Outbreak of contagious ecthyma caused by Orf virus (Parapoxvirus ovis) in a vaccinated sheep flock in Uruguay.

Orf virus (ORFV) causes contagious ecthyma (CE), a highly transmissible, zoonotic disease of small ruminants. CE most commonly affects lambs and unvaccinated sheep. This work reports epidemiologic, clinicopathologic, and virologic findings in a CE outbreak in a vaccinated sheep flock in Uruguay and failure to detect ORFV in a commercial vaccine.

Genetic diversity of Orf virus isolated from sheep in Uruguay.

Orf virus (ORFV) is the etiological agent of contagious ecthyma, a disease widely spread in the world that occasionally causes zoonotic infections. This work is the first molecular characterization of ORFV in Uruguay, where we analyzed twenty-one sheep samples, eighteen of which were recovered from thirteen ORFV outbreaks that occurred during 2004 to 2011 as well as three strains from a national vaccine. Phylogenetic analysis and the derived amino acid sequences from the B2L gene suggest that the Uruguayan virus do not form a unique cluster, with most of them displaying similarities with worldwide ORFV isolates as well as our vaccine strains.

Identification and molecular characterization of Orf virus in Argentina.

Orf virus (ORFV) is the etiological agent of contagious ecthyma (CE), a pustular dermatitis of sheep and goats. Outbreaks of ORFV have been observed in all geographical regions of the world, including Argentina. The origin and identity of Argentinian ORFVs are unknown, and no comparative or phylogenetic studies of these viruses have been performed. In this study, we described the sequencing and analysis of five ORFV molecular markers: a partial B2L gene (ORF011), VIR (ORF020), an envelope mature protein (ORF109), vIL10 (ORF127), and GIF (ORF117) from two particular Argentinian outbreaks of CE.

Pathogenesis in lambs and sequence analysis of putative virulence genes of Brazilian orf virus isolates.

The parapoxvirus orf virus (ORFV) is the agent of contagious ecthyma, an ubiquitous mucocutaneous disease of sheep and goats that may present variable clinical presentations. We herein studied the pathogenesis of ORFV infection in lambs and analyzed three putative virulence genes of four Brazilian ORFV isolates. Lambs inoculated in the labial commissures with each ORFV isolate (n=4, viral titer 10(5.6) TCID50/ml) developed classical orf lesions, characterized by a progressive course of erythema/macules, vesicles, pustules and proliferative scabs. Lesions lasted an average of 22.9 days (18-26) and virus shedding was detected for approximately 24.6 days (18-30). Two isolates (SV269/11 and SV820/10) produced more severe, long-lasting lesions resulting in highest clinical scores. Lambs inoculated with isolate SV581/11 developed lesions markedly milder (lower clinical scores [p<0.05]) and more limited than the other groups. Virus shedding by SV581/11 group, however, lasted similarly or even longer than the other groups. Sequence analysis of three virulence genes (VEGF, VIR and IL-10v) revealed amino acid deletions and mutations in VEGF and IL-10v genes of SV581/11 and SV252/11, the isolate(s) producing milder lesions. Additionally, the VEGF gene of isolate SV581/11 presented the lowest amino acid identity with the other isolates and with ORFV standard strain OV-IA82. Thus, these results demonstrate that ORFV isolates may display differential virulence in lambs and these differences might be associated with genetic changes in putative virulence genes.

Partial sequence analysis of B2L gene of Brazilian orf viruses from sheep and goats.

