Rodent control strategies and Lassa virus: some unexpected effects in Guinea, West Africa.

The Natal multimammate mouse (Mastomys natalensis) is the host of Lassa mammarenavirus, causing Lassa haemorrhagic fever in West Africa. As there is currently no operational vaccine and therapeutic drugs are limited, we explored rodent control as an alternative to prevent Lassa virus spillover in Upper Guinea, where the disease is highly endemic in rural areas. In a seven-year experiment, we distributed rodenticides for 10-30 days once a year and, in the last year, added intensive snap trapping for three months in all the houses of one village. We also captured rodents both before and after the intervention period to assess their effectiveness by examining alterations in trapping success and infection rates (Lassa virus RNA and IgG antibodies). We found that both interventions reduced the rodent population by 74-92% but swiftly rebounded to pre-treatment levels, even already six months after the last snap-trapping control. Furthermore, while we observed that chemical control modestly decreased Lassa virus infection rates annually (a reduction of 5% in seroprevalence per year), the intensive trapping unexpectedly led to a significantly higher infection rate (from a seroprevalence of 28% before to 67% after snap trapping control). After seven years, we conclude that annual chemical control, alone or with intensive trapping, is ineffective and sometimes counterproductive in preventing Lassa virus spillover in rural villages. These unexpected findings may result from density-dependent breeding compensation following culling and the survival of a small percentage of chronically infected rodents that may spread the virus to a new susceptible generation of mice.

Spatio-temporal spread of Lassa virus and a new rodent host in the Mano River Union area, West Africa.

The spread of Lassa virus (LASV) in Guinea, Liberia and Sierra Leone, which together are named the Mano River Union (MRU) area, was examined phylogeographically. To provide a reliable evolutionary scenario, new rodent-derived, whole LASV sequences were included. These were generated by metatranscriptomic next-generation sequencing from rodents sampled between 2003 and 2020 in 21 localities of Guinea and Sierra Leone. An analysis was performed using BEAST to perform continuous phylogeographic inference and EvoLaps v36 to visualize spatio-temporal spread. LASV was identified as expected in its primary host reservoir, the Natal multimammate mouse (Mastomys natalensis), and also in two Guinean multimammate mice (Mastomys erythroleucus) in northern Sierra Leone and two rusty-bellied brush-furred mice (Lophuromys sikapusi) in southern Sierra Leone. This finding is consistent with the latter two species being secondary host reservoirs. The strains in these three species were very closely related in LASV lineage IV. Phylogenetic analysis indicated that the most recent common ancestor of lineage IV existed 316-374 years ago and revealed distinct, well-supported clades from Sierra Leone (Bo, Kabala and Kenema), Guinea (Faranah, Kissidougou-Guekedou and Macenta) and Liberia (Phebe-Ganta). The phylogeographic scenario suggests southern Guinea as the point of origin of LASV in the MRU area, with subsequent spread to towards Mali, Liberia and Sierra Leone at a mean speed of 1.6 to 1.1 km/year.

A validated protocol to UV-inactivate SARS-CoV-2 and herpesvirus-infected cells.

Downstream analysis of virus-infected cell samples, such as reverse transcription polymerase chain reaction (RT PCR) or mass spectrometry, often needs to be performed at lower biosafety levels than their actual cultivation, and thus the samples require inactivation before they can be transferred. Common inactivation methods involve chemical crosslinking with formaldehyde or denaturing samples with strong detergents, such as sodium dodecyl sulfate. However, these protocols destroy the protein quaternary structure and prevent the analysis of protein complexes, albeit through different chemical mechanisms. This often leads to studies being performed in over-expression or surrogate model systems. To address this problem, we generated a protocol that achieves the inactivation of infected cells through ultraviolet (UV) irradiation. UV irradiation damages viral genomes and crosslinks nucleic acids to proteins but leaves the overall structure of protein complexes mostly intact. Protein analysis can then be performed from intact cells without biosafety containment. While UV treatment protocols have been established to inactivate viral solutions, a protocol was missing to inactivate crude infected cell lysates, which heavily absorb light. In this work, we develop and validate a UV inactivation protocol for SARS-CoV-2, HSV-1, and HCMV-infected cells. A fluence of 10,000 mJ/cm2 with intermittent mixing was sufficient to completely inactivate infected cells, as demonstrated by the absence of viral replication even after three sequential passages of cells inoculated with the treated material. The herein described protocol should serve as a reference for inactivating cells infected with these or similar viruses and allow for the analysis of protein quaternary structure from bona fide infected cells.

Experimental Morogoro Virus Infection in Its Natural Host, Mastomys natalensis.

