Rift Valley fever virus structural and nonstructural proteins: recombinant protein expression and immunoreactivity against antisera from sheep

The Rift Valley fever virus (RVFV) encodes the structural proteins nucleoprotein (N), aminoterminal glycoprotein (Gn), carboxyterminal glycoprotein (Gc), and L protein, 78-kD, and the nonstructural proteins NSm and NSs. Using the baculovirus system, we expressed the full-length coding sequence of N, NSs, NSm, Gc, and the ectodomain of the coding sequence of the Gn glycoprotein derived from the virulent strain of RVFV ZH548. Western blot analysis using anti-His antibodies and monoclonal antibodies against Gn and N confirmed expression of the recombinant proteins, and in vitro biochemical analysis showed that the two glycoproteins, Gn and Gc, were expressed in glycosylated form. Immunoreactivity profiles of the recombinant proteins in western blot and in indirect enzyme-linked immunosorbent assay against a panel of antisera obtained from vaccinated or wild type (RVFV)-challenged sheep confirmed the results obtained with anti-His antibodies and demonstrated the suitability of the baculo-expressed antigens for diagnostic assays. In addition, these recombinant proteins could be valuable for the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA)

Investigation of Ebolavirus exposure in pigs presented for slaughter in Uganda

In 2008, an outbreak ofReston ebolavirus(RESTV) in pigs in the Philippines expanded our understanding of the host range of ebolaviruses. Subsequent experimental infections with the human-pathogenic speciesZaire ebolavirus(EBOV) confirmed that pigs are susceptible to African species of ebolaviruses. Pig keeping has become an increasingly important livelihood strategy throughout parts of sub-Saharan Africa, driven by increasing demand for pork. The growth in pig keeping is particularly rapid in Uganda, which has the highest per capita pork consumption in East Africa and a history of sporadic human outbreaks of Ebola virus disease (EVD). Using a systematic sampling protocol, we collected sera from 658 pigs presented for slaughter in Uganda between December 2015 and October 2016. Forty-six pigs (7%) were seropositive based on ELISA tests at two different institutions. Seropositive pigs had antibodies that bound to Sudan NP (n = 27), Zaire NP (Kikwit;n = 8) or both NPs (n = 11). Sera from 4 of the ELISA-positive pigs reacted in Western blot (EBOV NP = 1; RESTV NP = 2; both NPs = 2), and one sample had full neutralizing antibody againstSudan ebolavirus(SUDV) in virus neutralization tests. Pigs sampled in June 2016 were significantly more likely to be seropositive than pigs sampled in October 2016 (p = .03). Seropositive pigs were sourced from all regions except Western region. These observed temporal and spatial variations are suggestive of multiple introductions of ebolaviruses into the pig population in Uganda. This is the first report of exposure of pigs in Uganda to ebolaviruses and the first to employ systematic abattoir sampling for ebolavirus surveillance during a non-outbreak period. Future studies will be necessary to further define the role pigs play (if any) in ebolavirus maintenance and transmission so that potential risks can be mitigated

Susceptibility of Pigs and Chickens to SARS Coronavirus

An outbreak of severe acute respiratory syndrome (SARS) in humans, associated with a new coronavirus, was reported in Southeast Asia, Europe, and North America in early 2003. To address speculations that the virus originated in domesticated animals, or that domestic species were susceptible to the virus, we inoculated 6-week-old pigs and chickens intravenously, intranasally, ocularly, and orally with 106 PFU of SARS-associated coronavirus (SARS-CoV). Clinical signs did not develop in any animal, nor were gross pathologic changes evident on postmortem examinations. Attempts at virus isolation were unsuccessful; however, viral RNA was detected by reverse transcriptase-polymerase chain reaction in blood of both species during the first week after inoculation, and in chicken organs at 2 weeks after inoculation. Virus-neutralizing antibodies developed in the pigs. Our results indicate that these animals do not play a role as amplifying hosts for SARS-CoV

Nipah Virus Infects Specific Subsets of Porcine Peripheral Blood Mononuclear Cells

Nipah virus (NiV), a zoonotic paramyxovirus, is highly contagious in swine, and can cause fatal infections in humans following transmission from the swine host. The main viral targets in both species are the respiratory and central nervous systems, with viremia implicated as a mode of dissemination of NiV throughout the host. The presented work focused on the role of peripheral blood mononuclear cells (PBMC) in the viremic spread of the virus in the swine host. B lymphocytes, CD4−CD8−, as well as CD4+CD8− T lymphocytes were not permissive to NiV, and expansion of the CD4+CD8− cells early post infection was consistent with functional humoral response to NiV infection observed in swine. In contrast, significant drop in the CD4+CD8− T cell frequency was observed in piglets which succumbed to the experimental infection, supporting the hypothesis that antibody development is the critical component of the protective immune response. Productive viral replication was detected in monocytes, CD6+CD8+ T lymphocytes and NK cells by recovery of infectious virus in the cell supernatants. Virus replication was supported by detection of the structural N and the non-structural C proteins or by detection of genomic RNA increase in the infected cells. Infection of T cells carrying CD6 marker, a strong ligand for the activated leukocyte cell adhesion molecule ALCAM (CD166) highly expressed on the microvascular endothelial cell of the blood-air and the blood-brain barrier may explain NiV preferential tropism for small blood vessels of the lung and brain

