Growth properties of recombinant viruses.

<p><b>A.</b> Viral growth curves in BHK-21 and A549 cells infected with rMP12 or rMP12:S-Swap (MOI of 0.01 or 5 as indicated). <b>B.</b> The effect of multiplicity of infection on viral yield in BHK-21 cells. Cells were infected with rMP12 or rMP12:S-Swap at multiplicities from 0.0005 to 5 PFU/cell. Viral supernatants were harvested at 72 h p.i. and titrated by plaque assay. Graphs are presented for one representative experiment. <b>C.</b> Viral growth curves in mosquito cells. <i>A. albopictus</i> C6/36 and U4.4, and <i>A. aegypti</i> Ae, cells were infected with rMP12, rMP12:S-Swap, rMP12ΔNSs:eGFP or rMP12:S-SwapΔNSs:eGFP at MOI of 1; BHK-21 cells were similarly infected as a control.</p

Oligonucleotides used in reverse transcription and qRT-PCR reactions.

a<p>From Rift Valley fever virus strain MP-12 segment S, DQ380154.</p>b<p>From Rift Valley fever virus strain MP-12 segment M, DQ380208.</p><p>Tagged oligonucleotides were used for strand specific reverse transcription. The TAG sequence is underlined.</p

Serial passage of mosquito cells infected with rMP12 or rMP12:S-Swap.

<p><i>A. albopictus</i> C6/36, U4.4 cells or <i>A. aegypti</i> Ae were infected with rMP12 or rMP12:S-Swap at a MOI of 0.01. Cell monolayers were passaged (split ratio 1∶5) every 5–7 days (when 100% confluency was observed). Cell extracts were prepared from each passage, proteins fractionated SDS-PAGE, transferred to a membrane, and blots probed with anti-N, anti-NSs and anti-tubulin antibodies as indicated. C3/36 cells infected with rMP12:S-Swap died after passage 3.</p

Protein expression by recombinant viruses.

<p><b>A.</b> Western blot analysis of proteins synthesised in BHK-21 cells infected with rMP12 (M), rMP12:S-Swap (S), rMP12ΔNSs:eGFP (MΔ) or rMP12:S-SwapΔNSs:eGFP (SΔ) at MOI of 1. Cell lysates, prepared at the indicated h p.i., were fractionated by SDS-PAGE, and after transfer, membranes were reacted with rabbit antibodies specific for N, NSs, Gn or eGFP as indicated. Anti-tubulin antibodies were used as a loading control. <b>B.</b> eGFP fluorescence in BHK-21 infected with rMP12ΔNSs:eGFP or rMP12:S-SwapΔNSs:eGFP as above. <b>C.</b> Western blot analysis of infected mosquito cells. <i>A.albopictus</i> C6/36, U4.4 or <i>A. aegypti</i> Ae cells were infected with recombinant viruses (MOI of 1) and lysates prepared at different times post infection. Fractionated proteins were probed with the indicated antibodies. <b>D.</b> eGFP fluorescence in mosquito cells infected with rMP12ΔNSs:eGFP or rMP12:S-SwapΔNSs:eGFP as above. Note that eGFP fluorescence in parts B and D was recorded first and then the same cells were harvested for the western blotting.</p

Creation of rMP12:S-Swap.

<p><b>A.</b> Schematic of the transcription and replication products of the S segments of rMP12 and rMP12:S-Swap. The sites at which oligonucleotides 1 and 2 anneal are indicated. <b>B.</b> Agarose gel showing RT-PCR products to confirm structure of S segment. BHK-21 cells were infected with rMP12 (MP12) or rMP12:S-Swap (SWAP) viruses at an MOI of 1. Total cellular RNA was extracted at 48 h p.i., and S-segment RT-PCR was performed. As a control, PCR on the appropriate cDNA-containing plasmids was performed with the same primers. <b>C.</b> Titres of recombinant virus stocks from multiple independent preparations were determined by plaque assay in BHK-21 cells. The mean titre and standard error of n = 4 preparations of each recombinant virus stock are shown (* p>0.05) <b>D.</b> Comparison of plaque sizes of rMP12, rMP12:S-Swap, rMP12ΔNSs:eGFP or rMP12:S-SwapΔNSs:eGFP on BHK-21 cells. Cell monolayers were fixed at 96 h p.i. with 4% paraformaldehyde and stained with Giemsa solution.</p

Summary of qRT-PCR analysis.

a<p>, significantly different, p<0.05 (see Methods for details).</p>b<p>, not significantly different,</p

Human immune cell engraftment does not alter development of severe acute Rift Valley fever in mice

<div><p>Rift Valley fever (RVF) in humans is usually mild, but, in a subset of cases, can progress to severe hepatic and neurological disease. Rodent models of RVF generally develop acute severe clinical disease. Here, we inoculated humanized NSG-SGM3 mice with Rift Valley fever virus (RVFV) to investigate whether the presence of human immune cells in mice would alter the progression of RVFV infection to more closely model human disease. Despite increased human cytokine expression, including responses mirroring those seen in human disease, and decreased hepatic viral RNA levels at terminal euthanasia, both high- and low-dose RVFV inoculation resulted in lethal disease in all mice with comparable time-to-death as unengrafted mice.</p></div

Human and mouse cytokine expression in RVFV-inoculated humanized mice.

