GV and NSDV infection interfere with the tyrosine phosphorylation of STAT1 and STAT2 in response to IFNα or IFNγ.

<p>Vero cells were infected with GV or NSDV at an MOI of 1 TCID₅₀ unit per cell or left uninfected (mock). At the indicated time post-infection cells were treated with or without IFNα (a) or IFNγ (b) for 30 min. The cells were harvested and the levels of STATs and tyrosine-phosphorylated STAT proteins were determined by Western blot analysis with the corresponding specific antibodies. The data shown are from a representative experiment.</p

NSDV and GV suppress transcription from the IFNβ promoter.

<p>Vero cells were transfected with 500 ng each pIFNβ-luc and pJATLacZ. After 24 hours of transfection cells were infected with GV and NSDV at an MOI of 1 TCID₅₀ unit per cell or left uninfected. Eight hours after infection with NSDV or GV, cells were superinfected with Newcastle disease virus (NDV) at an MOI of 1 TCID₅₀ unit per cell or left uninfected, and finally lysed four hours after infection with NDV. The luciferase and β-galactosidase activities of the cell extracts were determined. Results from three separate experiments were combined by setting the RLUs induced by NDV in uninfected cells to 100%; all experiments were performed in triplicate wells. Error bars show standard errors of the normalised data.</p

Delayed IFNβ induction in GV and NSDV-infected cells.

<p>Vero cells were transfected with 400 ng of pIFNβ-luc and 200 ng pJATLacZ. After 24 hours of transfection, the cells were infected with (a) NSDV or GV, or (b) NDV at an MOI of 1 TCID₅₀ unit per cell, or left uninfected. At the indicated time points cells were lysed and assayed for luciferase and β-galactosidase activities. The ratio of these two activities was taken as the relative luciferase activity (in RLU). Shown are the data from a representative experiment in triplicates; error bars represent one standard error of the mean.</p

GV and NSDV inhibit the induction of expression from IFN-responsive promoters.

<p>Vero cells were transfected with 500 ng pJATLacZ and (a) 500 ng pGL3-Mx-1-luc or (b) pGAS-luc. 30 hours post transfection cells were infected with GV or NSDV at an MOI of 1 or left uninfected. 18 hours post infection cells were treated for 6 hours with IFNα (a) or IFNγ (b), lysed, and the luciferase and β-galactosidase activities determined. Results from two (for IFNα) or three (for IFNγ) separate experiments were combined by setting the RLUs induced by IFNα or IFNγ in uninfected cells to 100%; all experiments were performed in triplicate wells. Error bars show standard errors of the normalised data.</p

GV and NSDV inhibit ubiquitination and ISG15ylation.

<p>(a) Vero cells were transfected with 500 ng pHA-Ub. 14 hours post transfection cells were infected with NSDV or GV at an MOI of 1 TCID₅₀ unit per cell. After a further 12 h cells were lysed in SDS PAGE sample buffer and total protein ubiquitination was analysed by Western blot using anti-HA. Lane 1: Untransfected cells; Lanes 2-4:transfected with pUb-HA; Lane 3: GV-infected; Lane 4: NSDV-infected. (b) Vero cells were transfected with 250 ng pHis-mISG15 with 750 ng empty vector (Lanes 5–7) or with 250 ng each HA-mHerc6, mUBE1L-HA and UbcM8 (Lanes 2-4) or left untransfected (Lane 1). After 14 hours of transfection cells were infected with GV (Lane 3, 6) or NSDV (Lane 4, 7) at an MOI of 1 TCID₅₀ unit per cell or left uninfected (Lanes 1, 2, 5). Twelve hours after infection, samples were analysed by Western blot using anti-6His antibody. Samples were also probed for the presence of viral protein (N) to confirm infection. (c) 293FT cells were transfected with 400 ng pUb-HA and with 500 ng empty vector (Lane 2) or with 500 ng of plasmid expressing CCHFV L1-354-HA (Lane 3) or GV L1-169-V5 (Lane 4) or left untransfected (Lane 1). 30 hours post transfection cells were lysed and total protein ubiquitination was determined by Western blot using anti-HA. The expression of GV L1-169 was detected by using anti-V5. (d) 293FT cells were transfected with 250 ng each pHis-mISG15, HA-mHerc6, mUBE1L-HA, UbcM8 alone (Lane 2) or together with 250 ng of plasmid expressing CCHFV L1-354-HA (Lane 4) or GV L1-169-V5 (Lane 5) or left untransfected (Lane 1). As extra negative control cells were transfected with 250 ng pHis-mISG15 and 750 ng empty plasmid (Lane 3). After 30 h of transfection cells were lysed and total ISG15ylation was analyzed by Western blot using anti-6His. The expression level of GV L1-169 and CCHFV L1-354 were assayed by using anti-V5 and anti-HA respectively. PCNA levels served as loading control in all experiments.</p

GV L protein inhibits transcription from IFN-responsive promoters.

