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1.
Virulence ; 15(1): 2398171, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-39258802

RESUMO

Orf virus (ORFV) is an acute contact, epitheliotropic, zoonotic, and double-stranded DNA virus that causes significant economic losses in the livestock industry. The objective of this study is to design an immunoinformatics-based multi-epitope subunit vaccine against ORFV. Various immunodominant cytotoxic T lymphocytes (CTL), helper T lymphocytes (HTL), and B-cell epitopes from the B2L, F1L, and 080 protein of ORFV were selected and linked by short connectors to construct a multi-epitope subunit vaccine. Immunogenicity was enhanced by adding an adjuvant ß-defensin to the N-terminal of the vaccine using the EAAAK linker. The vaccine exhibited a significant degree of antigenicity and solubility, without allergenicity or toxicity. The 3D formation of the vaccine was subsequently anticipated, improved, and verified. The optimized model exhibited a lower Z-score of -4.33, indicating higher quality. Molecular docking results demonstrated that the vaccine strongly binds to TLR2 and TLR4. Molecular dynamics results indicated that the docked vaccine-TLR complexes were stable. Immune simulation analyses further confirmed that the vaccine can induce a marked increase in IgG and IgM antibody titers, and elevated levels of IFN-γ and IL-2. Finally, the optimized DNA sequence of the vaccine was cloned into the vector pET28a (+) for high expression in the E.coli expression system. Overall, the designed multi-epitope subunit vaccine is highly stable and can induce robust humoral and cellular immunity, making it a promising vaccine candidate against ORFV.


Assuntos
Epitopos de Linfócito B , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Vírus do Orf , Vacinas de Subunidades Antigênicas , Vacinas Virais , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/química , Animais , Vírus do Orf/imunologia , Vírus do Orf/genética , Vacinas Virais/imunologia , Vacinas Virais/química , Vacinas Virais/genética , Camundongos , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/química , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/química , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/química , Ectima Contagioso/prevenção & controle , Ectima Contagioso/imunologia , Ectima Contagioso/virologia , Camundongos Endogâmicos BALB C , Feminino , Linfócitos T Citotóxicos/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia
2.
Virol J ; 21(1): 181, 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-39118175

RESUMO

The orf virus (ORFV) poses a serious threat to the health of domestic small ruminants (i.e., sheep and goats) and humans on a global scale, causing around $150 million in annual losses to livestock industry. However, the host factors involved in ORFV infection and replication are still elusive. In this study, we compared the RNA-seq profiles of ORFV-infected or non-infected sheep testicular interstitial cells (STICs) and identified a novel host gene, potassium voltage-gated channel subfamily E member 4 (KCNE4), as a key host factor involved in the ORFV infection. Both RNA-seq data and RT-qPCR assay revealed a significant increase in the expression of KCNE4 in the infected STICs from 9 to 48 h post infection (hpi). On the other hand, the RT-qPCR assay detected a decrease in ORFV copy number in both the STICs transfected by KCNE4 siRNA and the KCNE4 knockout (KO) HeLa cells after the ORFV infection, together with a reduced fluorescence ratio of ORFV-GFP in the KO HeLa cells at 24 hpi, indicating KCNE4 to be critical for the ORFV infection. Furthermore, the attachment and internalization assays showed decreased ORFV attachment, internalization, replication, and release by the KO HeLa cells, demonstrating a potential inhibition of ORFV entry into the cells by KCNE4. Pretreatment with the KCNE4 inhibitors such as quinidine and fluoxetine significantly repressed the ORFV infection. All our findings reveal KCNE4 as a novel host regulator of the ORFV entry and replication, shedding new insight into the interactive mechanism of ORFV infection. The study also highlights the K+ channels as possible druggable targets to impede viral infection and disease.


