Reverse genetics system for shuni virus, an emerging orthobunyavirus with zoonotic potential

The genus Orthobunyavirus (family Peribunyaviridae, order Bunyavirales) comprises over 170 named mosquito- and midge-borne viruses, several of which cause severe disease in animals or humans. Their three-segmented genomes enable reassortment with related viruses, which may result in novel viruses with altered host or tissue tropism and virulence. One such reassortant, Schmallenberg virus (SBV), emerged in north-western Europe in 2011. Shuni virus (SHUV) is an orthobunyavirus related to SBV that is associated with neurological disease in horses in southern Africa and recently caused an outbreak manifesting with neurological disease and birth defects among ruminants in Israel. The zoonotic potential of SHUV was recently underscored by its association with neurological disease in humans. We here report a reverse genetics system for SHUV and provide first evidence that the non-structural (NSs) protein of SHUV functions as an antagonist of host innate immune responses. We furthermore report the rescue of a reassortant containing the L and S segments of SBV and the M segment of SHUV. This novel reverse genetics system can now be used to study SHUV virulence and tropism, and to elucidate the molecular mechanisms that drive reassortment events.The Dutch Ministry of Agriculture, Nature and Food Quality of the Netherlands and the European Union’s Horizon 2020 research and innovation programme under LEAP-Agri grant agreement No 727715.http://www.mdpi.com/journal/viruseshj2020Medical Virolog

Quantifying Rift Valley fever virus transmission efficiency in a lamb-mosquito-lamb model

Rift Valley fever virus (RVFV) is a (re)emerging mosquito-borne pathogen impacting human and animal health. How RVFV spreads through a population depends on population-level and individual-level interactions between vector, host and pathogen. Here, we estimated the probability for RVFV to transmit to naive animals by experimentally exposing lambs to a bite of an infectious mosquito, and assessed if and how RVFV infection subsequently developed in the exposed animal. Aedes aegypti mosquitoes, previously infected via feeding on a viremic lamb, were used to expose naive lambs to the virus. Aedes aegypti colony mosquitoes were used as they are easy to maintain and readily feed in captivity. Other mosquito spp. could be examined with similar methodology. Lambs were exposed to either 1-3 (low exposure) or 7-9 (high exposure) infectious mosquitoes. All lambs in the high exposure group became viremic and showed characteristic signs of Rift Valley fever within 2-4 days post exposure. In contrast, 3 out of 12 lambs in the low exposure group developed viremia and disease, with similar peak-levels of viremia as the high exposure group but with some heterogeneity in the onset of viremia. These results suggest that the likelihood for successful infection of a ruminant host is affected by the number of infectious mosquitoes biting, but also highlights that a single bite of an infectious mosquito can result in disease. The per bite mosquito-to-host transmission efficiency was estimated at 28% (95% confidence interval: 15 - 47%). We subsequently combined this transmission efficiency with estimates for life traits of Aedes aegypti or related mosquitoes into a Ross-McDonald mathematical model to illustrate scenarios under which major RVFV outbreaks could occur in naïve populations (i.e., R0 >1). The model revealed that relatively high vector-to-host ratios as well as mosquitoes feeding preferably on competent hosts are required for R0 to exceed 1. Altogether, this study highlights the importance of experiments that mimic natural exposure to RVFV. The experiments facilitate a better understanding of the natural progression of disease and a direct way to obtain epidemiological parameters for mathematical models

Effect of NSR infection on intracellular and extracellular CD83 levels.

<p>(A) The levels of soluble CD83 in supernatants from cells harvested 24 h after stimulation with LPS, infection with NSR, or from cells mock infected with NSRmock were determined by ELISA. Bars represent average CD83 concentrations ±SD. Results from one of two independently performed experiments with similar results are shown. (B) Detection of CD83 in cell lysates by Western blot at 24 hpi. The different treatments are shown above the top panel and the probed proteins are depicted at the right. The positions of molecular weight standard proteins are shown at the left. The top blot was stripped and re-probed with antibodies against GAPDH and GFP, which served as loading control and control to confirm NSR infection, respectively. Results from one of two independent experiments with cells from two donors are shown.</p

Visualization of GAPDH, CD80 and CD83 mRNAs in infected cells using FISH.