Natural hosts of most arenaviruses are rodents. The human-pathogenic Lassa virus and several non-pathogenic arenaviruses such as Morogoro virus (MORV) share the same host species, namely Mastomys natalensis (M. natalensis). In this study, we investigated the history of infection and virus transmission within the natural host population. To this end, we infected M. natalensis at different ages with MORV and measured the health status of the animals, virus load in blood and organs, the development of virus-specific antibodies, and the ability of the infected individuals to transmit the virus. To explore the impact of the lack of evolutionary virus-host adaptation, experiments were also conducted with Mobala virus (MOBV), which does not share M. natalensis as a natural host. Animals infected with MORV up to two weeks after birth developed persistent infection, seroconverted and were able to transmit the virus horizontally. Animals older than two weeks at the time of infection rapidly cleared the virus. In contrast, MOBV-infected neonates neither developed persistent infection nor were able to transmit the virus. In conclusion, we demonstrate that MORV is able to develop persistent infection in its natural host, but only after inoculation shortly after birth. A related arenavirus that is not evolutionarily adapted to M. natalensis is not able to establish persistent infection. Persistently infected animals appear to be important to maintain virus transmission within the host population.

Validation of Inactivation Methods for Arenaviruses.

Several of the human-pathogenic arenaviruses cause hemorrhagic fever and have to be handled under biosafety level 4 conditions, including Lassa virus. Rapid and safe inactivation of specimens containing these viruses is fundamental to enable downstream processing for diagnostics or research under lower biosafety conditions. We established a protocol to test the efficacy of inactivation methods using the low-pathogenic Morogoro arenavirus as surrogate for the related highly pathogenic viruses. As the validation of chemical inactivation methods in cell culture systems is difficult due to cell toxicity of commonly used chemicals, we employed filter devices to remove the chemical and concentrate the virus after inactivation and before inoculation into cell culture. Viral replication in the cells was monitored over 4 weeks by using indirect immunofluorescence and immunofocus assay. The performance of the protocol was verified using published inactivation methods including chemicals and heat. Ten additional methods to inactivate virus in infected cells or cell culture supernatant were validated and shown to reduce virus titers to undetectable levels. In summary, we provide a robust protocol for the validation of chemical and physical inactivation of arenaviruses in cell culture, which can be readily adapted to different inactivation methods and specimen matrices.

Discovery and genetic characterization of a novel orthonairovirus in Ixodes ricinus ticks from Danube Delta.

Different arthropod species are vectors of a wide array of arboviruses (arthropod-borne viruses) and have likely been central to viral evolution. To better understand the extent of arthropod-borne pathogens, as well as their origin and evolutionary history, it is crucial to uncover the full range of microbial agents, including viruses associated with arthropods. In this study, a collection of ticks obtained in 2016 directly from mammal and bird hosts from several rural and natural sites of Danube Delta was subjected to transcriptome sequencing and amplification assays. Vector surveillance revealed the presence of a novel orthonairovirus species, designated Sulina virus, in Ixodes ricinus ticks. Phylogenetic clustering of each viral protein consistently placed the new virus in the Orthonairovirus genus as a new genogroup closely related to Tamdy orthonairovirus, a genogroup comprising both pathogenic and tick-associated orthonairoviruses. The serological testing of engorged ticks and blood of infected hosts, along with the inoculation of vertebrate cells and mice found no specific antibodies or viral replication, suggesting that Sulina virus is an orthonairovirus associated with the virome of Ixodes ricinus. Finally, the characterization of a novel orthonairovirus identified using high throughput sequencing will advance our knowledge of interactions between viruses and tick vectors, expanding our perspective on fundamental questions regarding orthonairovirus evolution, diversity, ecology and potential of emergence as pathogens.

Lassa fever in Benin: description of the 2014 and 2016 epidemics and genetic characterization of a new Lassa virus.

We report two outbreaks of Lassa fever that occurred in Benin in 2014 and 2016 with 20 confirmed cases and 50% (10/20) mortality. Benin was not previously considered to be an endemic country for Lassa fever, resulting in a delay to diagnose the disease and its human transmission. Molecular investigations showed the viral genomes to be similar to that of the Togo strain, which is genetically very different from other known strains and confirms the existence of a new lineage. Endemic circulation of Lassa virus in a new territory and the genetic diversity thus confirm that this virus represents a growing threat for West African people. Given the divergence of the Benin strain from the prototypic Josiah Sierra Leone strain frequently used to generate vaccine candidates, the efficacy of vaccine candidates should also be demonstrated with this strain.

Households as hotspots of Lassa fever? Assessing the spatial distribution of Lassa virus-infected rodents in rural villages of Guinea.

