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There is strong evidence that equine parvovirus-hepatitis (EqPV-H) is associated with the onset of Theiler’s disease, an acute hepatic necrosis, in horses. However, the impact of this virus on other hepatopathies remains unknown. The objective of this retrospective study was to evaluate the prevalence and quantify the viral loads of EqPV-H in formalin-fixed, paraffin-embedded equine and donkey livers with various histopathologic abnormalities. The pathologies included cirrhosis, circulatory disorders of the liver, toxic and metabolic hepatic diseases as well as neoplastic and inflammatory diseases (n = 84). Eight normal liver samples were included for comparison as controls. EqPV-H DNA was qualitatively and quantitatively measured by real-time PCR and digital PCR, respectively. The virus was detected in two livers originating from horses diagnosed with abdominal neoplasia and liver metastasis (loads of 5 × 103 and 9.5 × 103 genome equivalents per million cells). The amount of viral nucleic acids measured indicates chronic infection or persistence of EqPV-H, which might have been facilitated by the neoplastic disease. In summary, this study did not provide evidence for EqPV-H being involved in hepatopathies other than Theiler’s disease.

There is growing evidence that equine papillomavirus type 2 (EcPV2) infection is etiologically associated with the development of genital squamous cell carcinoma (SCC) and precursor lesions in equids. However, the precise mechanisms underlying neoplastic progression remain unknown. To allow the study of EcPV2-induced carcinogenesis, we aimed to establish a primary equine cell culture model of EcPV2 infection. Three-dimensional (3D) raft cultures were generated from equine penile perilesional skin, plaques and SCCs. Using histological, molecular biological and immunohistochemical methods, rafts versus corresponding natural tissue sections were compared with regard to morphology, presence of EcPV2 DNA, presence and location of EcPV2 gene transcripts and expression of epithelial, mesenchymal and tumor/proliferation markers. Raft cultures from perilesional skin harboring only a few EcPV2-positive (EcPV2+) cells accurately recapitulated the differentiation process of normal skin, whilst rafts from EcPV2+ penile plaques were structurally organized but showed early hyperplasia. Rafts from EcPV2+ SCCs exhibited pronounced hyperplasia and marked dysplasia. Raft levels of EcPV2 oncogene transcription (E6/E7) and expression of tumor/proliferation markers p53, Ki67 and MCM7 expression positively correlated with neoplastic progression, again reflecting the natural situation. Three-dimensional raft cultures accurately reflected major features of corresponding ex vivo material, thus constituting a valuable new research model to study EcPV2-induced carcinogenesis.

Background There is growing evidence that equine papillomavirus type 2 (EcPV2) infection is causally associated with the development of equine genital squamous cell carcinomas (SCCs). Early stages of disease present clinically as plaques or wart-like lesions which can gradually progress to tumoural lesions. Histologically these lesions are inconsistently described as benign hyperplasia, papilloma, penile intraepithelial neoplasia (PIN), carcinoma in situ (CIS) or SCC. Guidelines for histological classification of early SCC precursor lesions are not precisely defined, leading to potential misdiagnosis. The aim of this study was to identify histologic criteria and diagnostic markers allowing for a more accurate diagnosis of EcPV2-associated equine penile lesions. Results A total of 61 archived equine penile lesions were histologically re-assessed and classified as benign hyperplasia, papilloma, CIS or SCC. From these, 19 representative lesions and adjacent normal skin were comparatively analysed for the presence of EcPV2 DNA and transcripts using PCR and RNA in situ hybridisation (RISH). All lesional samples were positive by EcPV2 PCR and RISH, while adjacent normal skin was negative. RISH analysis yielded signal distribution patterns that allowed distinction of early (hyperplasia, papilloma) from late stage lesions (CIS, SCC). Subsequently, the 19 lesions were further assessed for expression of p53, Ki67, MCM7 and MMP1 by immunohistochemistry (IHC). All four proteins were expressed in both normal and lesional tissue. However, p53 expression was up-regulated in basal keratinocyte layers of papillomas, CIS and SCCs, as well as in upper keratinocyte layers of CIS and SCCs. MCM7 expression was only up-regulated in upper proliferating keratinocyte layers of papillomas, CIS and SCCs. Conclusion This study proposes combining a refined histological protocol for analysis of equine penile lesions with PCR- and/or RISH based EcPV2-screening and p53/MCM7 IHC to more accurately determine the type of lesion. This may help to guide the choice of optimum treatment strategy, especially at early stages of disease.

Equine parvovirus hepatitis (EqPV‐H) was first described in 2018 in a fatal case of Theiler's disease which followed the administration of an equine‐origin biological product. The virus has since been frequently identified in serum and liver tissue of horses affected by Theiler's disease—an acute, severe hepatitis characterised by fulminant hepatic necrosis with a fatal outcome in most cases. EqPV‐H is hepatotropic, appears to be associated with subclinical to severe hepatitis in horses, and is a likely cause of Theiler's disease. Although this disease is most frequently reported following the administration of equine‐origin biological products, it can also occur among in‐contact horses. Horizontal transmission may be iatrogenic, via contaminated equine‐origin biological products such as equine serum, botulism or tetanus antitoxin, and mesenchymal stem cells or by means of the oral route of infection. Other horizontal transmission routes, for example, arthropod vectors, warrant further investigation. A worldwide prevalence of EqPV‐H antibodies and DNA has been reported in asymptomatic horses. EqPV‐H‐positive horses suffering from acute, severe hepatitis have reportedly developed clinical signs including icterus, lethargy, inappetence, and neurological abnormalities and have had increased liver‐associated biochemistry parameters recorded. The most common histopathological abnormalities of the liver have been hepatocellular necrosis, collapse of the lobular architecture, and lymphocytic infiltration. Most horses infected experimentally with EqPV‐H have developed subclinical hepatitis, and close temporal associations between peak viraemia, seroconversion, and the onset of hepatitis have been observed. Based on strong evidence indicating that EqPV‐H causes hepatitis in horses, veterinarians should consider this virus an important differential diagnosis in such cases. Potential risks associated with the administration of equine‐origin biological products must be emphasised.

