Molecular investigation and genetic characterization of feline leukemia virus (FeLV) in cats referred to a veterinary teaching hospital in Northern Italy.

Feline leukemia virus (FeLV) is responsible for feline leukemia syndrome in domestic cats. The prevention and control of disease caused by FeLV are primarily based on vaccination and identification and isolation of infected subjects. Antigen diagnostic methods, which are the most widely used in clinical practices, can be associated to molecular tests to characterize the FeLV detected. In this study, a quantitative SYBR Green Real-Time PCR (qPCR) assay was used to detect FeLV proviral DNA in blood samples from antigen positive cats referred to a veterinary teaching hospital in Northern Italy in 2018-2021. To genetically characterize the identified viruses, a portion of the viral envelope (env) gene was amplified using six different end-point PCRs and sequenced. Twenty-two of 26 (84.6%) cats included in the study tested positive by qPCR assay. This suggests a high performance of the qPCR adopted but further studies are required to investigate the cause of discordant results between the antigen test and qPCR in four cats. From env gene analysis, 15/22 qPCR-positive cats were infected by FeLV subtype A and 5/15 shown coinfection with subtype B.

Fibroblast-associated protein-α expression and BPV nucleic acid distribution in equine sarcoids.

Sarcoids are the most common cutaneous tumor of equids and are caused by bovine papillomavirus (BPV). Different clinical subtypes of sarcoids are well characterized clinically but not histologically, and it is not known whether viral activity influences the clinical or histological appearance of the tumors. The aim of this study was to verify whether the development of different clinical types of sarcoids or the presence of certain histological features were associated with BPV distribution within the tumor. The presence of BPV was assessed by polymerase chain reaction (PCR) and visualized in histological sections by chromogenic in situ hybridization (CISH) in 74 equine sarcoids. Furthermore, to better characterize the molecular features of neoplastic cells, immunohistochemistry for S100, smooth muscle actin-α (αSMA), and fibroblast-associated protein-α (FAPα) was performed. The presence of BPV was confirmed in all tissues examined by either or both PCR and CISH (72/74, 97% each). Of 70/74 CISH-positive cases, signal distribution appeared as either diffuse (61/70, 87%) or subepithelial (9/70, 13%); the latter was more frequently observed in the verrucous subtype. However, no statistically significant association was found between clinical subtypes and specific histological features or hybridization pattern. Moreover, CISH signal for BPV was not detected in the epidermis overlying sarcoids nor in the tissue surrounding the neoplasms. By immunohistochemistry, αSMA confirmed the myofibroblastic differentiation of neoplastic cells in 28/74 (38%) sarcoids. Using tissue microarrays, FAPα labelling was observed in neoplastic fibroblasts of all sarcoids, suggesting this marker as a potential candidate for the immunohistochemical diagnosis of sarcoids.

Bovine Papillomatosis Hiding a Zoonotic Infection: Epitheliotropic Viruses in Bovine Skin Lesions.

We describe two cases of skin co-infections with epitheliotropic viruses, detected in two cattle during lumpy skin disease (LSD) surveillance in northern Italy. A diagnostic protocol including different molecular methods as well as negative staining electron microscopy was applied to detect the most common viral agents belonging to the family Papillomaviridae, Poxviridae and Herpesviridae which cause skin diseases in cattle. Two specimens were collected from cases clinically diagnosed as papillomatosis and pseudo-LSD. Both skin lesions were shown to harbor more than one viral species. This case report shows, for the first time, co-infection of zoonotic parapoxvirus with bovine papillomavirus and herpesvirus in skin lesions of cattle. In particular, the simultaneous presence of virions morphologically referable to parapoxvirus and papillomavirus confirms that the replication of both viruses in the same lesion can happen and the so-called papillomatosis can bear zoonotic viruses.

Bovine Papillomavirus 1 Gets Out of the Flock: Detection in an Ovine Wart in Sicily.

A proliferative cauliflower lesion was excised from the udder of a sheep. Histological investigation confirmed the macroscopic classification of the lesion as a papilloma, without any fibroblastic proliferation. PCR revealed the presence of bovine papillomavirus (BPV), which was further confirmed by the identification of a Deltapapillomavirus 4 by Next Generation Sequencing analysis. This was subsequently classified as bovine papillomavirus type 1. Negative staining electron microscopy (EM) analyses produced negative test results for papillomavirus particles. RNA in situ hybridization (ISH) confirmed the presence of BPV-1. The results further confirm the ability of BPVs belonging to the Deltapapillomavirus genus to infect distantly related species and to cause lesions that are different from sarcoids.