We herein describe the partial nucleotide sequencing and phylogenetic analysis of the B2L gene of seventeen Brazilian orf viruses (ORFV). Seventeen viruses were recovered from outbreaks of contagious ecthyma in sheep and goats in four states in Southern and Northeast country, and three from commercial vaccines. Most analyzed viruses were associated with outbreaks of classical contagious ecthyma, with lip, nostrils and labial commissure involvement, yet udder/teat, feet, vulvar and disseminated lesions were also reported in some cases. Nucleotide sequence analysis revealed a high degree of B2L similarity among sheep sequences (>99%) regardless the geographic origin, and a remarkable high identity for the two goat isolates (>99.8%), with similarity dropping to below 99% when comparing viruses from the two species. A phylogenetic tree grouped most sheep and goat viruses on different branches. In addition, sequence alignment allowed the identification of up to six scattered nucleotide changes that were predominant and more consistent in goat isolates, including a number of sequences from other continents. Thus, in spite of the high nucleotide similarity, different degrees of similarity and discrete nucleotide changes in the B2L gene may help in grouping ORFV viruses according to host species.

Looking back: a genetic retrospective study of Brazilian Orf virus isolates.

Orf virus (ORFV), the prototype of the genus Parapoxvirus, is the aetiological agent of contagious ecthyma (CE), a pustular dermatitis that afflicts domestic and wild small ruminants. CE is one of the most widespread poxvirus diseases in the world, causing public health impacts. Outbreaks of ORFV have been observed in all geographical regions of Brazil, affecting ovine and caprine herds. The origins, epidemiology and identity of Brazilian ORFVs are unknown, and no comparative or phylogenetic studies of these viruses have been performed. In the present study, we revisited CE outbreaks which occurred until 32 years ago, and we assessed, genetically, five viral isolates. We performed the sequencing and analysis of the three ORFV molecular markers: B2L gene, virus interferon resistance gene (VIR) and the vascular endothelial growth factor gene. Nucleotide and amino acid analysis of the analysed genes demonstrated that Brazilian ORFVs do not form a unique cluster, and presented more similarity to other worldwide ORFV samples than with each other. These data raise the questions of whether there are different worldwide ORFVs circulating in Brazil, or if all the Brazilian ORFV samples are of the same virus taken at distinct time points.

Multifocal cutaneous ORF virus infection in goats in the Amazon region, Brazil.

Orf virus is the etiological agent of contagious ecthyma, a severe exanthematic disease that affects small ruminants. Orf virus is zoonosis that is associated with occupational contact with infected animals in human disease. Clinically, contagious ecthyma is characterized by the appearance of vesicles, pustules, ulcers, and papillomatous proliferative lesions on the skin of the lips and nostrils. Here we describe a case of lethal cutaneous multifocal Orf virus infection in goats in the Amazon region of Brazil. Exanthematic lesions were collected and epidemiological and clinical data were obtained. Orf virus was detected using PCR amplification of the whole B2L, VIR, and VEGF open reading frame. Phylogenetic analysis revealed that this virus clustered together with the Orf virus samples isolated during classical contagious ecthyma. The present work is the first to report a severe proliferative Orf virus case in South America.

Virological and clinico-pathological features of orf virus infection in experimentally infected rabbits and mice.

Many aspects of the biology of orf virus (ORFV) infection remain poorly understood and attempts to establish animal models have yielded conflicting and non-reproducible results. We herein describe the characterization of ORFV infection and disease in rabbits and mice. A protocol of intradermal inoculation was employed to inoculate 10(8.5)TCID50/mL of ORFV strain IA-82 in the skin of ears, of the back and labial commissures. All inoculated rabbits presented a clinical course characterized by erythema, macules, papules/vesicles or pustules that eventually dried originating scabs. Local signs started around days 3 and 4 post-inoculation (pi) and lasted 3-10 days. Virus was recovered from lesions between days 2 and 14pi. Histological examination of lesions revealed focal proliferative dermatitis with ballooning degeneration and eosinophilic intracytoplasmic inclusion bodies in keratinocytes, histological hallmarks of contagious ecthyma in sheep. A similar, albeit milder clinical course occurred in 5/10 inoculated mice; virus was recovered from lesions from three animals. Inoculated lambs - used as controls - developed severe lesions of contagious ecthyma. VN tests performed at day 28pi failed to detect neutralizing antibodies in all inoculated animals. In contrast, convalescent rabbit sera were positive by ELISA at dilutions from 100 to 400. These results show that rabbits are susceptible to ORFV infection and thus may be used to study selected aspects of ORFV biology.