The Natal multimammate mouse (Mastomys natalensis) is the reservoir host of Lassa virus (LASV), an arenavirus that causes Lassa haemorrhagic fever in humans in West Africa. While previous studies suggest that spillover risk is focal within rural villages due to the spatial behaviour of the rodents, the level of clustering was never specifically assessed. Nevertheless, detailed information on the spatial distribution of infected rodents would be highly valuable to optimize LASV-control campaigns, which are limited to rodent control or interrupting human-rodent contact considering that a human vaccine is not available. Here, we analysed data from a four-year field experiment to investigate whether LASV-infected rodents cluster in households in six rural villages in Guinea. Our analyses were based on the infection status (antibody or PCR) and geolocation of rodents (n = 864), and complemented with a phylogenetic analysis of LASV sequences (n = 119). We observed that the majority of infected rodents were trapped in a few houses (20%) and most houses were rodent-free at a specific point in time (60%). We also found that LASV strains circulating in a specific village were polyphyletic with respect to neighbouring villages, although most strains grouped together at the sub-village level and persisted over time. In conclusion, our results suggest that: (i) LASV spillover risk is heterogeneously distributed within villages in Guinea; (ii) viral elimination in one particular village is unlikely if rodents are not controlled in neighbouring villages. Such spatial information should be incorporated into eco-epidemiological models that assess the cost-efficiency of LASV control strategies.

Cytokine Profile Distinguishes Children With Plasmodium falciparum Malaria From Those With Bacterial Blood Stream Infections.

BACKGROUND: Malaria presents with unspecific clinical symptoms that frequently overlap with other infectious diseases and is also a risk factor for coinfections, such as non-Typhi Salmonella. Malaria rapid diagnostic tests are sensitive but unable to distinguish between an acute infection requiring treatment and asymptomatic malaria with a concomitant infection. We set out to test whether cytokine profiles could predict disease status and allow the differentiation between malaria and a bacterial bloodstream infection. METHODS: We created a classification model based on cytokine concentration levels of pediatric inpatients with either Plasmodium falciparum malaria or a bacterial bloodstream infection using the Luminex platform. Candidate markers were preselected using classification and regression trees, and the predictive strength was calculated through random forest modeling. RESULTS: Analyses revealed that a combination of 7-15 cytokines exhibited a median disease prediction accuracy of 88% (95th percentile interval, 73%-100%). Haptoglobin, soluble Fas-Ligand, and complement component C2 were the strongest single markers with median prediction accuracies of 82% (with 95th percentile intervals of 71%-94%, 62%-94%, and 62%-94%, respectively). CONCLUSIONS: Cytokine profiles possess good median disease prediction accuracy and offer new possibilities for the development of innovative point-of-care tests to guide treatment decisions in malaria-endemic regions.

Phylogeography of Lassa Virus in Nigeria.

Lassa virus is genetically diverse with several lineages circulating in West Africa. This study aimed at describing the sequence variability of Lassa virus across Nigeria and inferring its spatiotemporal evolution. We sequenced and isolated 77 Lassa virus strains from 16 Nigerian states. The final data set, including previous works, comprised metadata and sequences of 219 unique strains sampled between 1969 and 2018 in 22 states. Most of this data originated from Lassa fever patients diagnosed at Irrua Specialist Teaching Hospital, Edo State, Nigeria. The majority of sequences clustered with the main Nigerian lineages II and III, while a few sequences formed a new cluster related to Lassa virus strains from Hylomyscus pamfi Within lineages II and III, seven and five sublineages, respectively, were distinguishable. Phylogeographic analysis suggests an origin of lineage II in the southeastern part of the country around Ebonyi State and a main vector of dispersal toward the west across the Niger River, through Anambra, Kogi, Delta, and Edo into Ondo State. The frontline of virus dispersal appears to be in Ondo. Minor vectors are directed northeast toward Taraba and Adamawa and south toward Imo and Rivers. Lineage III might have spread from northern Plateau State into Kaduna, Nasarawa, Federal Capital Territory, and Bauchi. One sublineage moved south and crossed the Benue River into Benue State. This study provides a geographic mapping of lineages and phylogenetic clusters in Nigeria at a higher resolution. In addition, we estimated the direction and time frame of virus dispersal in the country.IMPORTANCE Lassa virus is the causative agent of Lassa fever, a viral hemorrhagic fever with a case fatality rate of approximately 30% in Africa. Previous studies disclosed a geographical pattern in the distribution of Lassa virus strains and a westward movement of the virus across West Africa during evolution. Our study provides a deeper understanding of the geography of genetic lineages and sublineages of the virus in Nigeria. In addition, we modeled how the virus spread in the country. This knowledge allows us to predict into which geographical areas the virus might spread in the future and prioritize areas for Lassa fever surveillance. Our study not only aimed to generate Lassa virus sequences from across Nigeria but also to isolate and conserve the respective viruses for future research. Both isolates and sequences are important for the development and evaluation of medical countermeasures to treat and prevent Lassa fever, such as diagnostics, therapeutics, and vaccines.