Assuntos
Vírus do Orf , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Internalização do Vírus , Animais , Humanos , Ovinos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Células HeLa , Vírus do Orf/genética , Vírus do Orf/fisiologia , Replicação Viral , Interações Hospedeiro-Patógeno , Masculino , Ectima Contagioso/virologia
3.
J Wildl Dis ; 60(4): 912-923, 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-39021057

RESUMO

Contagious ecthyma is a common, worldwide, and highly transmissible viral zoonotic skin disease caused by the orf virus (ORFV). It mainly affects farmed small ruminants, but it has also been described in a broad range of wild and domestic mammals, with Caprinae species most susceptible. Between November 2019 and January 2020, adults, juveniles, yearlings, and kids from an Iberian ibex (Capra pyrenaica) population established in the Montgrí Massif Natural Park (Catalonia, northeastern Spain) were observed with clinical signs and lesions compatible with ORFV infection. The carcass of an adult male with severe disease enabled sample collection for histopathology and molecular studies, confirming ORFV DNA in the skin lesions. Sequence analyses indicated that the ORFV strain detected had high homology (>98%) with strains previously obtained from other European wild ruminant species. The outbreak peaked in December 2019, with an estimated prevalence of 68.97% (95% confidence interval, 53.35-84.59). From February 2020 forward, no individuals with lesions were observed. The yearly counts for population monitoring corroborated the apparently negligible ORFV impact on the ibex population of the Montgrí Natural Park. There are no previous reports of contagious ecthyma in a free-ranging Iberian ibex population.


Assuntos
Surtos de Doenças , Ectima Contagioso , Doenças das Cabras , Cabras , Animais , Espanha/epidemiologia , Doenças das Cabras/epidemiologia , Doenças das Cabras/virologia , Masculino , Ectima Contagioso/epidemiologia , Ectima Contagioso/virologia , Surtos de Doenças/veterinária , Animais Selvagens/virologia , Vírus do Orf/isolamento & purificação , Parques Recreativos , Feminino
4.
Adv Exp Med Biol ; 1451: 171-181, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38801578

RESUMO

Despite being common worldwide, parapoxvirus infections are regarded as neglected zoonoses because their incidence is either unknown or grossly overestimated. In ruminants all throughout the world, parapoxvirus produces oral lesions and infectious pustular dermatitis. The pathogen is typically spread directly via items contaminated with parapoxvirus and indirectly via a near contact with dermatological lesions that contain the virus on affected animals. Animals infected with the parapoxvirus typically exhibit no clinical symptoms, and the mode of parapoxvirus transmission is occasionally unclear. For accurate etiological diagnosis and appropriate therapy of patients affected by zoonotic infections, the significance of adopting a "One Health" approach and cross-sector collaboration between human and veterinary medicine should be emphasized. The causative pathogen of ecthyma contagiosum in general people is the orf virus, which mostly infects various animals, either pets or wildlife species. The illness primarily affects minute wild ruminants, sheep, cattle, deer, and goats, and it can spread to people through contact with infected animals or contaminated meats anywhere in the world. Taxonomically speaking, the virus belongs to the parapoxvirus genus. Thus pathogen can be detected from crusts for a very long period (several months to several years), and the virus is found to be resistant to inactivation with a hot or dry atmosphere. In immunocompetent individuals, the lesions often go away on their own with a period as long 2 months. Nevertheless, it necessitates the applying of diverse strategies, such as antiviral, immunological modulator, or modest surgical excisions in immunosuppressed patients. The interaction of the virus with various host populations aids in the development of a defense mechanism against the immune system. The parapoxvirus illness in humans is covered in this chapter. The orf illness, a significant known human parapoxvirus infection, is given specific attention.


Assuntos
Ectima Contagioso , Vírus do Orf , Ectima Contagioso/virologia , Ectima Contagioso/transmissão , Ectima Contagioso/diagnóstico , Ectima Contagioso/epidemiologia , Animais , Humanos , Vírus do Orf/patogenicidade , Vírus do Orf/isolamento & purificação , Vírus do Orf/genética , Zoonoses/virologia , Zoonoses/transmissão , Parapoxvirus/genética , Parapoxvirus/isolamento & purificação
5.
Vet Pathol ; 61(5): 803-814, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38613413

