Christian E. Lange

Clinically Healthy Skin of Dogs Is a Potential Reservoir for Canine Papillomaviruses

ABSTRACT Papillomaviruses have been linked to several skin disorders in the dog. In order to have a suitable diagnostic tool for canine papillomavirus detection, eight PCRs with published primer combinations were evaluated. The most sensitive PCR was used to demonstrate that papillomavirus DNA can be detected on nonlesional skin of dogs.

Three novel canine papillomaviruses support taxonomic clade formation

More than 100 human papillomaviruses (HPVs) have been identified and had their whole genomes sequenced. Most of these HPVs can be classified into three distinct genera, the alpha-, beta- and gamma-papillomaviruses (PVs). Of note, only one or a small number of PVs have been identified for each individual animal species. However, four canine PVs (CPVs) (COPV, CPV2, CPV3 and CPV4) have been described and their entire genomic sequences have been published. Based on their sequence similarities, they belong to three distinct clades. In the present study, circular viral DNA was amplified from three dogs showing signs of pigmented plaques, endophytic papilloma or in situ squamous cell carcinoma. Analysis of the DNA sequences suggested that these are three novel viruses (CPV5, CPV6 and CPV7) whose genomes comprise all the conserved sequence elements of known PVs. The genomes of these seven CPVs were compared in order properly classify them. Interestingly, phylogenetic analyses, as well as pairwise sequence alignments of the putative amino acid sequences, revealed that CPV5 grouped well with CPV3 and CPV4, whereas CPV7 grouped with CPV2 but neither group fitted with other classified PVs. However, CPV6 grouped with COPV, a lambda-PV. Based on this evidence, allocation of CPVs into three distinct clades could therefore be supported. Thus, similar to HPVs, it might be that the known and currently unknown CPVs are related and form just a few clades or genera.

Identification of two novel equine papillomavirus sequences suggests three genera in one cluster.

The number of recognized papillomavirus (PV) species and potential PV genera has dramatically been increasing throughout the past decade. It seems that every host species might potentially harbour a large set of PVs, while the PVs of each species appear to belong to only a few genera. In horses at least three conditions beside the equine sarcoid have been described, being supposedly PV induced namely classical equine papillomas, genital papillomas and aural plaques. We were able to identify the DNA of novel equine PVs (EcPVs) in the two latter disorders where PV involvement had been predicted. Both PV genomes were entirely cloned and sequenced. Both EcPV genomes, one derived from a penile papilloma, the other derived from an ear papilloma contain the characteristic open reading frames (ORFs) E6, E7, E1, E2, L2 and L1, a large non-coding region between the late and early region as well as a small non-coding region between the early and the late region. The viruses were consequently designated as EcPV2 and EcPV3. The genomes of the three equine PVs were analysed and compared with each other and further PVs. Upon phylogenetic analyses the equine PVs group well together. Pairwise alignment of the L1 nucleotide sequences reveals that EcPV1 shares 54.9% identities with EcPV2 and 53.2% with EcPV3. EcPV2 and EcPV3 share 51.3% identities. As the three EcPVs share less than 60% of nucleotide identities in L1, they may be regarded as belonging to different genera.

Novel snake papillomavirus does not cluster with other non-mammalian papillomaviruses

Papillomaviruses (PVs) are associated with the development of neoplasias and have been found in several different species, most of them in humans and other mammals. We identified, cloned and sequenced PV DNA from pigmented papilloma-like lesions of a diamond python (Morelia spilota spilota). This represents the first complete PV genome discovered in a Squamata host (MsPV1). It consists of 7048 nt and contains the characteristic open reading (ORF) frames E6, E7, E1, E2, L1 and L2. The L1 ORF sequence showed the highest percentage of sequence identities to human PV5 (57.9%) and Caribbean manatee (Trichechus manatus) PV1 (55.4%), thus, establishing a new clade. According to phylogenetic analysis, the MsPV1 genome clusters with PVs of mammalian rather than sauropsid hosts.

Four novel papillomavirus sequences support a broad diversity among equine papillomaviruses

Papillomaviruses appear to be species-specific pathogens, and it was suggested that each animal species might harbour its own set of papillomaviruses. However, all approaches addressing the underlying evolutionary phenomena still suffer from very limited data about animal papillomaviruses. In case of the horse for example, only three equine papillomaviruses (EcPVs) have been identified. To further address the situation in this host, suspected papillomavirus-associated lesions were tested for EcPV DNA. Four novel EcPV types were detected and their genomes entirely cloned and sequenced. They display the characteristic organization, with early (E) and late (L) regions harbouring the seven classical open reading frames divided by non-coding regions. They were named EcPVs 4, 5, 6 and 7, according to their dissimilarity to other papillomaviruses. Most L1 nucleotide identities were shared with EcPV2 in case of EcPV4 (62 %) and EcPV5 (60 %) or with EcPV3 in case of EcPV6 (70 %) and EcPV7 (71 %). Thus, EcPVs 4 and 5 may establish novel species within the genus Dyoiota, while EcPVs 6 and 7 might fit into the genus Dyorho and belong to the same species as EcPV3. They were found in genital plaques (EcPV4), aural plaques (EcPV5, EcPV6) or penile masses (EcPV7). Interestingly, PCR analysis revealed the DNA of EcPV2 and EcPV4 as well as of EcPV3 and EcPV6 together in the same tissue samples, respectively. In conclusion, the DNA of four novel EcPV types was identified and cloned. They cluster with the known types and support broad genetic EcPV diversity in at least two of the known clades. Furthermore, PCR assays also provide evidence for EcPV co-infections in horses.