<p>DCs were stimulated for 24 h with LPS, infected with NSR or left untreated and then fixed and subjected to FISH. Shown are (A) representative cells of each treatment condition from three independently performed experiments with cells from three different donors and (B) average ±SD spot counts of cells probed for GAPDH, CD80 and CD83. Relevant statistical significance is indicated with an asterisk.</p

Analysis of CD83, CD80, GAPDH and PPIA mRNA levels.

<p>DC RNA samples, prepared 24 h after treatment (as indicated), were analysed by qRT-PCR. Bars represent average Ct values from triplicates ±SD with cells from one donor. The experiment is a representative of 3 independently performed experiments with cells from 3 different donors. Statistical significance is indicated with an asterisk.</p

Cytokine secretion by NSR-infected DCs.

<p>Supernatants of infected or control-treated DCs were harvested at 24 hpi and analysed with a luminex-based cytokine assay. Bars represent the mean cytokine concentrations ± SD of triplicates with cells from one donor. Statistical significance between infected (NSR) and mock-infected (NSRmock) conditions is indicated.</p

Expression of CD83 after inhibition of cellular protein degradation routes.

<p>(A) Flow cytometry analysis of CD83 surface expression after inhibition of the proteasome. DCs were stimulated with NSRmock, LPS+NSRmock or LPS+NSR for 8 h and then clasto Lactacystin β-lactone (CLBL) was added at two concentrations, as indicated. Control cells were left untreated or were treated with DMSO. Cells were analysed at 24 hpi for CD83 expression. Bars represent average MFI ±SD from two experiments with cells from one donor (B) Detection of total amounts of CD83 in cell lysates by Western blot. Cells were stimulated as described under point “A” and treatments are indicated above the image. (C) Inhibition of endocytosis. DCs were stimulated with LPS+NSRmock, LPS+NSR or left unstimulated. Cytochalasin D (Cyt D) or the solvent DMSO were subsequently added at different time points. The moments of adding Cyt D/DMSO and harvesting of cells are indicated above each graph. Bars represent average fold change of the MFI relative to unstimulated cells treated with DMSO ±SD. Average values of three experiments with cells from one donor are depicted. Relevant statistical significances are shown.</p

Infection of DCs by NSR.

<p>(A) DCs were infected with NSR for 24 h and evaluated for expression of GFP, using an EVOS fluorescence microscope. (B) Infection efficiency under optimal conditions as determined by flow cytometry. (C) Viability and percentage of infected cells at different time points after infection. Cells were infected with NSR or mock-infected with NSRmock, harvested at the indicated time points, stained with 7AAD and analysed by flow cytometry. The percentage of GFP expressing cells (bars) and the viability after NSR or NSRmock infections (lines) is depicted. Viability of the cells was calculated relative to the viability at 8 hpi, which was set at 100%. The data depict average values from two experiments with cells from two different donors ±SD. (D) Morphology of DCs stimulated with the indicated stimuli at 24 h post treatment.</p

The macrophage scavenger receptor CD163 functions as an innate immune sensor for bacteria

The plasma membrane glycoprotein receptor CD163 is a member of the scavenger receptor cystein-rich (SRCR) superfamily class B that is highly expressed on resident tissue macrophages in vivo. Previously, the molecule has been shown to act as a receptor for hemoglobin-haptoglobin complexes and to mediate cell-cell interactions between macrophages and developing erythroblasts in erythroblastic islands. Here, we provide evidence for a potential role for CD163 in host defense. In particular, we demonstrate that CD163 can function as a macrophage receptor for bacteria. CD163 was shown to bind both Gram-positive and -negative bacteria, and a previously identified cell-binding motif in the second scavenger domain of CD163 was sufficient to mediate this binding. Expression of CD163 in monocytic cells promoted bacteria-induced proinflammatory cytokine production. Finally, newly generated antagonistic antibodies against CD163 were able to potently inhibit cytokine production elicited by bacteria in freshly isolated human monocytes. These findings identify CD163 as a macrophage receptor for bacteria and suggest that, during bacterial infection, CD163 on resident tissue macrophages acts as an innate immune sensor and inducer of local inflammatio