Epitheliotropic Infections in Wildlife Ruminants From the Central Alps and Stelvio National Park.

The mountain chain of the Alps, represents the habitat of alpine fauna where the red deer (Cervus elaphus) population is the outmost numerous, followed by the chamois (Rupicapra rupicapra) and the alpine ibex (Capra ibex) at higher altitudes. Previous reports showed the circulation of epitheliotropic viruses, belonging to the families Papillomaviridae and Poxviridae, causing skin and mucosal lesions in wild ruminants of the Stelvio National Park, situated in the area. To deepen our knowledge on the natural dynamics of the infections, a passive surveillance on all the cases of proliferative skin and mucosal lesions in wild ruminants was performed. Twenty-seven samples (11 chamois, 10 red deer and 6 ibex) collected from 2008 to 2018 were analyzed by negative staining electron microscopy, histology, and PCR followed by genome sequencing and phylogenetic analyses. Results confirmed the spread of Parapoxvirus of Red Deer in New Zealand (PVNZ) in Italy, and its ability to cause severe lesions i.e., erosions and ulcers in the mouth. We showed for the first time a PVNZ/CePV1v (C. elaphus papillomavirus 1 variant) co-infection identified in one red deer. This result supports previous evidence on the ability of papillomavirus and parapoxvirus to mutually infect the same host tissue. Interestingly two ibex and one chamois showing orf virus (OV) skin lesions were shown to be co-infected with bovine papillomavirus type 1 and 2. The presence of bovine papillomavirus, in orf virus induced lesions of chamois and ibex raises the question of its pathogenetic role in these animal species. For the first time, OV/CePV1v co-infection was demonstrated in another chamois. CePV1v is sporadically reported in red deer throughout Europe and is considered species specific, its identification in a chamois suggests its ability of cross-infecting different animal species. Poxviruses and papillomavirus have been simultaneously detected also in the skin lesions of cattle, bird and human suggesting a possible advantageous interaction between these viruses. Taken together, our findings add further information on the epidemiology and pathogenetic role of epitheliotropic viruses in wild ruminants living in the central Alps and in Stelvio National Park.

Cutaneous Fibropapilloma in a Red Deer (Cervus elaphus) Associated with Cervus elaphus Papillomavirus in Portugal.

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.

Molecular Detection of Bovine Papillomavirus DNA in the Placenta and Blood of Healthy Mares and Respective Foals.

Despite the characteristic species specificity of Papillomaviruses (PVs), the bovine papillomavirus (BPV) types 1, 2, and-more rarely-13, can cross-infect equids, where they are involved in the pathogenesis of sarcoid neoplasms. Sarcoids are locally invasive fibroblastic skin tumors that represent the most common skin neoplasms in horses worldwide. The transmission mechanism of BPV is still controversial in horses. Thus far, direct and indirect routes have been implicated, while vertical transmission has been suggested after the detection of viral DNA in the semen of healthy stallions. Testing of the blood and placenta of non-sarcoid baring mares and their respective foals revealed that the equine placenta can harbor BPV DNA, leading us to speculate a possible prenatal vertical DNA transmission in equids.

p16 Immunostaining of Canine Squamous Cell Carcinomas Is Not Associated with Papillomaviral DNA.

While papillomavirus (PVs) are an established cause of human cancer, few reports have supported a relationship between PV and canine squamous cell carcinomas (SCCs). Human oncogenic PVs lead to an increased expression of the p16 tumor suppressor protein, and the latter can be demonstrated immunohistochemically to support a likely causal relationship between tumor and PV infection. In the present study, archive samples of canine SCC from different anatomical locations were tested by polymerase chain reaction for the presence of PV DNA and by p16 immunohistochemistry. The aims were to investigate the relationship between p16 expression and presence of PV DNA, in order to assess the utility of p16 overexpression as a biomarker of PV infection in canine SCC. A total of 52 SCCs were included. Nine cases (17.3%) showed moderate p16 immunoreactivity, with no association with tumor degree of differentiation, histotype or mitotic activity. The canPVf/FAP64 primers amplified Canis familiaris PV-1 DNA from 3 out of 52 tumors (5.8%), one cutaneous, one oral and one tonsillar SCC. There was no association between PV presence and p16 immunostaining. These results do not support a significant role of PVs in the development of canine SCCs. Additionally, PV infection was apparently not the cause of the p16 immunostaining observed in a subset of canine SCCs. A better awareness of p16 level of expression and cellular function in canine cancer may help to define its diagnostic and prognostic role.