RESUMO

ORF virus (ORFV) causes contagious ecthyma ("ORF"), a disease of sheep and goats characterized by lesions ranging from vesicles and pustules to atypical papilloma-like and angiomatous lesions in the skin and mucosae. The authors investigated the molecular factors leading to the ORF-associated atypical tumor-like changes. Fifteen lambs, 15 kids, and an adult ram clinically affected by natural ORFV infection were enrolled in the study and examined by several methods. ORFV was detected by viral culture or real-time polymerase chain reaction (RT-PCR) in the lesioned tissues and in the blood of the clinically affected sheep and goats. Surprisingly, ORFV was also detected in the blood of healthy goats from an affected herd. Microscopically, they found a pseudo-papillomatous proliferation of the epithelium, while the dermis and lamina propria were expanded by a proliferating neovascular component that highly expressed the viral vascular endothelial growth factor (vVEGF) and its host receptor vascular endothelial growth factor receptor 2 (VEGFR2). Immunohistochemistry, immunofluorescence, and in situ hybridization for mRNA showed that epidermal growth factor receptor (EGFR) was expressed in the fibrovascular component, in the infiltrating CD163+ macrophages, and in the basal stratum of the epidermis. Confocal immunofluorescence microscopy demonstrated that CD163+ macrophages were associated with VEGF and VEGFR2. Finally, they found by quantitative RT-PCR the overexpression of the interleukin-6 and VEGFR2 genes in the lesioned tissues. These findings suggest that ORFV activates an inflammatory reaction characterized by CD163+ macrophages expressing EGFR and VEGFR2, which might play an oncogenic role through synergistic action with vVEGF signaling.


Assuntos
Ectima Contagioso , Receptores ErbB , Doenças das Cabras , Cabras , Inflamação , Vírus do Orf , Animais , Vírus do Orf/genética , Vírus do Orf/isolamento & purificação , Ovinos , Ectima Contagioso/patologia , Ectima Contagioso/virologia , Doenças das Cabras/virologia , Doenças das Cabras/patologia , Inflamação/veterinária , Inflamação/patologia , Inflamação/virologia , Receptores ErbB/metabolismo , Receptores ErbB/genética , Masculino , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Feminino , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Macrófagos/virologia , Macrófagos/patologia , Doenças dos Ovinos/virologia , Doenças dos Ovinos/patologia , Receptores de Superfície Celular
6.
Infect Genet Evol ; 98: 105220, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35066165

RESUMO

Poxviruses are a family of specialized cytoplasm-parasitic DNA viruses that replicate and assembly in virus factory. In Parapoxvirus (PPV) genus, with the orf virus (ORFV) as a representative species of this genus, their behaviors are significantly different from that of Orthopoxvirus, and the plots of viral practical solutions for evading host immunity are intricate and fascinating, particularly to anti-host and host's antiviral mechanisms. In order to protect the virus factory from immune elimination caused by infection, PPVs attempt to interfere with multiple stress levels of host, mainly by modulating innate immunity response (IIR) and adaptive immunity response (AIR). Given that temporarily constructed by virus infection, ORFV-HOST (OH) system accompanied by viral strategies is carefully managed in the virus factory, thus directing many life-critical events once undergoing the IIR and AIR. Evolutionarily, to reduce the risk of system destruction, ORFV have evolved into a mild-looking mode to avoid overstimulation. Moreover, the current version of development also focus on recognizing and hijacking more than eight antiviral security mechanisms of host cells, such as the 2',5'-oligoadenylate synthetase (OAS)/RNase L and PKR systems, the ubiquitin protease system (UPS), and so on. In summary, this review assessed inescapable pathways as mentioned above, through which viruses compete with their hosts strategically. The OH system provides a panoramic view and a powerful platform for us to study the PPV-Host interaction, as well as the corresponding implications on a great application potential in anti-virus design.


Assuntos
Imunidade Adaptativa , Ectima Contagioso/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Vírus do Orf/fisiologia , Animais , Bovinos , Ectima Contagioso/virologia , Humanos , Vírus do Orf/genética , Ovinos
7.
FEBS Lett ; 595(23): 2897-2908, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34778960

RESUMO

Cellular double-stranded RNA-binding proteins (DRBPs) play important roles in the regulation of innate immune responses and microRNA (miRNA) biogenesis. The current study aimed to understand whether OV20.0, a DRBP of orf virus (ORFV), is involved in cellular RNA biogenesis via association with host DRBPs. We found that OV20.0 interacts with DiGeorge syndrome critical region 8 (DGCR8), a subunit of the miRNA processor complex, and binds to primary- and precursor-miRNA. Additionally, OV20.0 regulates DGCR8 expression in multiple ways, including through interaction with the DGCR8 protein and binding to DGCR8 mRNA. Lastly, our data show that DGCR8 plays an antiviral role against ORFV infection, whereas it is beneficial for influenza virus propagation, indicating that the underlying mechanisms could be diverse among different viruses.