Equine sarcoids (ES) were diagnosed in 12 Somali wild asses (SWA) (Equus africanus somaliensis) from 10 different institutions of the SWA European Endangered Species Programme from 1976 to 2019. Samples of surgically excised masses, biopsies, or necropsy samples were submitted for histologic and virologic analysis. In addition, tissue samples from one onager (Equus hemionus onager), one kulan (Equus hemionus kulan), and two Hartmann's mountain zebras (HMZ) (Equus zebra hartmannae) were examined. Histology confirmed the diagnosis of ES exhibiting the typical microscopic features. Polymerase chain reaction detected bovine papillomavirus type 1 (BPV1) DNA in eight SWA samples and bovine papillomavirus type 2 (BPV2) DNA in one SWA sample. The onager, kulan, and one HMZ sample tested positive for BPV1. The other HMZ tested positive for BPV1 and BPV2. This is the first report of ES in an onager. Surgical excision was the treatment elected by most veterinarians. A follow-up survey of the cases over several years after clinical diagnosis and therapy revealed variable individual outcome with ES recurrence in four cases. Three SWA and the kulan were euthanized due to the severity of the lesions. Nine affected SWA were males with seven having a sarcoid located at the prepuce. Because a genetic disposition is a risk factor for the development of ES in horses, this may also be true for endangered wild equids with few founder animals in their studbook history. Innovative approaches regarding therapy and prevention of ES in wild equids are therefore highly encouraged.

Equine sarcoids (ES) were diagnosed in 12 Somali wild asses (SWA) (

Papillomavirus-specific DNA was detected in skin lesions collected from an okapi (Okapia johnstoni) in the Zoo Basel. According to the nucleotide sequence analysis, the virus belongs to the genus Deltapapillomavirus. Based on bioinformatics analysis, we propose to designate the newly identified virus as Okapia johnstoni Papillomavirus type 1 (OjPV1). OjPV1 is genetically most closely related to a recently described giraffe (Giraffa camelopardalis) -specific papillomavirus (GcPV1). Of note, the putative oncogenic E5 proteins from OjPV1 and GcPV1 are more conserved than the L1 proteins. This indicates, that the selection pressure on E5 may be more pronounced than that on the otherwise most conserved major capsid protein L1.

Several attempts have been made to categorize equid- and bovid-specific bovine papillomavirus 1 (BPV1) isolates based on sequence tags. This study includes newly determined sequence information from 33 BPV1 isolates of equine, asinine and bovine origin and investigates sequence bias due to host species. Twenty of the viral genomes were sequenced over their entire length and a further thirteen were sequenced, including flanking sequences, at two specific sites, the LCR and the E5 ORF. Alignment and analyses of the sequences did not reveal statistically significant site differences between the sequences of bovine and equid origin. None of the proposed sites of divergence noted by other authors demonstrated significant species-specific characteristics. Our results suggest that BPV1 is shared between equine, asinine and bovine host species, and that viral transfer between bovines and equids is a repeated and ongoing phenomenon.

Equine papillomavirus type 2 (EcPV2) was discovered only recently, but it is found consistently in the context of genital squamous cell carcinomas (SCCs). Since neither cell cultures nor animal models exist, the characterization of this potential disease agent relies on the analysis of patient materials. To analyse the host and viral transcriptome in EcPV2-affected horses, genital tissue samples were collected from horses with EcPV2-positive lesions as well as from healthy EcPV2-negative horses. It was determined by RNA-seq analysis that there were 1957 differentially expressed (DE) host genes between the SCC and control samples. These genes were most abundantly related to DNA replication, cell cycle, extracellular matrix (ECM)-receptor interaction and focal adhesion. By comparison to other cancer studies, MMP1 and IL8 appeared to be potential marker genes for the development of SCCs. Analysis of the viral reads revealed the transcriptional activity of EcPV2 in all SCC samples. While few reads mapped to the structural viral genes, the majority of reads mapped to the non-structural early (E) genes, in particular to E6, E7 and E2/E4. Within these reads a distinct pattern of splicing events, which are essential for the expression of different genes in PV infections, was observed. Additionally, in one sample the integration of EcPV2 DNA into the host genome was detected by DNA-seq and confirmed by PCR. In conclusion, while host MMP1 and IL8 expression and the presence of EcPV2 may be useful markers in genital SCCs, further research on EcPV2-related pathomechanisms may focus on cell cycle-related genes, the viral genes E6, E7 and E2/E4, and integration events.

We present the full-length genome sequence of a new papillomavirus detected in skin lesions collected from a boa (Boa constrictor). Based on the nucleotide sequence analysis, we propose to designate the newly identified virus as Boa constrictor papillomavirus type 1 (BcPV1), a new species in the genus Dyomupapillomavirus.