<p>(a) Vero cells were transfected essentially as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028594#pone-0028594-g005" target="_blank">Figure 5</a> except that 100 ng of the reporter gene plasmid pGL3Mx-1-lucwas used. All transfections were made up to the same amount of DNA using empty vector where required. After 40 hours of transfection cells were incubated with or without IFNα. After a further 8 hours the cells were lysed and the luciferase and β-galactosidase activities determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028594#s4" target="_blank">material and methods</a>. (b) Vero cells were transfected essentially as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028594#pone-0028594-g005" target="_blank">Figure 5</a> except that 400 ng of the reporter gene plasmid pGAS-luc was used. 40 hours post-transfection cells were treated with IFNγ. After a further 6 hours cells were lysed and the luciferase and β-galactosidase activities determined. Results from separate experiments were combined by setting the RLUs induced by IFNα in cells transfected with empty vector to 100%. Error bars show standard errors of normalised data.</p

Antibodies to the Core Proteins of Nairobi Sheep Disease Virus/Ganjam Virus Reveal Details of the Distribution of the Proteins in Infected Cells and Tissues

<div><p>Nairobi sheep disease virus (NSDV; also called Ganjam virus in India) is a bunyavirus of the genus <i>Nairovirus</i>. It causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%. The virus is closely related to the human pathogen Crimean-Congo haemorrhagic fever virus (CCHFV). Little is currently known about the biology of NSDV. We have generated specific antibodies against the virus nucleocapsid protein (N) and polymerase (L) and used these to characterise NSDV in infected cells and to study its distribution during infection in a natural host. Due to its large size and the presence of a papain-like protease (the OTU-like domain) it has been suggested that the L protein of nairoviruses undergoes an autoproteolytic cleavage into polymerase and one or more accessory proteins. Specific antibodies which recognise either the N-terminus or the C-terminus of the NSDV L protein showed no evidence of L protein cleavage in NSDV-infected cells. Using the specific anti-N and anti-L antibodies, it was found that these viral proteins do not fully colocalise in infected cells; the N protein accumulated near the Golgi at early stages of infection while the L protein was distributed throughout the cytoplasm, further supporting the multifunctional nature of the L protein. These antibodies also allowed us to gain information about the organs and cell types targeted by the virus <i>in vivo</i>. We could detect NSDV in cryosections prepared from various tissues collected post-mortem from experimentally inoculated animals; the virus was found in the mucosal lining of the small and large intestine, in the lungs, and in mesenteric lymph nodes (MLN), where NSDV appeared to target monocytes and/or macrophages.</p></div

Distribution of the N and L proteins in NSDV-infected cells.

<p>Vero cells were infected with the NSDVi isolate at a MOI of 1 TCID₅₀. After 8, 12 or 16 h cells were fixed with 4% PFA followed by ice cold methanol and were stained sequentially with affinity purified anti-L(CT) antibodies, Zenon AlexaFluor 594 (red) (Fab:antibody ratio 3.5) and pre-made complex of affinity purified rabbit anti-N antibodies with Zenon AlexaFluor 488 (green) (Fab:antibody ratio 3). Nuclei were counterstained using DAPI (blue). Representative focal planes from Z-stack series are shown. The ImarisColoc function of the Imaris x64 version 7.4.2 software was used to generate a colocalisation channel (D, I, N, S) for each time post infection using images composed of 6 (P-R), 10 (A-C) or 14 (F-H and K-M) focal planes through the thickness of an infected cell. To highlight areas where the L protein is present in the absence of the N protein, the colocalisation channel was subtracted from the L channel (red) (E, J, O, T). Bars correspond to 20 μm unless otherwise indicated. Dashed white boxes in (K-O) indicate the area shown enlarged in (P-T). Arrowheads in A highlight the punctate distribution of the N protein at 8 hpi.</p

Effect of NSDV infection on distribution of macrophages/monocytes in experimentally inoculated sheep.

<p>Cryosections were prepared as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124966#pone.0124966.g007" target="_blank">Fig 7</a> and stained with mouse monoclonal anti-calprotectin/L1 antibody (L1) and affinity-purified rabbit anti-NSDV N protein antibodies (NSDV N), followed by Alexa Fluor 488 goat anti-mouse IgG (green) and Alexa Fluor 568 goat anti-rabbit IgG (red). DAPI was used as a counterstain (blue). Scale bars indicate 40 μm.</p

NSDV N protein distribution in lymph node, spleen, liver and kidney of infected sheep.

<p>Cryosections were prepared and stained as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124966#pone.0124966.g007" target="_blank">Fig 7</a>. Scale bars indicate 40 μm (A, B, D, E, G, H, J, K) or 10 μm (C, F, I).</p