<p>Background control NSGS mice or Hu-NSG-SGM3 mice at 13 or 19 wk post engraftment were inoculated intramuscularly with 10⁴ TCID₅₀ (Hi) or 10¹ TCID₅₀ (Lo) of RVFV; samples were collected at terminal euthanasia (3 DPI for Lo; 2 DPI for Hi). Human (A) and mouse (B) cytokine expression in plasma of Hu-NSG-SGM3 determined by multiplex bead-based assays (mean ± SD). Illustrated is a subset of analytes that, in general, demonstrated the most pronounced increases in expression; complete human 25-plex and mouse 26-plex array data are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201104#pone.0201104.s001" target="_blank">S1 Table</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201104#pone.0201104.s002" target="_blank">S2 Table</a>, respectively. In addition to samples not obtained due to acute terminal disease, samples excluded due to insufficient volume from human cytokine analysis include Hi-13-wk-2 (ID 5083–08) and Hi-NSGS-2, and from mouse cytokine analysis Lo-13-wk-4 (5084–20), and Hi-13-wk-2 (ID 5083–08). Historical samples from mock-inoculated (control, open circles) Hu-NSG-SGM3 mice were used to determine baseline expression in both panels. Shading indicates areas outside of the dynamic range of the assay, as determined by the standard curve run in conjunction with samples.</p

Weight change, survival, and viral RNA levels in RVFV-inoculated humanized mice.

<p>(A) Weight change (mean ± SD) and (B) survival in unengrafted NSG-SGM3 (NSGS) mice and Hu-NSG-SGM3 mice inoculated intramuscularly with 10⁴ TCID₅₀ (Hi) or 10¹ TCID₅₀ (Lo) of RVFV. Humanized mice were inoculated either 13 or 19 weeks (wk) post engraftment, as indicated. (C) Viral RNA genome copy number (per μL of RNA) normalized to 18S in blood, liver, spleen, and brain collected at time of terminal euthanasia (NSGS mice: 2–3 DPI for Hi or 3–4 DPI for Lo; Hu-NSG-SGM3 mice: 2 DPI for Hi or 3 DPI for Lo) determined by qRT-PCR (mean ± SD). Blood was obtained from all but 3 mice (Lo-13-wk-1 [ID 5082–15], and one mouse each in the Hi- and Lo-NSGS groups). Significant at confidence of *p ≦ 0.05 (NSGS vs. Hu-NSG-SGM3 at same dose); **p < 0.0001 (mean in liver of all groups vs. in spleen or brain).</p

Development of a reverse genetics system for Sosuga virus allows rapid screening of antiviral compounds

<div><p>Sosuga virus (SOSV) is a recently discovered zoonotic paramyxovirus isolated from a single human case in 2012; it has been ecologically and epidemiologically associated with transmission by the Egyptian rousette bat (<i>Rousettus aegyptiacus</i>). Bats have long been recognized as sources of novel zoonotic pathogens, including highly lethal paramyxoviruses like Nipah virus (NiV) and Hendra virus (HeV). The ability of SOSV to cause severe human disease supports the need for studies on SOSV pathogenesis to better understand the potential impact of this virus and to identify effective treatments. Here we describe a reverse genetics system for SOSV comprising a minigenome-based assay and a replication-competent infectious recombinant reporter SOSV that expresses the fluorescent protein ZsGreen1 in infected cells. First, we used the minigenome assay to rapidly screen for compounds inhibiting SOSV replication at biosafety level 2 (BSL-2). The antiviral activity of candidate compounds was then tested against authentic viral replication using the reporter SOSV at BSL-3. We identified several compounds with anti-SOSV activity, several of which also inhibit NiV and HeV. Alongside its utility in screening for potential SOSV therapeutics, the reverse genetics system described here is a powerful tool for analyzing mechanisms of SOSV pathogenesis, which will facilitate our understanding of how to combat the potential public health threats posed by emerging bat-borne paramyxoviruses.</p></div