Identification and Characterization of Fringilla coelebs Papillomavirus 1 (FcPV1) in Free-living and Captive Birds in Italy.

A papillomavirus (PV) was identified by negative-staining electron microscopy in skin lesions of two bird species (Fringillidae) in Italy. Genetic analyses revealed an FcPV1 with a low genetic variability in the E6, E7, E1, E2, and L1 genes and the long control region when compared to the FcPV1 reference strain.

Bovine papillomavirus type 7 in Italy: complete genomes and sequence variants.

Two novel bovine papillomavirus type 7 (BPV-7) variants have been identified in teat cutaneous papillomas affecting dairy cows in northern Italy. The entire genome sequences of two BPV-7 Italian variants showed major sequence differences in the long control region (LCR) and in the L2 gene compared to the Japanese reference strain. In order to define the stability of these genetic variants, the L2 and LCR sequences of seven further BPV-7 positive isolates were characterized. An insertion of six amino acids in the L2 structural protein has been detected in all samples while different genetic variants have been identified for the LCR. These findings provide new insights on intra-type variability of BPVs and represent a starting point for future studies aimed at establishing the biological role of the different BPV genomic regions and investigating the pathogenic potential of papillomavirus variants.

Evidence of zoonotic Poxviridae coinfections in clinically diagnosed papillomas using a newly developed mini-array test.

Our study describes a newly developed mini-array test for the rapid detection of poxviruses in animals and humans. The method is based on detection that combines target nucleic acid amplification by polymerase chain reaction and specific hybridization, using enzyme-linked antibodies, allowing identification of zoonotic orthopoxviruses and parapoxviruses in animal and human biological samples. With 100% specificity, the test rules out the possibility of cross-reactions with viral agents causing look-alike diseases. The assay was employed in the field to investigate the causes of several outbreaks of a malignant proliferative skin disease that affected domestic ruminants in Sicily during 2011-2014. Due to specific aspects of the lesions, the animals were clinically diagnosed with papillomatosis. The mini-array test allowed the identification of coinfections caused by more than 1 viral species belonging to the Parapoxvirus and Orthopoxvirus genera, either in goats or in cattle. Our study suggests that the so-called "papillomatosis" can be the result of multiple infections with epitheliotropic viruses, including zoonotic poxviruses that cannot be properly identified with classical diagnostic techniques.

PAPILLOMAVIRUS IN HEALTHY SKIN AND MUCOSA OF WILD RUMINANTS IN THE ITALIAN ALPS.

We investigated healthy skin and mucosal specimens of wild ruminants in the Italian Alps. We identified bovine papillomavirus (BPV)-2 DNA in the healthy skin of wild ruminants and documented coinfection of BPV-1 and Cervus elaphus papillomavirus (CePV)-1 in a healthy red deer (Cervus elaphus). We also demonstrated cross-infections of BPVs of the genus Xipapillomavirus, both as single virus infection and also in association with Deltapapillomavirus types 1 and 2, confirming that host tropism of papillomaviruses is not as species-specific as previously thought. Our results suggest that subclinical infections could be linked to the presence of domestic ruminants sharing the same habitat with wild species and that the wildlife may act as a reservoir for papillomaviruses affecting domestic species.

E5 nucleotide polymorphisms suggest quasispecies occurrence in BPV-1 sub-clinically infected horses.

BPV-1 is known as the main causative agent of equine sarcoid, but the virus has also been detected in skin and blood of healthy horses. Previous reports demonstrated the presence of E5 variants in sarcoids of donkeys and horses; we investigated whether this genetic variability might be also found in BPV-1, PBMC associated, of sub-clinically infected horses. With this aim, we analyzed the E5 gene of 21 BPV-1 strains from diseased and sub-clinically infected horses. Our analyses lead us to demonstrate that multiple sequence variants can be present in the blood of sub-clinically infected horses, with alternative bases corresponding to either synonymous or non-synonymous codons in the E5 oncogene sequences. The results give support to the proposed existence of "equine adapted" BPV-1 strains with the occurrence of viral variants, resembling quasispecies, in clinically healthy horses with viremia.