Assuntos
Ectima Contagioso/virologia , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Células A549 , Animais , Cães , Ectima Contagioso/metabolismo , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , MicroRNAs/genética , Vírus do Orf/patogenicidade , Ligação Proteica , RNA Mensageiro/metabolismo
8.
J Virol ; 95(19): e0015321, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34287041

RESUMO

Orf virus (ORFV) is a highly epitheliotropic parapoxvirus with zoonotic significance that induces proliferative lesions in the skin of sheep, goats, and humans. Several viral proteins carried by ORFV, including nuclear factor-κB (NF-κB) inhibitors, play important roles in hijacking host-associated proteins for viral evasion of the host innate immune response. However, the roles of proteins with unknown functions in viral replication and latent infection remain to be explored. Here, we present data demonstrating that the ORF120, an early-late ORFV-encoded protein, activates the NF-κB pathway in the early phase of infection, which implies that ORFV may regulate NF-κB through a biphasic mechanism. A DUAL membrane yeast two-hybrid system and coimmunoprecipitation experiments revealed that the ORF120 protein interacts with Ras-GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1). The overexpression of the ORF120 protein can efficiently increase the expression of G3BP1 and nuclear translocation of NF-κB-p65 in primary ovine fetal turbinate (OFTu) and HeLa cells. The knockdown of G3BP1 significantly decreased ORF120-induced NF-κB activation, indicating that G3BP1 is involved in ORF120-induced NF-κB pathway activation. A dual-luciferase reporter assay revealed that ORF120 could positively regulate the NF-κB pathway through the full-length G3BP1 or the domain of G3BP1RRM+RGG. In conclusion, we demonstrate, for the first time, that the ORF120 protein is capable of positively regulating NF-κB signaling by interacting with G3BP1, providing new insights into ORFV pathogenesis and a theoretical basis for antiviral drug design. IMPORTANCE As part of the host innate response, the nuclear factor-κB (NF-κB) pathway plays a partial antiviral role in nature by regulating the innate immune response. Thus, the NF-κB pathway is probably the most frequently targeted intracellular pathway for subversion by anti-immune modulators that are carried by a wide range of pathogens. Various viruses, including poxviruses, carry several proteins that prepare the host cell for viral replication by inhibiting cytoplasmic events, leading to the initiation of NF-κB transcriptional activity. However, NF-κB activity is hypothesized to facilitate viral replication to a great extent. The significance of our research is in the exploration of the activation mechanism of NF-κB induced by the Orf virus (ORFV) ORF120 protein interacting with G3BP1, which helps not only to explain the ability of ORFV to modulate the immune response through the positive regulation of NF-κB but also to show the mechanism by which the virus evades the host innate immune response.


Assuntos
DNA Helicases/metabolismo , Ectima Contagioso/virologia , NF-kappa B/metabolismo , Vírus do Orf/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , DNA Helicases/química , Células HeLa , Humanos , Vírus do Orf/genética , Vírus do Orf/crescimento & desenvolvimento , Vírus do Orf/patogenicidade , Proteínas de Ligação a Poli-ADP-Ribose/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Helicases/química , Proteínas com Motivo de Reconhecimento de RNA/química , Ovinos , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Transcrição Gênica , Ativação Transcricional , Proteínas Virais/genética , Virulência
9.
Vet Microbiol ; 257: 109066, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33866062

RESUMO

The parapoxvirus orf virus (ORFV) encodes several immunomodulatory proteins (IMPs) that modulate host innate and pro-inflammatory responses to infection. Using the ORFV IA82 strain as the parental virus, recombinant viruses with individual deletions in the genes encoding the IMPs chemokine binding protein (CBP; ORFV112), inhibitor of granulocyte-monocyte colony-stimulating factor and IL-2 (GIF, ORFV117) and interleukin 10 homologue (vIL-10; ORFV127) were generated and characterized in vitro and in vivo. The replication properties of the individual gene deletion viruses in cell culture was not affected comparing with the parental virus. To investigate the effect of the individual gene deletions in ORFV infection and pathogenesis, groups of four lambs were inoculated with each virus and were monitored thereafter. Lambs inoculated with either recombinant or with the parental ORFV developed characteristic lesions of contagious ecthyma. The onset, nature and severity of the lesions in the oral commissure were similar in all inoculated groups from the onset (3 days post-inoculation [pi]) to the peak of clinical lesions (days 11-13 pi). Nonetheless, from days 11-13 pi onwards, the oral lesions in lambs inoculated with the recombinant viruses regressed faster than the lesions produced by the parental virus. Similarly, the amount of virus shed in the lesions were equivalent among lambs of all groups up to day 15 pi, yet they were significantly higher in the parental virus group from day 16-21 pi. In conclusion, individual deletion of these IMP genes from the ORFV genome resulted in slight reduction in virulence in vivo, as evidenced by a reduction in the duration of the clinical disease and virus shedding.