Evaluation of rodent control to fight Lassa fever based on field data and mathematical modelling.

The Natal multimammate mouse (Mastomys natalensis) is the reservoir host of Lassa virus, an arenavirus that causes Lassa haemorrhagic fever in humans in West Africa. Because no vaccine exists and therapeutic options are limited, preventing infection through rodent control and human behavioural measures is currently considered to be the only option. In order to assess the efficacy of rodent control, we performed a 4-year field experiment in rural Upper Guinea and developed a mathematical model to simulate different control strategies (annual density control, continuous density control, and rodent vaccination). For the field study, rodenticide baits were placed each year in three rural villages, while three other villages were used as controls. Rodents were trapped before and after every treatment and their antibody status and age were determined. Data from the field study were used to parameterize the mathematical model. In the field study, we found a significant negative effect of rodent control on seroprevalence, but this effect was small especially given the effort. Furthermore, the rodent populations recovered rapidly after rodenticide application, leading us to conclude that an annual control strategy is unlikely to significantly reduce Lassa virus spillover to humans. In agreement with this finding, the mathematical model suggests that the use of continuous control or rodent vaccination is the only strategy that could lead to Lassa virus elimination. These field and model results can serve as a guide for determining how long and frequent rodent control should be done in order to eliminate Lassa virus in rural villages.

Kinetics of Soluble Mediators of the Host Response in Ebola Virus Disease.

Background: The pathophysiology of Ebola virus disease (EVD) is still poorly understood. This study aimed at identifying soluble biomarkers that inform on disease mechanisms. Methods: Fifty-four soluble mediators of the immune, coagulation, and endothelial system were measured in baseline and follow-up samples from hospitalized patients with EVD, using Luminex technology. Cross-sectional expression levels and changes over time were correlated with outcome. Results: Levels of circulating proinflammatory cytokines and chemokines, as well as markers of endothelial dysfunction and coagulopathy, were elevated on admission to hospital in patients who died from EVD as compared to survivors. These markers further increased in patients who died and/or decreased over time in survivors. In contrast, markers of gut integrity and T-cell response were higher in survivors and increased until discharge. Conclusions: Inflammatory response, endothelial integrity, gastric tissue protection, and T cell immunity play a role in EVD pathophysiology.

Widespread arenavirus occurrence and seroprevalence in small mammals, Nigeria.

BACKGROUND: Lassa fever, killing thousands of people annually, is the most reported viral zoonotic disease in Nigeria. Recently, different rodent species carrying diverse lineages of the Lassa virus (LASV) in addition to a novel Mobala-like genetic sequence were detected within the country. Here, screening 906 small mammal specimens from 11 localities for IgG antibodies and incorporating previous PCR detection data involving the same populations, we further describe arenavirus prevalence across Nigeria in relation to host species and geographical location. METHODS: Small mammals were trapped during the period 2011-2015 according to geographical location (endemic and non-endemic zones for Lassa fever), season (rainy and dry seasons between 2011 and 2012 for certain localities) and habitat (indoors, peridomestic settings and sylvatic vegetation). Identification of animal specimens from genera such as Mastomys and Mus (Nannomys) was assisted by DNA sequencing. Small mammals were tested for LASV IgG antibody using an indirect immunofluorescence assay (IFA). RESULTS: Small mammals were infected in both the endemic and non-endemic zones for Lassa fever, with a wider range of species IgG-positive (n = 8) than those which had been previously detected to be PCR-positive (n = 3). IgG-positive species, according to number of infected individuals, were Mastomys natalensis (n = 40), Mastomys erythroleucus (n = 15), Praomys daltoni (n = 6), Mus baoulei (n = 5), Rattus rattus (n = 2), Crocidura spp. (n = 2), Mus minutoides (n = 1) and Praomys misonnei (n = 1). Multimammate mice (Mastomys natalensis and M. erythroleucus) were the most ubiquitously infected, with animals testing positive by either PCR or IgG in 7 out of the 11 localities sampled. IgG prevalence in M. natalensis ranged from 1% in Abagboro, 17-36 % in Eguare Egoro, Ekpoma and Ngel Nyaki, up to 52 % in Mayo Ranewo. Prevalence according to locality, season and age was not, however, statistically significant for M. natalensis in Eguare Egoro and Ekpoma, localities that were sampled longitudinally. CONCLUSIONS: Overall, our study demonstrates that arenavirus occurrence is probably more widely distributed geographically and in extent of host taxa than is currently realized. This expanded scope should be taken into consideration in Lassa fever control efforts. Further sampling should also be carried out to isolate and characterize potential arenaviruses present in small mammal populations we found to be seropositive.

