Isolation and Characterization of Monoclonal Antibodies Against a Virion Core Protein of Orf Virus Strain NA1/11 As Potential Diagnostic Tool for Orf Viruses

Wang, Xiaoping; Zhang, Jiafeng; Hao, Wenbo; Peng, Yongzheng; Li, Hong; Li, Wei; Li, Ming; Luo, Shuhong

doi:10.1089/mab.2014.0101

Cited by 7 publications

(11 citation statements)

References 46 publications

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“…Higher molecular weight forms of ORFV073 in EEV suggest possible covalent modification of virion-incorporated ORFV073 during particle maturation and morphogenesis. As a control, the virion core protein ORFV086 was detected as a predominant 21 kDa band together with previously described higher molecular weight forms in both EEV and IMV fractions [ 42 ] ( Fig 7A , bottom panel).…”

Section: Resultssupporting

confidence: 71%

“…Whole cell lysates (10 μg) from mock and OV-IA82RV073 Flag infected cells (MOI = 10) (24 h p.i) and purified OV-IA82Δ073 and OV-IA82RV073 Flag EEV and IMV virion proteins (10 μg) were resolved by SDS-PAGE, blotted to nitrocellulose membrane and probed with primary antibody against flag (Catalog no. A00187-200; Genscript) or ORFV086 structural protein [ 42 ]. Blots were developed with HRP-conjugated goat anti mouse secondary antibody (sc-2031; Santa Cruz) and chemiluminescent reagent (Super Signal West Femto, Thermo Fischer).…”

Section: Methodsmentioning

confidence: 99%

“…At 30 min and 1, 2, 6, 8, 12, 16 and 24 h p.i., cells were fixed with 4% formaldehyde for 20 min, permeabilized with 0.2% Triton-X for 10 min, blocked with 1% bovine serum albumin for 1 h and then incubated with primary mouse monoclonal antibody against flag (no. A00187-200; Genscript) or ORFV086 [ 42 ] overnight at 4°C. Cells were then incubated with Alexa Fluor 488-labeled secondary goat anti mouse antibody (no.…”

Section: Methodsmentioning

confidence: 99%

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A parapoxviral virion protein inhibits NF-κB signaling early in infection

et al. 2017

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Poxviruses have evolved unique proteins and mechanisms to counteract the nuclear factor κB (NF-κB) signaling pathway, which is an essential regulatory pathway of host innate immune responses. Here, we describe a NF-κB inhibitory virion protein of orf virus (ORFV), ORFV073, which functions very early in infected cells. Infection with ORFV073 gene deletion virus (OV-IA82Δ073) led to increased accumulation of NF-κB essential modulator (NEMO), marked phosphorylation of IκB kinase (IKK) subunits IKKα and IKKβ, IκBα and NF-κB subunit p65 (NF-κB-p65), and to early nuclear translocation of NF-κB-p65 in virus-infected cells (≤ 30 min post infection). Expression of ORFV073 alone was sufficient to inhibit TNFα induced activation of the NF-κB signaling in uninfected cells. Consistent with observed inhibition of IKK complex activation, ORFV073 interacted with the regulatory subunit of the IKK complex NEMO. Infection of sheep with OV-IA82Δ073 led to virus attenuation, indicating that ORFV073 is a virulence determinant in the natural host. Notably, ORFV073 represents the first poxviral virion-associated NF-κB inhibitor described, highlighting the significance of viral inhibition of NF-κB signaling very early in infection.

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Section: Resultssupporting

confidence: 71%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A parapoxviral virion protein inhibits NF-κB signaling early in infection

et al. 2017

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“…Virus-containing bands were collected, and virus was recovered by centrifugation and resuspended in 10 mM TrisHcl. Whole cell extracts (60 μg) from OV-IA82-RV119 Flag infected OFTu cells, and purified EEV and IMV virion proteins (10 μg) were resolved by SDS-PAGE, blotted to nitrocellulose membranes and probed with primary antibodies against Flag (Genscript, Cat # A00187), ORFV structural protein ORFV086 or Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) (sc-25778; Santa Cruz) overnight at 4°C [ 49 ]. Blots were incubated with appropriate HRP-labeled secondary antibodies (1:15,000) (anti-mouse, sc-2031 and anti-rabbit, sc-2004; Santa Cruz) for 1 h at room temperature (RT) and membranes were developed using chemiluminescent reagents (SuperSignal West Femto, Thermo Fischer Scientific).…”

Section: Methodsmentioning

confidence: 99%

A parapoxviral virion protein targets the retinoblastoma protein to inhibit NF-κB signaling

et al. 2017

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Poxviruses have evolved multiple strategies to subvert signaling by Nuclear Factor κB (NF-κB), a crucial regulator of host innate immune responses. Here, we describe an orf virus (ORFV) virion-associated protein, ORFV119, which inhibits NF-κB signaling very early in infection (≤ 30 min post infection). ORFV119 NF-κB inhibitory activity was found unimpaired upon translation inhibition, suggesting that virion ORFV119 alone is responsible for early interference in signaling. A C-terminal LxCxE motif in ORFV119 enabled the protein to interact with the retinoblastoma protein (pRb) a multifunctional protein best known for its tumor suppressor activity. Notably, experiments using a recombinant virus containing an ORFV119 mutation which abrogates its interaction with pRb together with experiments performed in cells lacking or with reduced pRb levels indicate that ORFV119 mediated inhibition of NF-κB signaling is largely pRb dependent. ORFV119 was shown to inhibit IKK complex activation early in infection. Consistent with IKK inhibition, ORFV119 also interacted with TNF receptor associated factor 2 (TRAF2), an adaptor protein recruited to signaling complexes upstream of IKK in infected cells. ORFV119-TRAF2 interaction was enhanced in the presence of pRb, suggesting that ORFV119-pRb complex is required for efficient interaction with TRAF2. Additionally, transient expression of ORFV119 in uninfected cells was sufficient to inhibit TNFα-induced IKK activation and NF-κB signaling, indicating that no other viral proteins are required for the effect. Infection of sheep with ORFV lacking the ORFV119 gene led to attenuated disease phenotype, indicating that ORFV119 contributes to virulence in the natural host. ORFV119 represents the first poxviral protein to interfere with NF-κB signaling through interaction with pRb.

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“…Serology is useless since it cannot distinguish Orf virus from other parapoxviruses (eg, pseudocowpox) virus and no serologic tests are routinely performed for Orf. 9 To date, noninvasive diagnosis of Orf was limited to dermoscopic observation: several aspecific features were described, variously combined to the clinical stage. 10,11 In the regenerative stage, which is the most characteristic stage presenting with a well-defined nodule with a central crust, the most commonly reported dermoscopic features are white structureless areas, white-grayish ring, white shiny streaks, dotted/hairpin/polymorphic vessels, scales, and black dots.…”

Section: Editormentioning

confidence: 99%