a novel PV, AplaPV2. Despite the proximity of the mass of the digit to the articular cartilage of an interphalangeal joint, neither mass appeared to have arisen directly from cartilage based on histologic examination, although chondrocytes of the articular cartilage of the distal phalangeal joint cannot be ruled out as cells of origin. Alternatively, the cell of origin for the masses may be synovial or perivascular mesenchymal stem cells. The neoplasms in the mallard were reminiscent of a mesenchymal neoplasm with cartilaginous differentiation described in a northern fulmar (Procellariiformes). ⁵ In the neoplasm from the fulmar, a PV was detected (FgPV1) that was also by far the closest phylogenetic relative to the highly divergent PV identified in our case. ⁵

The virions found within the IBs averaged 44.0 nm (SD 3.0 nm) diameter, smaller than the reported 50–55 nm size range of PVs and more consistent with the 45–50 nm size of polyomaviruses. ¹² However, formalin fixation may have caused shrinkage of the particles. ¹⁸ The whole viral genome of AplaPV2 was recovered from the mass on the digit, but only a partial, although identical, viral genome sequence (154 bp) was recovered from the axillary mass, both of high sequence quality, and all but confirming that the same virus was present in both masses.

All established PV genomes contain the core ORFs L1 and L2 for structural proteins, as well as E1 and E2 for nonstructural proteins. ⁴ The GC content in AplaPV2 (48.6%) is similar to FgPV1 and within the 47.2–52.9% range of avian PVs. ¹⁴ The arrangement of the genome is also consistent with those of established PVs.^20,21,25 The upstream regulatory region regulates viral replication and transcription of the early and late proteins; its presence in the genome of this novel virus further confirms that this is in fact a PV.

Traditionally, comparison of PVs is based on the L1 gene sequence. ²⁵ AplaPV2 was determined to be a novel PV given that the L1 codon region shared only 59.49% nucleotide identity with FgPV1. ²⁵ AplaPV2 clustered in a different genus than reported duck PVs. ⁴ The PV classification criteria approved by ICTV are based on a combination of nucleotide identity cutoffs (60% for genera, 70% for species) for L1 ORF and the L1 phylogenetically supported taxa. ²⁵ Based on these criteria, AplaPV2 should be assigned as a new species and share the genus Treiszetapapillomavirus with FgPV1. The E9 ORF is absent in AplaPV2, FgPV1, and turtle PVs (Chelonia mydas papillomavirus 1, Caretta caretta papillomavirus 1).^3,6

FgPV1 and AplaPV2 are unusual PVs that infect mesenchymal cells and are associated with mesenchymal proliferations with chondroid differentiation. PVs infect epithelial cells typically, leading to hyperplasia of the strata granulosum and spinosum and neoplasia of cutaneous stratified epithelium.^12,15,16 It is unknown which viral characteristics allow both AplaPV2 and FgPV1 to infect mesenchymal cells and whether the virus induces chondroid differentiation. PVs passively use cellular maturation pathways to assemble complete virions. The latter has been described in papilloma-viral warts in which INIBs are present in more mature epithelial cells of the strata granulosum and corneum rather than the strata basale and spinosum. ¹⁶ Concurrent mesenchymal and epithelial proliferation is a feature of sarcoids and bovine fibropapillomas; no epithelial proliferation was noted with the masses associated with AplaPV2 or FgPV1. ⁵

Our case highlights atypical biological behavior of a novel PV. The fact that FgPV1, the only genetically closely related member of the avian PVs, is able to productively infect differentiated mesenchymal cells suggests that specific genetic variations enabled these viruses to do so. Further investigation of the genetic make-up of these viruses is warranted to understand this unusual biological behavior.