Assuntos
Genes Virais/imunologia , Vírus do Orf/genética , Vírus do Orf/patogenicidade , Doenças dos Ovinos/virologia , Fatores Etários , Animais , Citocinas/genética , Citocinas/imunologia , Ectima Contagioso/imunologia , Ectima Contagioso/virologia , Genoma Viral , Mutação , Vírus do Orf/imunologia , Ovinos/virologia , Doenças dos Ovinos/imunologia , Transdução de Sinais , Virulência , Replicação Viral/genética , Eliminação de Partículas Virais , Sequenciamento Completo do Genoma
10.
Viral Immunol ; 33(6): 468-476, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32315577

RESUMO

As a zoonotic disease, ovine contagious pustular dermatitis (Orf) is a serious threat to sheep as well as humans. Orf virus (ORFV) interferon resistance protein (VIR) is the principal virulence protein that encodes a dsRNA-binding protein to inhibit host antiviral response. p53 is one of the key proteins of the host antiviral innate immunity. It not only enhances type I interferon secretion but also induces apoptosis in infected cells, and plays a crucial role in the immune response against various viral infections. However, it remains to be elucidated what role p53 plays in ORFV replication and whether ORFV's own protein VIR regulates p53 expression to promote self-replication. In this study, we showed that p53 has an antiviral effect on ORFV and can inhibit ORFV replication. In addition, ORFV nonstructural protein VIR interacts with p53 and degrades p53, which inhibits p53-mediated positive regulation of downstream antiviral genes. This study provides new insight into the immune evasion mediated by ORFV and identifies VIR as an antagonistic factor for ORFV to evade the antiviral response.


Assuntos
Interações entre Hospedeiro e Microrganismos/genética , Vírus do Orf/genética , Proteína Supressora de Tumor p53/genética , Proteínas Virais/genética , Replicação Viral/genética , Animais , Linhagem Celular , Cricetinae , Ectima Contagioso/virologia , Fibroblastos/imunologia , Fibroblastos/virologia , Regulação Viral da Expressão Gênica , Cabras , Evasão da Resposta Imune/genética , Imunidade Inata , Rim/citologia , Vírus do Orf/fisiologia , Ovinos , Pele/citologia , Proteínas Virais/metabolismo
11.
Virus Res ; 281: 197908, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32126295

RESUMO

Orf, a poxviral skin infection of small ruminants is caused by orf virus (ORFV) of the genus Parapoxvirus of the Poxviridae family. Vascular endothelial growth factor (VEGF) is an important virulence factor that is responsible for proliferative lesions in parapoxviral infections. VEGF gene shows high intra- and inter-species variability. Two variants of VEGF have been described globally in ORFV, viz. NZ2- and NZ7-like. In the present study, ORFV isolates of different geographic regions of India were analysed on the basis of the VEGF gene. Indian ORFV isolates showed 95.7-100 % nucleotide (nt) and 78.4-99.3 % amino acid (aa) identity with each other, except ORFV-Assam/LK/14 and ORFV-Meghalaya/03 which shared 85.1-88.35 % and 79.1-81.8 % identity, at nt and aa levels, respectively with other Indian ORFV isolates. All Indian ORFVs under the study demonstrated 83.5-99.1 % nt and 80.5-97.9 % aa identity with NZ7-like VEGF as compared to 41.2-44.8 % nt and 30.7-38.4 % aa identity with NZ2-like VEGF on comparison with global ORFV strains. Phylogenetic analysis based on the VEGF gene showed two clusters of ORFV in which the Indian ORFVs clustered with NZ7-like VEGF from global ORFV strains, mostly from China. Despite the considerable variation, VEGF protein from Indian ORFV strains showed conserved VEGF homology domain with eight cysteine residues. Homology modeling of Indian ORFV strains predicted the presence of extended Loop 3 similar to NZ7-like VEGF. Therefore, the present study showed the circulation of ORFV strains with comparatively less variable NZ7-like VEGF in India which implicates its importance in the epidemiology of ORFV infections in the country.