Framework for Leadership and Training of Biosafety Level 4 Laboratory Workers

One-sentence summary for table of contents: Training should include theoretical consideration of biocontainment principles, practical hands-on training, and mentored on-the-job experience

Innate Immune Response to Rift Valley Fever Virus in Goats

Rift Valley fever (RVF), a re-emerging mosquito-borne disease of ruminants and man, was endemic in Africa but spread to Saudi Arabia and Yemen, meaning it could spread even further. Little is known about innate and cell-mediated immunity to RVF virus (RVFV) in ruminants, which is knowledge required for adequate vaccine trials. We therefore studied these aspects in experimentally infected goats. We also compared RVFV grown in an insect cell-line and that grown in a mammalian cell-line for differences in the course of infection. Goats developed viremia one day post infection (DPI), which lasted three to four days and some goats had transient fever coinciding with peak viremia. Up to 4% of peripheral blood mononuclear cells (PBMCs) were positive for RVFV. Monocytes and dendritic cells in PBMCs declined possibly from being directly infected with virus as suggested by in vitro exposure. Infected goats produced serum IFN-γ, IL-12 and other proinflammatory cytokines but not IFN-α. Despite the lack of IFN-α, innate immunity via the IL-12 to IFN-γ circuit possibly contributed to early protection against RVFV since neutralising antibodies were detected after viremia had cleared. The course of infection with insect cell-derived RVFV (IN-RVFV) appeared to be different from mammalian cell-derived RVFV (MAM-RVFV), with the former attaining peak viremia faster, inducing fever and profoundly affecting specific immune cell subpopulations. This indicated possible differences in infections of ruminants acquired from mosquito bites relative to those due to contact with infectious material from other animals. These differences need to be considered when testing RVF vaccines in laboratory settings

Comparison of T cell subpopulations between pigs that died during acute infection versus survivors.

<p>The differences in CD4+ and CD8+ cell subpopulations during the acute infection with NiV up to 7 dpi. Values, based on flow cytometry analysis, were arbitrarily set as 50% at 0 dpi. The solid line represents piglets that died at 7 dpi, the dashed line represents the survivors. Standard Error is represented as error bars for 2 pigs per each group/mean value. Survivors had significantly higher values for the CD4−CD8+ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030855#pone-0030855-g008" target="_blank"><b>Fig. 8.A</b></a>) at 7 dpi compare to the piglets which died at that day. Marked difference was observed for the CD4+CD8− T lymphocytes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030855#pone-0030855-g008" target="_blank"><b>Fig. 8.B</b></a>). The down-trend starting almost immediately post infection for this cell subpopulation in pigs that died due to NiV infection was especially pronounced. In contrast, there was an up-regulation of CD4+CD8− T helper cells at 2 dpi in the survivors.</p

Flow chart of sorting and staining of PBMC.

<p>Sorting of cell subpopulations from peripheral blood mononuclear cells (PBMC) by magnetic beads coated with antibodies against selected markers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030855#pone-0030855-g001" target="_blank"><b>Fig. 1A</b></a>), and staining of PBMC for CD4 and CD8 markers for analysis by flow cytometry (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030855#pone-0030855-g001" target="_blank"><b>Fig. 1B</b></a>). (^hi) indicates high density expression of a specific marker, and (^lo) indicates low density expression.</p

T cell subpopulation frequencies in NiV infected pigs during the acute phase of the infection.

<p>Changes in CD4D8 cell subpopulation frequencies in PBMC of pigs infected with NiV based on flow cytometry analysis. The values obtained at 0 dpi were arbitrarily set at 50%. The data (mean and standard error) are based on six infected (solid line) and 4 control (dashed line) animals. Notably, significant changes with opposite trends were observed for CD4−CD8+ T cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030855#pone-0030855-g007" target="_blank"><b>Fig. 7.A</b></a>), and CD4+ CD8− T cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030855#pone-0030855-g007" target="_blank"><b>Fig. 7.B</b></a>). The changes in CD4+CD8+ T cell frequency, although statistically significant, were only minor, perhaps with slight decline toward the 7 dpi in the infected piglets compared to the controls (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030855#pone-0030855-g007" target="_blank"><b>Fig. 7.C</b></a>).</p