A pedunculated cauliflower-like mass was detected on the left posterior limb of a subadult male red deer (Cervus elaphus) after a hunt in Portugal. Histologically the lesion was classified as cutaneous fibropapilloma. The identification of Cervus elaphus papillomavirus (CePV-1 variant) was based on sequencing of the L1 gene. The L2 sequence revealed a nine-nucleotide deletion, as already reported in the Italian and Hungarian CePV-1, further supporting the theory that this is a distinctive genomic characteristic of this viral variant, as this feature has been found in distinct cases from geographically distant countries. In addition, a coinfection with bovine papillomavirus was evidenced by amplification and sequencing of the E5 gene, confirming the ability of Delta papillomaviruses to cross-infect different animal species and providing more evidence that wildlife may act as reservoir for papillomaviruses affecting domestic species. Papillomavirus infection in red deer has been sporadically described in different European countries; in this work, we describe the identification of a CePV-1 variant infection associated with a red deer fibropapilloma in Portugal.
Skin fibropapillomas and papillomas are benign exophytic tumors that have been associated with papillomavirus (PV) infection (Sundberg and Nielsen 1981). Papillomaviruses are small nonenveloped DNA viruses belonging to the family Papillomaviridae, comprising 49 genera with 183 and 330 PV types currently listed as reference genomes for human and animals, respectively, in the Papillomavirus Episteme (National Institute of Allergy and Infectious Diseases 2019). Some PVs cause asymptomatic infections in the skin and mucosa, whereas others produce different lesions, from highly productive self-limited warts to invasive cancers (Bravo and Félez-Sánchez 2015). Even though human PVs have been the most studied because of their association with some human cancers, in the last 14 yrs animal PVs have been identified in 80 different host species. Research has been focused mainly on macaques (Macaca spp.), domestic cows (Bos taurus), horses (Equus caballus), and dogs (Canis lupus familiaris; Rector and Van Ranst 2013), but an increasing number of PV types have been studied more deeply in order to better understand their evolution and the different diseases that they can cause. Twelve PVs belonging to three different genera have been isolated from cervids and entirely sequenced in Europe, North America, and New Zealand (Groff and Lancaster 1985; Erdélyi et al. 2008; Mengual-Chuliá et al. 2018a, b). Red deer (Cervus elaphus) can be infected by Cervus elaphus PV (CePV-1), belonging to the Epsilon genus, causing pigmented plaque lesions similar to squamous papilloma (Munday et al. 2016), and, from a variant of the Capreolus capreolus PV (CcaPV-1), the Delta PV already-named variant CePV-1v, causing fibropapillomas (Scagliarini et al. 2013). Viral fibropapillomas in red deer have been sporadically described in different European countries, such as Italy, Spain, France, England, Austria, and Hungary (Erdélyi et al. 2009). In particular, CcaPV-1 infection has been identified as an endemic disease in roe deer (Capreolus capreolus) populations of the Carpathian basin in Central Europe (Hungary, Austria, and Croatia), the Czech Republic (Král et al. 2015), and Slovakia (Rajský et al. 2016) and CePV-1v has been reported in two single cases in Italy and Austria (Erdélyi et al. 2009; Scagliarini et al. 2013). We report a cutaneous fibropapilloma in red deer associated with a CePV-1v infection in Portugal.
During the postmortem examination of hunted animals in Rosmaninhal, Idanha-aNova, Portugal, a subadult male red deer presented with a mass on the left posterior limb near the hock. The lesion consisted of a pedunculated cauliflower-like mass with 4×3×3 cm major dimensions and 15-g mass (Fig. 1). The animal had an excellent body condition and the inspection did not reveal other relevant macroscopic lesions in lymph nodes or in internal organs. The whole lesion was fixed in 10% neutral buffered formalin. Tissue was embedded in paraffin, sectioned at 3–4 µm, and stained with H&E, Masson's trichrome, and Van Gieson staining (Mescher 2013). Subsequently, total DNA was extracted from formalin-fixed paraffin-embedded tissue sample. Briefly, 20 mg of tissue section was deparaffinized with Diaphane (Histo-Line Laboratories, Milan, Italy) and the DNA was extracted from the obtained tissue pellet using the NucleoSpin tissue kit (Macherey-Nagel, Dueren, Germany) following the manufacturer's instructions. We checked DNA integrity by amplification of a fragment of the C. elaphus cytochrome oxidase subunit I (Table 1). The PCR reactions were performed using primers specifically designed on the CePV-1v sequence (GenBank no. JQ744282.1) for the amplification of the entire L1 and a segment of the L2 genes (Table 1). Additionally, amplification of the bovine PV 1 (BPV-1) E5 gene was performed as described (Brandt et al. 2008). The obtained amplicons were Sanger sequenced in both strands with the corresponding primers.
The tumor was diagnosed as cutaneous fibropapilloma. Histopathologic examination showed pseudocarcinomatous hyperplasia of the epidermis with orthokeratotic hyperkeratosis and hypergranulosis. Additionally, koilocytes with large, mildly acidophilic vacuolated cytoplasm and intranuclear basophilic inclusions were present in the stratum spinosum. The basal layer of the epidermis was not altered. The dermis had extensive proliferation of fibroblasts and connective tissue with occasional multifocal mononuclear inflammatory infiltrate (Fig. 2). Masson's trichrome stains dermis collagen fibers with a blue color and keratin with a red color. Dermal collagen was stained red with Van Gieson staining. The CoxI gene positivity demonstrated the viability of the genomic DNA of the sample. We identified CePV-1v based on the L1 gene sequencing (GenBank no. MK113078) that showed 99.7% nucleotide and 99.8% amino acid identity with the Italian variant CePV-IT1127 (GenBank no. JQ744282). Sequencing of L2 (GenBank no. MK113079) revealed a nine-nucleotide deletion, causing the lack of three amino acids of the encoded capsid protein. The presence of BPV-1 was evidenced by amplification and sequencing of the E5 oncogenic gene (GenBank no. MK113080).
The viral etiology of fibropapillomas in cervids has already been reported in different European countries but not previously in Portugal. The current rule for the classification of PVs into types and genera is based on nucleotide sequence identity of the L1 gene (de Villiers et al. 2014). In the case described here, the viral origin was confirmed by sequencing of the L1 gene demonstrating the presence of the CePV-1v. Identification of L2 gene deletion in our sample further supports the theory that this is a distinctive genomic characteristic of the CePV-1v, already classified as a variant of the CcaPV-1 infecting roe deer, as this deletion has been found in three different cases from geographically distant countries, Portugal, Italy, and Austria (Erdélyi et al. 2009; Scagliarini et al. 2013). This genomic feature may be used for diagnostic purposes to rapidly distinguish the two viral variants in deer. Also, a coinfection with BPV-1 was detected in the case reported here, supporting the cross-species transmission ability of Delta PV and providing more evidence that wildlife may act as a reservoir for PVs affecting domestic species (Scagliarini et al. 2013; Savini et al. 2016). The identification of cutaneous fibropapilloma in red deer seems relatively rare with only sporadic reports that may be due to difficulties in sample collection. The case reported here confirms that CePV-1v is species specific and is currently circulating throughout Europe.
We thank Rui Miranda and J. Serejo Proença for helping in the collection of the samples.