Assuntos
Surtos de Doenças/veterinária , Ectima Contagioso , Vírus do Orf , Animais , DNA Viral/genética , Ectima Contagioso/epidemiologia , Ectima Contagioso/virologia , Genes Virais/genética , Humanos , Índia/epidemiologia , Vírus do Orf/classificação , Vírus do Orf/genética , Filogenia , Filogeografia , Ovinos , Proteínas Virais/genética
13.
BMC Biotechnol ; 19(1): 102, 2019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31870349

RESUMO

BACKGROUND: Interferon-gamma (IFN-γ) is an important mediator of type I immune response and has antiviral, immunoregulatory and anti-tumor properties, plays a wide range of roles in inflammation and autoimmune diseases. The aim of this study was to obtain monoclonal antibody (mAb) against caprine IFN-γ by immunizing of BALB/c mice with the purified rIFN-γ. RESULTS: Recombinant caprine IFN-γ was expressed in Escherichia coli strain BL21 (DE3) and monoclonal antibodies against caprine IFN-γ were produced by immunizing of BALB/c mice with rIFN-γ. One hybridoma secreting mAb was screened by enzyme-linked immunosorbent assay (ELISA) which was designated as 2C. MAb secreted by this cell line were analyzed through ELISA, western blot and application of the mAb was evaluated by immunofluorescence analysis using goat lip tissues infected with Orf virus. ELISA analysis revealed that mAb 2C can specifically recognize rIFN-γ protein and culture supernatant of goat peripheral blood mononuclear cells (PBMCs) stimulated by concanavalin A (Con A) but cannot recognize the fusion tag protein of pET-32a. Western blot analysis showed that mAb 2C can specifically react with the purified 34.9 kDa rIFN-γ protein but does not react with the fusion tag protein of pET-32a. Immunofluorescence results demonstrated that mAb 2C can detect IFN-γ secreted in histopathological sites of goats infected with Orf virus. CONCLUSIONS: A caprine IFN-γ-specific mAb was successfully developed in this study. Further analyses showed that the mAb can be used to detect IFN-γ expression level during contagious ecthyma in goats.


Assuntos
Anticorpos Monoclonais/análise , Interferon gama/análise , Interferon gama/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Western Blotting , Ectima Contagioso/sangue , Ectima Contagioso/imunologia , Ectima Contagioso/virologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Doenças das Cabras/sangue , Doenças das Cabras/imunologia , Doenças das Cabras/virologia , Cabras , Hibridomas/metabolismo , Interferon gama/sangue , Interferon gama/genética , Leucócitos Mononucleares/imunologia , Camundongos Endogâmicos BALB C , Vírus do Orf/fisiologia
14.
Sci Rep ; 9(1): 16262, 2019 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-31700161

RESUMO

Orf is a highly contagious zoonotic disease of small ruminants caused by Parapoxvirus. Kisspeptin, encoded by the KISS1 gene with its cognate receptor GPR-54 is recognized as an upstream orchestrator in the hypothalamic-pituitary-gonadal axis. This study was designed to construct a DNA vaccine that produces a fused peptide composed of a major immunodominant protein of the orf virus (B2L) and kisspeptin-54, a neuropeptide with recognized roles in mammalian reproductive biology. The administration of this recombinant vaccine is shown to produce a significant antibody and cell-mediated immune response directed against B2L compared to the control group (p < 0.05). Furthermore, we found that rats inoculated with PBK-asd vaccine up-regulated antigen-mediated splenocyte proliferation and significantly raised antigen-specific tumor necrosis factor-alpha (TNFα-), interferon-gamma (IFN-ϒ) and interleukin (IL-2) compared to the control group (p < 0.05). This recombinant vaccine also stimulated antibody responses to kisspeptin and decreased serum luteinizing hormone and testosterone levels. Moreover, the current recombinant vaccine caused testicular atrophy and arrested spermatogenesis. It is concluded that this recombinant B2L and Kisspeptin-54 vaccine could be a promising approach for construction of bivalent orf virus and immunocastration vaccine. Furthermore, we concluded that the orf virus envelope protein (B2L) could be used as an immunomodulator for kisspeptin-54 to produce a strong antibody response.