Complete canine papillomavirus life cycle in pigmented lesions

Canine papillomaviruses (CPVs) have been identified in various benign and malignant neoplastic skin disorders. The most frequent manifestations of CPV infections are classical warts and pigmented plaques. Although the etiology of canine oral papillomatosis is well established, knowledge about CPVs role in the development of pigmented plaques remains vague. Indeed, as CPV DNA may frequently be found on clinically healthy canine skin, its mere detection in lesions cannot be regarded as a sufficient indicator of causality. Whether CPVs are actually active in pigmented plaques, a requirement for any conceivable involvement, is consequently an open question. To inquire such viral activity, two distinct clinical cases of canine pigmented lesions were evaluated in greater detail. The histological findings in the two cases supported the clinical diagnosis of pigmented viral plaques. Sequencing of amplified DNA from these lesions revealed the genomes of two novel CPV types, i.e. CPV9 and CPV14, both putatively belonging to the genus Chi. Furthermore, transcription and splicing of corresponding CPV mRNA could be shown by RT-PCR in the respective lesions. Finally, viral particles were detected by electron microscopy in homogenates as well as in nuclei of keratinocytes in pigmented lesions. In conclusion, the results link clinical signs of pigmented plaques to histological changes, the presence of CPV specific DNA, viral gene transcription, and the presence of viral particles in and from the lesions. Thus, the findings outline the entire replicative cycle of CPVs in pigmented plaques, which might help understanding the relationship between these viruses and the associated disorders.

A case of a canine pigmented plaque associated with the presence of a Chi-papillomavirus

The seven fully described canine papillomaviruses (CPVs) have been allocated by sequence comparison and other genetic features into three phylogenetic clades. This largely reflects clinical findings, so each sequence of a newly discovered CPV in combination with clinical and pathological details is a valuable piece of evidence. We hypothesize that the genomic sequence of a new CPV can help to predict clinical features and progression, and that this can be tested in subsequent cases. In this case, a 2-year-old female dachshund-mix presented with papillomatosis clinically and histologically characterized as pigmented viral plaques. PCRs using primers evaluated for CPVs successfully amplified papillomavirus (PV) DNA. Sequencing of the products revealed an unknown PV putatively belonging to the PV genus Chi. Rolling circle amplification was used to amplify the entire viral genome. Sequencing revealed a novel PV, designated as CPV8, which was most closely related (63% homology) to the recently discovered CPV4. CPV4 is associated with benign pigmented plaques in pugs. Phylogenetic analysis based on the nucleotide sequences of four viral genes showed that the novel virus was closest to CPV3, CPV4 and CPV5. The presence of viral DNA was confirmed in the lesions by in situ hybridization using specific probes. CPV8 may consequently be regarded as the fourth member of the Chi-papillomavirus genus. All viruses belonging to this genus induce pigmented plaques in dogs. These findings support the hypothesis that genomic sequences can be useful in predicting the clinical features of CPV infection.

Entire Genomic Sequence of Novel Canine Papillomavirus Type 13

ABSTRACT Papillomaviruses are associated with benign and malignant neoplasias of the skin and mucous membranes. The sequence of a novel canine papillomavirus was determined from DNA detected in the oral cavity of a dog. The sequence of the novel virus canine papillomavirus type 13 (CPV13) shares the highest levels of similarity with the Tau papillomaviruses CPV2 and CPV7.

MULTIPLE PAPILLOMAS IN A DIAMOND PYTHON, MORELIA SPILOTA SPILOTA

A 4-yr-old male diamond python (Morelia spilota spilota) was evaluated for multiple black papillated exophytic skin proliferations and signs of pneumonia. The histopathologic structure of the skin biopsy specimens led to the diagnosis of a benign papilloma-like neoplasia. In this case, papillomavirus DNA could be amplified from a biopsy sample with a broad range polymerase chain reaction. Nested pan-herpes polymerase chain reaction was negative, and herpesvirus inclusion bodies were not found. Because of the histologically benign nature of the papilloma, the skin proliferations were left untreated. Ten mo after the first presentation, the skin lesions had regressed almost completely; 34 mo later, only scars from the biopsies were left.

Novel canine papillomavirus type 18 found in pigmented plaques

A variety of neoplasias of the skin and mucous membranes in humans and animals can be ascribed to papillomavirus infections. Some of these papillomavirus induced neoplasias can undergo malignant transformation, while most remain benign. Papillomavirus infections are often even asymptomatic. Therefore the understanding of these viruses is important for appropriate prevention, diagnostic and therapeutic approaches. The entire genomic sequence of a new canine papillomavirus was determined from DNA detected in pigmented plaques in a pug. The novel canine papillomavirus type 18 (CPV18) falls into the genus Chipapillomavirus whose members have all been isolated form dogs with pigmented plaques, some of them also with squamous cell carcinomas. A small partial sequence of a 2009 isolate from a dog in California is identical with CPV18. Papillomaviruses (PVs) are non-enveloped, double-stranded DNA viruses with a circular genome of about 8.000 base pairs (bp). They are generally host species-specific with some exceptions. Numerous of the known PVs are associated with benign and malignant neoplasias of the skin and mucous membranes in humans and animals, but there is evidence, that asymptomatic infections are more common [1,9,14]. More than 200 human and 140 animal PVs have been characterized, illustrating broad genetic diversity (http://pave.niaid.nih.gov/) [2,6,9]. The identification and study of new animal PVs are important because new PVs are potential pathogens in veterinary medicine, some animal PVs may serve as models for human disease and tools for basic biology, and the analysis of animal PVs helps provide insights into PV evolution. Canine PVs (CPVs) have been found associated with classical exophytic papillomas such as the common canine oral papillomatosis, with endophytic papillomas, with pigmented plaques and in rare