Expression of the M, H, and F proteins from peste des petitsruminants virus (PPRV) and virus-like particles (VLPs).

<p>TheVLPs were purified using sucrose gradient centrifugation, and the protein composition was determined by western blot analysis using a mouse anti-PPRV polyclonal antibody. Lane 1, Sf21 cells (negative control);lane 2, PPRV-H VLPs; lane 3,PPRV-F VLPs; lane 4, molecular weight marker;lane 5, PPRV (positive control).</p

Primer sequences used for RT-PCR amplification of M, H, and F genes of peste des petits ruminants (PPRV) and identification of recombinant bacmids.

<p>The primers used for RT-PCR amplification of the M, H, and F genes of PPRV. The primer pairs M1/M2, H1/H2, and F1/F2 were used to amplify the M, H, and F genes, respectively. The underlined nucleotidesin the forward primers are a Kozak sequence added to optimize the expression of the target foreign gene. Restriction enzyme sites were introduced at the respective 5′-termini (shown in bold); the relevant enzymes are indicated in the last column. The primer pairs M13 For/M13 Rev were used to identify the recombinant bacmids.</p

Peste des petitsruminants virus (PPRV) neutralizing antibody titers and lymphocyte proliferation responses in goats immunized with PPRV virus-like particles (VLPs) or native PPRV particles.

<p>PPRV neutralizing antibody titers in goats immunized with the PPRV VLPs, PPRV, and PBS. Serum samples were collected at weeks 0,1,2, 3, 5, 7, 11, and 15 after primary vaccination. Proliferation responses of peripheral blood lymphocytes in goats were tested at week 2 after boost immunization.</p><p>PBS  =  phosphate-buffered saline, as negative control; PPRV  =  Peste des petits virus; VLP  =  virus-like particles.</p><p>*Different superscript letters indicate that the differences were significant (p<0.05), same superscript letters indicate that the differences were not significant (p>0.05). (according to one-way ANOVA, using GraphPad).</p

Peste des petitsruminants virus (PPRV)-specific serum IgG1 and IgG2a antibody responses.

<p>Sera were taken from immunized mice at 3 weeks after primary (P) and booster (B) inoculation. PPRV-specific IgG1(black columns) or IgG2a(white columns) antibodies were determined by enzyme-linked immunosorbent assay (ELISA). The ratios of IgG1 to IgG2a subclasses are given above the columns. Bars indicate standard deviations.</p

Experimental schedule and peste des petitsruminants virus (PPRV)-specific IgG antibody responses to PPRV-H or PPRV-F virus-like particles (VLPs).

<p>Groups of mice were immunized subcutaneously, twice, as indicated, with 4-week intervals. Three mice from each group were sacrificed and splenocytes were pooled for the lymphocyte proliferation assay. Enzyme-linked immunosorbent assay (ELISA) plates were coated with whole PPRV, as indicated in the Materials and Methods section. Serially diluted sera were used after primary and booster vaccinations. Total immunoglobulin G levels were determined from mice immunized with PPRV VLPs or PPRVNig75/1.</p

Photograph of a sucrose gradient and transmission electron microscopy (TEM) of virus-like particles (VLPs).

<p>The culture supernatant of Sf21 insect cells at 72-h post-infection with recombinant baculoviruses (rBVs) was subjected to sucrose gradient centrifugation; the opalescent band (A, indicated by the arrow) was collected for TEM after staining with 1% uranyl acetate. a trace of residual baculoviruses(D, indicated by the white arrow) were consistently detected in VLP preparations(D, indicated by the black arrow). Both PPRV-H VLPs (F) and PPRV-F VLPs (G) isolated in this manner showed spherical shapes upon TEM, with spikes on their surfaces, and with a diameter of approximately 80–100 nm were observed. Native peste des petits ruminants virus (PPRV) propagated in Vero cells was also purified and negatively stained (E). Ultrathin sections of Sf21 insect cells showing budding of PPRV-H and PPRV-F VLPs (B and C) from the plasma membrane at 48 h after infection with rBVs.</p

Schematic diagram and identification of recombinant pFastBac vectors.

<p>The M gene of peste des petits ruminants virus (PPRV) was inserted under the control of the baculovirus p10 promoter and the PPRVH or F genes were controlled by the baculovirusP_Hpromoter, as described in the Materials and Methods section.</p

Specific interferon (IFN)-γproduction in response to peste des petits ruminants virus (PPRV) proteins in splenocytes from mice, as detected by ELISPOT assay.

<p>Splenocytes from mice inoculated with PPRV virus-like particles (VLPs), PPRV per se, or phosphate-buffered saline (PBS) were isolated and cultured for 16h in the presence of inactivated PPRV. The production of IFN-γwas measured using an ELISPOT assay. Samples were tested in triplicate and mean IFN-γvalues for each mouse are shown.</p

Experimental designs used for the animal studies.

<p>PBS  =  phosphate-buffered saline; PPRV  =  Peste des petits ruminants virus; S.C.  =  subcutaneous, TCID₅₀  = 50% tissue culture infective dose; VLP  =  virus-like particles.</p

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<p>Bovine tuberculosis (bTB) is primarily caused by infection with Mycobacterium bovis, which belongs to the Mycobacterium tuberculosis complex. The airborne route is considered the most common for transmission of M. bovis, and more than 15% of cattle with bTB shed the Mycobacterium, which can be detect by nested PCR to amplify mycobacterial mpb70 from a nasal swab from a cow. To screen for cytokines fostering early and accurate detection of bTB, peripheral blood mononuclear cells were isolated from naturally M. bovis-infected, experimentally M. bovis 68002-infected, and uninfected cattle, then these cells were stimulated by PPD-B, CFP-10-ESAT-6 (CE), or phosphate-buffered saline (PBS) for 6 h. The levels of interferon gamma (IFN-γ), IFN-γ-induced protein 10 (IP-10), IL-6, IL-12, IL-17A, and tumor necrosis factor alpha mRNA were measured using real-time PCR. To explore the cytokines associated with different periods of M. bovis infection, cattle were divided into three groups: PCR-positive, PCR-negative, and uninfected using the tuberculin skin test, CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test, IFN-γ release assay (IGRA), CFP-10/ESAT-6 (CE)-based IGRA, and nested PCR. The expression of IP-10, IL-17A, and IFN-γ proteins induced by PPD-B, CE, or PBS was detected by ELISA. The results showed that levels of PPD-B-stimulated IL-17A and IP-10 (mRNA and protein), and CE-induced IP-10 (mRNA and protein) were significantly higher in cattle naturally or experimentally infected with M. bovis than in those that were uninfected. The levels of PPD-B- or CE-induced IL-17A and IP-10 (protein) could be used to differentiate M. bovis-infected calves from uninfected ones for 6 to 30 weeks post-infection, whereas PPD-B- and CE-induced IP-10 and IL-17A mRNA expression could be used to differentiate M. bovis-infected calves from uninfected ones between 6 and 58 weeks post-infection. However, CE-induced IL-17A (protein) was not a reliable indicator of M. bovis infection in cattle that were confirmed positive for infection by nested PCR. Furthermore, the levels of PPD-B- or CE-induced IP-10 and IL-17A protein were lower than IFN-γ in M. bovis-infected cattle. Therefore, IL-17A and IP-10 protein are not suitable biomarkers for bTB. Antigen-induced IP-10 mRNA should be analyzed further for their potential to be used in the diagnosis of bTB.</p