Federica Savini

Cervus elaphus papillomavirus (CePV1): New insights on viral evolution in deer

We identified a novel papillomavirus (CePV1) in a fibropapilloma of a 1.5 year old male red deer (Cervus elaphus) shot in the Italian Alps in Brescia province. PV particles were first observed by electron microscopy and PV DNA was then identified by PCR using degenerate primers. Subsequently we cloned the entire genome and determined its complete sequence. CePV1 genome is 8009 bp long and contains all 9 ORFs and the long untranslated regulatory region characteristic for Delta-papillomaviruses. Pairwise nucleotide alignments and phylogenetic analyses based on concatenated E1-E2-L1 ORFs allowed to determine the highest similarity with the Capreolus caprelus papillomavirus CcaPV1. The analysis of the host-parasite phylogenetic tree interactions suggest the co-divergence of CePV1 and C. elaphus while the identified topological incongruences leading us to speculate that CcaPV1 could eventually be the result of an earlier host switch event.

Orf in South Africa: Endemic but neglected

A survey amongst sheep and goat producers and veterinarians was undertaken to collect epidemiological data on orf in South Africa. Previous epidemiological studies on the presence of the disease in the country have not been documented and this report is the first descriptive epidemiological study of orf in South Africa. A seven-month investigation, realised by direct and indirect interviews and field observation, enabled us to outline incidence and risk factors of this disease and to better understand how the local farmers in rural areas relate to it. The results may contribute to better management of the disease in rural areas. By means of molecular analyses the phylogenetic relationships between field isolates from different areas have been identified. The findings gave a first important contribution to the general assessment of the economic impact of orf virus infections and the extent of the risk to human health.

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.

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.

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.

Treatment of equine sarcoids

WE have read with interest the paper by Stadler and others (2011), in which the authors showed the efficacy of topical aciclovir to treat equine sarcoids. We have recently treated three horses showing lesions resembling sarcoids with the antiviral drug cidofovir (1 per cent [w/w]) (Vistide; Pfizer), in combination with sucralfate gel (Sonvico and others 2009). Sarcoid diagnosis was first established by clinical examination: horse 1 (a quarter horse) had a single occult sarcoid lesion in the pastern, …

Molecular and phylogenetic analysis of the filarial nematode Micipsella numidica from the hare Lepus europaeus in Italy

The genus Micipsella comprises three species of filariae to date identified in lagomorphs only, whereas the other genera belonging to the subfamily Splendidofilariinae are described as parasites of birds, reptiles and mammals. In the present study seven specimens of Micipsella numidica (Seurat, 1917), collected from the hare Lepus europaeus in Italy, were characterized genetically by molecular amplification of the mitochondrial genes (12S rDNA; cox1) and the 5S rDNA gene spacer region. Phylogenetic trees inferred using available sequences from filariae and those identified in this study evidenced a close relationship between M. numidica and Splendidofilariinae of other mammals and reptiles (Rumenfilaria andersoni and Madathamugadia hiepei). The present findings, apart from adding new data about the hosts in Italy, support the taxonomic position of M. numidica and highlight the substantial biological and molecular differences existing between Splendidofilariinae and other Onchocercidae. The study also contributes to our knowledge of the molecular/genetic diagnosis of filarial parasites of veterinary and medical concern in any vertebrate or invertebrate host.

Regressing Multiple Viral Plaques and Skin Fragility Syndrome in a Cat Coinfected with FcaPV2 and FcaPV3

Feline viral plaques are uncommon skin lesions clinically characterized by multiple, often pigmented, and slightly raised lesions. Numerous reports suggest that papillomaviruses (PVs) are involved in their development. Immunosuppressed and immunocompetent cats are both affected, the biological behavior is variable, and the regression is possible but rarely documented. Here we report a case of a FIV-positive cat with skin fragility syndrome and regressing multiple viral plaques in which the contemporary presence of two PV types (FcaPV2 and FcaPV3) was demonstrated by combining a quantitative molecular approach to histopathology. The cat, under glucocorticoid therapy for stomatitis and pruritus, developed skin fragility and numerous grouped slightly raised nonulcerated pigmented macules and plaques with histological features of epidermal thickness, mild dysplasia, and presence of koilocytes. Absolute quantification of the viral DNA copies (4555 copies/microliter of FcaPV2 and 8655 copies/microliter of FcaPV3) was obtained. Eighteen months after discontinuation of glucocorticoid therapy skin fragility and viral plaques had resolved. The role of the two viruses cannot be established and it remains undetermined how each of the viruses has contributed to the onset of VP; the spontaneous remission of skin lesions might have been induced by FIV status change over time due to glucocorticoid withdraw and by glucocorticoids withdraw itself.

Bovine papillomatosis: First detection of bovine papillomavirus types 6, 7, 8, 10 and 12 in Italian cattle herds.

Limited information about the distribution of different bovine papillomavirus (BPV) types in Italy is available; therefore, this study aimed to investigate the presence of BPVs in bovine lesions in the Emilia Romagna region. Sixty-four proliferative lesions were collected between december 2011 and december 2014, and subsequently analysed by qualitative PCR with genus- and type-specific primer pairs, as well as rolling circle amplification (RCA). The results demonstrated, for the first time in Italy, the presence of BPV 6, 7, 8, 10 and 12 and also types previously described elsewhere. In addition, the high prevalence of viral co-infections in this sample set provides new information about viral tropism.