Persistence and clearance of Ebola virus RNA from seminal fluid of Ebola virus disease survivors: a longitudinal analysis and modelling study

Background By January, 2016, all known transmission chains of the Ebola virus disease (EVD) outbreak in west Africa had been stopped. However, there is concern about persistence of Ebola virus in the reproductive tract of men who have survived EVD. We aimed to use biostatistical modelling to describe the dynamics of Ebola virus RNA load in seminal fl uid, including clearance parameters. Methods In this longitudinal study, we recruited men who had been discharged from three Ebola treatment units in Guinea between January and July, 2015. Participants provided samples of seminal fl uid at follow-up every 3–6 weeks, which we tested for Ebola virus RNA using quantitative real-time RT-PCR. Representative specimens from eight participants were then inoculated into immunodefi cient mice to test for infectivity. We used a linear mixed-eff ect model to analyse the dynamics of virus persistence in seminal fl uid over time. Findings We enrolled 26 participants and tested 130 seminal fl uid specimens; median follow up was 197 days (IQR 187–209 days) after enrolment, which corresponded to 255 days (228–287) after disease onset. Ebola virus RNA was detected in 86 semen specimens from 19 (73%) participants. Median duration of Ebola virus RNA detection was 158 days after onset (73–181; maximum 407 days at end of follow-up). Mathematical modelling of the quantitative time-series data showed a mean clearance rate of Ebola virus RNA from seminal fl uid of –0·58 log units per month, although the clearance kinetic varied greatly between participants. Using our biostatistical model, we predict that 50% and 90% of male survivors clear Ebola virus RNA from seminal fl uid at 115 days (90% prediction interval 72–160) and 294 days (212–399) after disease onset, respectively. We also predicted that the number of men positive for Ebola virus RNA in aff ected countries would decrease from about 50 in January 2016, to fewer than 1 person by July, 2016. Infectious virus was detected in 15 of 26 (58%) specimens tested in mice. Interpretation Time to clearance of Ebola virus RNA from seminal fl uid varies greatly between individuals and could be more than 13 months. Our predictions will assist in decision-making about surveillance and preventive measures in EVD outbreaks

Establishment of Recombinant Trisegmented Mopeia Virus Expressing Two Reporter Genes for Screening of Mammarenavirus Inhibitors

Highly pathogenic Arenaviruses, like the Lassa Virus (LASV), pose a serious public health threat in affected countries. Research and development of vaccines and therapeutics are urgently needed but hampered by the necessity to handle these pathogens under biosafety level 4 conditions. These containment restrictions make large-scale screens of antiviral compounds difficult. Therefore, the Mopeia virus (MOPV), closely related to LASV, is often used as an apathogenic surrogate virus. We established for the first time trisegmented MOPVs (r3MOPV) with duplicated S segments, in which one of the viral genes was replaced by the reporter genes ZsGreen (ZsG) or Renilla Luciferase (Rluc), respectively. In vitro characterization of the two trisegmented viruses (r3MOPV ZsG/Rluc and r3MOPV Rluc/ZsG), showed comparable growth behavior to the wild type virus and the expression of the reporter genes correlated well with viral titer. We used the reporter viruses in a proof-of-principle in vitro study to evaluate the antiviral activity of two well characterized drugs. IC50 values obtained by Rluc measurement were similar to those obtained by virus titers. ZsG expression was also suitable to evaluate antiviral effects. The trisegmented MOPVs described here provide a versatile and valuable basis for rapid high throughput screening of broadly reactive antiviral compounds against arenaviruses under BSL-2 conditions

Evaluation of antiviral efficacy of ribavirin, arbidol, and T-705 (favipiravir) in a mouse model for Crimean-Congo hemorrhagic fever.

BACKGROUND: Mice lacking the type I interferon receptor (IFNAR-/- mice) reproduce relevant aspects of Crimean-Congo hemorrhagic fever (CCHF) in humans, including liver damage. We aimed at characterizing the liver pathology in CCHF virus-infected IFNAR-/- mice by immunohistochemistry and employed the model to evaluate the antiviral efficacy of ribavirin, arbidol, and T-705 against CCHF virus. METHODOLOGY/PRINCIPAL FINDINGS: CCHF virus-infected IFNAR-/- mice died 2-6 days post infection with elevated aminotransferase levels and high virus titers in blood and organs. Main pathological alteration was acute hepatitis with extensive bridging necrosis, reactive hepatocyte proliferation, and mild to moderate inflammatory response with monocyte/macrophage activation. Virus-infected and apoptotic hepatocytes clustered in the necrotic areas. Ribavirin, arbidol, and T-705 suppressed virus replication in vitro by ≥3 log units (IC50 0.6-2.8 µg/ml; IC90 1.2-4.7 µg/ml). Ribavirin [100 mg/(kg×d)] did not increase the survival rate of IFNAR-/- mice, but prolonged the time to death (p<0.001) and reduced the aminotransferase levels and the virus titers. Arbidol [150 mg/(kg×d)] had no efficacy in vivo. Animals treated with T-705 at 1 h [15, 30, and 300 mg/(kg×d)] or up to 2 days [300 mg/(kg×d)] post infection survived, showed no signs of disease, and had no virus in blood and organs. Co-administration of ribavirin and T-705 yielded beneficial rather than adverse effects. CONCLUSIONS/SIGNIFICANCE: Activated hepatic macrophages and monocyte-derived cells may play a role in the proinflammatory cytokine response in CCHF. Clustering of infected hepatocytes in necrotic areas without marked inflammation suggests viral cytopathic effects. T-705 is highly potent against CCHF virus in vitro and in vivo. Its in vivo efficacy exceeds that of the current standard drug for treatment of CCHF, ribavirin

Treatment of CCHFV-infected IFNAR^−/− mice with ribavirin and arbidol hydrochloride.