Assuntos
Ectima Contagioso/imunologia , Kisspeptinas/imunologia , Vírus do Orf/imunologia , Espermatogênese/imunologia , Vacinas de DNA/imunologia , Proteínas Virais/imunologia , Animais , Biomarcadores , Citocinas/metabolismo , Ectima Contagioso/virologia , Hormônios , Imunofenotipagem , Masculino , Ratos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Testículo/imunologia , Testículo/metabolismo , Vacinas de DNA/administração & dosagem
16.
Emerg Infect Dis ; 25(12): 2197-2204, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31742503

RESUMO

Zoonotic transmission of parapoxvirus from animals to humans has been reported; clinical manifestations are skin lesions on the fingers and hands after contact with infected animals. We report a human infection clinically suspected as being ecthyma contagiosum. The patient, a 65-year-old woman, had 3 nodules on her hands. She reported contact with a sheep during the Aïd-el-Fitr festival in France during 2017. We isolated the parapoxvirus orf virus from these nodules by using a nonconventional cell and sequenced the orf genome. We identified a novel orf virus genome and compared it with genomes of other orf viruses. More research is needed on the genus Parapoxvirus to understand worldwide distribution of and infection by orf virus, especially transmission between goats and sheep.


Assuntos
Ectima Contagioso/diagnóstico , Ectima Contagioso/virologia , Genoma Viral , Vírus do Orf/genética , Biópsia , DNA Viral , Ectima Contagioso/epidemiologia , Ectima Contagioso/história , França/epidemiologia , História do Século XXI , Humanos , Vírus do Orf/classificação , Vírus do Orf/isolamento & purificação , Vírus do Orf/ultraestrutura , Filogenia , Reação em Cadeia da Polimerase , Vigilância da População , Sequenciamento Completo do Genoma
17.
Acta Virol ; 63(3): 270-277, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31507192

RESUMO

Orf, also called contagious ecthyma or contagious pustular dermatitis, is a significant zoonotic disease that primarily affects goat and sheep globally. Currently, the infection by orf virus (ORFV) has been observed in different host species worldwide, including China. Here, a suspected outbreak of orf infection in a goat farm in Anhui Province in 2018 was investigated. Through PCR, electron microscopy, and cell culture techniques, we confirmed that the outbreak was caused by ORFV. Consequently, the orf virus strain was named the AH/LA/2018 strain. The amplified and sequenced ORFV011 (B2L) and ORFV059 (F1L) genes were used to construct phylogenetic trees to elucidate the genetic characteristics of the ORFV and the molecular epidemiology of orf. The present study is the first systematic evolution analysis of the ORFV strain isolated in Anhui Province. The results of this study will be helpful to better understand the characteristics of ORFV, to help prevent and control the transmission of ORFV at an early stage in China. Keywords: Anhui Province; goat; orf virus; phylogenetic analysis.


Assuntos
Ectima Contagioso , Vírus do Orf , Filogenia , Animais , Células Cultivadas , China/epidemiologia , Ectima Contagioso/virologia , Genes Virais/genética , Doenças das Cabras/epidemiologia , Doenças das Cabras/virologia , Cabras , Microscopia Eletrônica de Transmissão , Vírus do Orf/classificação , Vírus do Orf/ultraestrutura , Reação em Cadeia da Polimerase , Ovinos , Doenças dos Ovinos/epidemiologia , Doenças dos Ovinos/virologia
18.
Emerg Infect Dis ; 25(8): 1585-1586, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31310206

RESUMO

Orf (ecthyma contagiosum) is an infection of the skin caused by a DNA virus belonging to the genus Parapoxvirus. We recently observed 7 cases of orf in Muslim men living in the metropolitan area of Milan, Italy, who acquired the infection after the Feast of Sacrifice.