Detection and phylogenetic analysis of Orf virus from sheep in Brazil: a case report.

BACKGROUND: Orf virus (ORFV), the prototype of the genus Parapoxvirus (PPV), is the etiological agent of contagious ecthyma, a severe exanthematic dermatitis that afflicts domestic and wild small ruminants. Although South American ORFV outbreaks have occurred and diagnosed there are no South American PPV major membrane glycoprotein B2L gene nucleotide sequences available. CASE PRESENTATION: an outbreak of ovine contagious ecthyma in Midwest Brazil was investigated. The diagnosis was based on clinical examinations and molecular biology techniques. The molecular characterization of the virus was done using PCR amplification, cloning and DNA sequencing of the B2L gene. The phylogenetic analysis demonstrated a high degree of identity with ORFV strains, and the isolate was closest to the ORFV-India 82/04 isolate. Another Brazilian ORFV isolate, NE1, was sequenced for comparative analysis and also showed a high degree of identity with an Asian ORFV strain. CONCLUSION: Distinct ORFV strains are circulating in Brazil. This is the first report on the phylogenetic analysis of an ORFV in South America.

A study of the process of apoptosis in animals infected with the contagious ecthyma virus.

Contagious ecthyma virus (CEV) is a disease caused by a parapoxvirus, also is a potent genetic carrier with the capacity for regulating apoptosis in the cells of infected skin, a mechanism that serves for evading the immune response of the host. It has been suggested that the virus may remain in the skin and be able to cause repeated infections in the same flock. The effect of infection as well as the presence of contagious ecthyma virus was evaluated in terms of lesions and apoptosis in the skin of animals, infected both naturally and experimentally. Samples used were obtained from a naturally infected sheep, 5 goats inoculated with CEV and a negative control. Samples obtained were longitudinally sectioned and processed using photon and electron microscopy, and embedded in paraffin and araldite. Samples embedded in paraffin were sectioned in 5 microm of thickness and dyed with orange eosin-hematoxilin G and Gomori's trichrom stain, apoptosis was demonstrated by the TUNEL assay, the viral antigen was revealed using polyclonal antibodies, and the presence of lymphocytes CD4+ and CD8+, with monoclonal antibodies. The samples processed in resin were cut to obtain semi-fine sections and dyed with toluidine blue-borax, and the ultra-fine sections were impregnated with lead citrate and uranyl acetate. Observations were similar in both, the natural infected animal and the experimental group. Infiltration was observed as well as images suggestive of a process of apoptosis. The TUNEL assay demonstrated that the number of epithelial cells undergoing apoptosis diminished during the process and increased among defense cells, until they almost disappeared at the beginning of healing. Cells undergoing apoptosis were located near the sebaceous glands and pilose follicles. The infiltrated lymphocytes gradually diminished. The viral antigen was observed in cells with morphology suggestive of apoptosis, located in sebaceous glands and pilose follicles. Using electron microscopy, cells with morphology compatible with that of lymphocytes were observed to be undergoing apoptosis, but there was little evidence of viral particles.

Molecular characterization of Brazilian isolates of orf virus.

Outbreaks of an epidermic disease suggesting parapox virus infections have been observed in all major herds of sheep and goats from different geographical areas of Brazil. Clinical samples (dried scabs) were collected and orf virus was isolated and characterized by electron microscopy in previous work. In order to characterize these viruses at the molecular level, a modified methodology for genomic DNA extraction directly from scabs was used and such DNA was used to derive the restriction enzyme digestion patterns for clinical samples from three distinct geographic origins. Pulsed field gel electrophoresis was used to separate restriction enzyme DNA fragments and heterogeneity among isolates from different geographic areas could be observed on stained gels. The HindIII-G DNA fragment from orf-A virus genome was cloned and hybridized to DNA of other orf virus isolates. Further heterogeneity was confirmed by these hybridizations.