<p>Mice were inoculated i.p. with 10, 100, or 1,000 FFU of virus. Ribavirin was administered i.p. once daily. Animals received a ribavirin dose of 100/(kg×d) or 0.9% NaCl as a placebo. Treatment was commenced 1 h p.i. and continued until death or day 8. Arbidol was administered once daily per os using a stomach probe. Animals received an arbidol dose of 150 mg/(kg×d) or 0.5% methylcellulose as a placebo. Treatment was commenced 1 day before infection and continued until death or day 8. Organ titers were determined in animals that succumbed to the infection or had to be euthanized due to the severity of the disease. In addition, three animals from the ribavirin-treated group were randomly euthanized at day 3 p.i. to determine the virus titer in organs (weight, AST, ALT, and viremia data obtained from these mice until day 3 were included in the respective graphs). Mean and standard deviation are shown for weight and log-transformed organ titers. Vertical bars in the graphs for AST and ALT (note the log scale of the y-axis) and the log-transformed virus titers in blood represent the mean values. The duration of treatment in the survival plots, the range of viremia below the detection limit of the immunofocusassay as well as the normal reference range of AST and ALT in mice <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002804#pntd.0002804-Zhou1" target="_blank">[59]</a> are shaded in grey. Notes. 100 FFU placebo group: The animal, which died at day 6, showed no AST/ALT elevation and viremia at day 4. 100 FFU ribavirin group: The surviving animal did not show detectable viremia, but AST elevation. Viremia was not determined at day 11. 10 FFU arbidol group: The surviving animal had no AST elevation and viremia at day 4. Some values for days 4, 8, and, 11 were not determined due to insufficient amount of blood.</p

Testing of the antiviral activity of ribavirin, arbidol hydrochloride, and T-705 in cell culture.

<p>Vero E6 cells were inoculated with CCHFV at a MOI of 0.01 and compound was added 1–4 days p.i. by immunofocus assay. Cell growth and viability under compound treatment was determined by the MTT method. Dose–response curves were fitted to the data. The graphs show representative experiments with data points representing mean and standard deviation of ≥3 replicates. The IC₅₀, IC₉₀, and IC₉₉ values were calculated from the sigmoidal functions of 2–3 independent experiments and are shown below the graphs (mean and range).</p

Survival, body weight, AST, ALT, and virus titer in blood and organs of IFNAR^−/− mice infected with different doses of CCHFV.

<p>Mice were inoculated i.p. with 10, 100, 1,000, or 10,000 FFU of virus. Organ titers were determined in animals that succumbed to the infection or had to be euthanized due to the severity of the disease. Mean and standard deviation are shown for weight and log-transformed organ titers. Vertical bars in the graphs for AST and ALT (note the log scale of the y-axis) and the log-transformed virus titers in blood represent the mean values. The data for the 100 and 1,000 FFU groups of naïve animals were pooled with corresponding data from placebo controls shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002804#pntd-0002804-g005" target="_blank">Fig. 5</a> to provide more reliable estimates for the parameters. The range of viremia below the detection limit of the immunofocusassay as well as the normal reference range of AST and ALT in mice <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002804#pntd.0002804-Zhou1" target="_blank">[59]</a> are shaded in grey. Notes. 10 FFU group: The 2 surviving animals showed neither AST/ALT elevation nor viremia at day 4 (the ALT value for one animal was not determined). 100 FFU group: The animal, which died at day 6, showed no AST/ALT elevation and viremia at days 2 and 4.</p

IHC for markers of inflammation, apoptosis, and proliferation in the liver of naïve and CCHFV-infected IFNAR^−/− mice and effect of treatment with ribavirin and T-705.

<p>Animals were inoculated i.p. with 100 FFU of CCHFV. Liver was collected (i) from naïve animals (ii) from animals that succumbed to the infection without treatment at day 3 p.i., (iii) that succumbed to the infection at day 9 p.i. following ribavirin treatment with 100 mg/(kg×d), and (iv) that were euthanized at day 3 p.i. during T-705 treatment with 300 mg/(kg×d). Treatment with ribavirin or T-705 was commenced 1 h p.i. and continued until death or day 8. Sections were stained with antibodies against CD3, B220, Ly6G, Iba-1, iNOS, cleaved caspase-3, and Ki67. T cells are marked with arrows, as the antibody against CD3 shows some spurious crossreactivity. Histopathological findings are representative for 2–3 animals that were analyzed per group. Scale bar = 50 µm.</p