Assuntos
Ectima Contagioso/transmissão , Ectima Contagioso/virologia , Vírus do Orf , Animais , Ectima Contagioso/diagnóstico , Ectima Contagioso/epidemiologia , Humanos , Itália/epidemiologia , Vírus do Orf/classificação , Vírus do Orf/genética , Vírus do Orf/isolamento & purificação , Zoonoses
19.
Artigo em Inglês | MEDLINE | ID: mdl-31300096

RESUMO

Orf virus (ORFV) causes contagious skin disease that mainly affects sheep and goats with zoonotic potential. However, there is not enough information about the association between ORFV and occurrence of skin disease in cattle. The present study describes outbreaks of ORFV infection in cattle in different provinces that are located in the Aegean, Central Anatolian and Mediterranean regions of Turkey. During the months of June and August 2017, vesicular fluid and scab samples were collected from cattle which had proliferative skin lesions. First, presence of lumpy skin disease virus (LSDV) and bovine herpesvirus 2 (BoHV-2, known as the causative agent of pseudo-lumpy skin disease) were investigated by real time PCR and PCR, respectively. Then, samples tested for the presence of parapoxviruses by PCR using primers specific to major envelope protein gene (B2L). Parapoxvirus DNA was detected in investigated samples whereas LSDV and BoHV-2 DNA were not detected. The analysis of the B2L gene sequences revealed that cattle were infected with ORFV. The isolates in the present study shared 100% sequence identity at the nucleotide and amino acid level when compared with previously characterised Turkish field ORFV isolates from goats in 2016. Results of the study show unusual infection of cattle with ORFV, and suggest that ORFV jumps the host species barrier from goats to cattle.


Assuntos
Doenças dos Bovinos/virologia , Surtos de Doenças/veterinária , Ectima Contagioso/epidemiologia , Vírus do Orf/isolamento & purificação , Animais , Bovinos/virologia , Doenças dos Bovinos/epidemiologia , DNA Viral , Ectima Contagioso/virologia , Cabras/virologia , Vírus do Orf/genética , Filogenia , Análise de Sequência de DNA , Pele/patologia , Turquia/epidemiologia , Proteínas Virais/genética
20.
Virus Genes ; 55(4): 490-501, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31030330

RESUMO

Orf virus (ORFV), a typical member of the Parapoxvirus genus within the family Poxviridae, which is the causative agent of Orf, a common epitheliotropic viral disease of sheep, goats, wild ruminants, and humans. In the present study, we sequenced the complete genomic sequences of two ORFV strains (ORFV-SY17, isolated from sheep, and ORFV-NA17, isolated from goat) and conducted the comparative analysis of multiple ORFVs. The complete genomic sequence of ORFV-SY17 was at length of 140,413 bp, including 131 potential open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) of 4267 bp at both ends. The ORFV-NA17 strain displayed the similar genome structure with ORFV-SY17. The whole genomic sequence of ORFV-NA17 strain was 139,287 bp in length and contained 132 ORFs flanked by ITRs of 3974 bp. The overall G+C contents of ORFV-SY17 and ORFV-NA17 genome sequences were about 63.8% and 63.7%, respectively. The ITR sequences analysis showed that ORFV-SY17 and ORFV-NA17 contained the terminal BamHI sites and conserved telomere resolution sequences at both ends of their genome. In addition, comparative analysis of ORFs among ORFV-SY17, ORFV-NA17, and other ORFV strains revealed several sequence variations caused by insertions or deletions, especially in ORFs 005 and 116, which were very likely associated with host species. Phylogenetic analysis based on the complete genome sequences revealed that ORFV-SY17 was genetically closely related to NA1/11 and HN3/12 strains derived from sheep, while ORFV-NA17 was closely related to YX strain derived from goat. The multiple alignment of deduced amino acid sequences further revealed the genetic relationship between host species and genetic variations of ORFV strains. Taken together, the availability of genomic sequences of ORFV-SY17 and ORFV-NA17 strains from Jilin Province will aid in our understanding of the genetic diversity and evolution of ORFV strains in this region and can assist in distinguishing between ORFV strains that originate in sheep and goats.


Assuntos
Ectima Contagioso/virologia , Genoma Viral , Doenças das Cabras/virologia , Vírus do Orf/genética , Vírus do Orf/isolamento & purificação , Doenças dos Ovinos/virologia , Animais , China , Cabras , Humanos , Vírus do Orf/classificação , Vírus do Orf/ultraestrutura , Filogenia , Ovinos , Sequenciamento Completo do Genoma
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