New Lineage of Lassa Virus, Togo, 2016.

We describe a strain of Lassa virus representing a putative new lineage that was isolated from a cluster of human infections with an epidemiologic link to Togo. This finding extends the known range of Lassa virus to Togo.

Control measures following a case of imported Lassa fever from Togo, North Rhine Westphalia, Germany, 2016.

In a patient transferred from Togo to Cologne, Germany, Lassa fever was diagnosed 12 days post mortem. Sixty-two contacts in Cologne were categorised according to the level of exposure, and gradual infection control measures were applied. No clinical signs of Lassa virus infection or Lassa specific antibodies were observed in the 62 contacts. Thirty-three individuals had direct contact to blood, other body fluids or tissue of the patients. Notably, with standard precautions, no transmission occurred between the index patient and healthcare workers. However, one secondary infection occurred in an undertaker exposed to the corpse in Rhineland-Palatinate, who was treated on the isolation unit at the University Hospital of Frankfurt. After German authorities raised an alert regarding the imported Lassa fever case, an American healthcare worker who had cared for the index patient in Togo, and who presented with diarrhoea, vomiting and fever, was placed in isolation and medevacked to the United States. The event and the transmission of Lassa virus infection outside of Africa underlines the need for early diagnosis and use of adequate personal protection equipment (PPE), when highly contagious infections cannot be excluded. It also demonstrates that larger outbreaks can be prevented by infection control measures, including standard PPE.

Detection and characterization of a novel rhabdovirus in Aedes cantans mosquitoes and evidence for a mosquito-associated new genus in the family Rhabdoviridae.

Thanks to recent advances in random amplification technologies, metagenomic surveillance expanded the number of novel, often unclassified viruses within the family Rhabdoviridae. Using a vector-enabled metagenomic (VEM) tool, we identified a novel rhabdovirus in Aedes cantans mosquitoes collected from Germany provisionally named Ohlsdorf virus (OHSDV). The OHSDV genome encodes the canonical rhabdovirus structural proteins (N, P, M, G and L) with alternative ORF in the P gene. Sequence analysis indicated that OHSDV exhibits a similar genome organization and characteristics compared to other mosquito-associated rhabdoviruses (Riverside virus, Tongilchon virus and North Creek virus). Complete L protein based phylogeny revealed that all four viruses share a common ancestor and form a deeply rooted and divergent monophyletic group within the dimarhabdovirus supergroup and define a new genus, tentatively named Ohlsdorfvirus. Although the Ohlsdorfvirus clade is basal within the dimarhabdovirus supergroup phylogeny that includes genera of arthropod-borne rhabdoviruses, it remains unknown if viruses in the proposed new genus are vector-borne pathogens. The observed spatiotemporal distribution in mosquitoes suggests that members of the proposed genus Ohlsdorfvirus are geographically restricted/separated. These findings increase the current knowledge of the genetic diversity, classification and evolution of this virus family. Further studies are needed to determine the host range, transmission route and the evolutionary relationships of these mosquito-associated viruses with those infecting vertebrates.

Interferon-γ-Driven iNOS: A Molecular Pathway to Terminal Shock in Arenavirus Hemorrhagic Fever.

Arenaviruses such as Lassa virus (LASV) cause hemorrhagic fever. Terminal shock is associated with a systemic cytokine storm, but the mechanisms are ill defined. Here we used HLA-A2-expressing mice infected with a monkey-pathogenic strain of lymphocytic choriomeningitis virus (LCMV-WE), a close relative of LASV, to investigate the pathophysiology of arenavirus hemorrhagic fever (AHF). AHF manifested as pleural effusions, edematous skin swelling, and serum albumin loss, culminating in hypovolemic shock. A characteristic cytokine storm included numerous pro-inflammatory cytokines and nitric oxide (NO) metabolites. Edema formation and terminal shock were abrogated in mice lacking inducible nitric oxide synthase (iNOS), although the cytokine storm persisted. iNOS was upregulated in the liver in a T cell- and interferon-γ (IFN-γ)-dependent fashion. Accordingly, blockade of IFN-γ or depletion of T cells repressed hepatic iNOS and prevented disease despite unchecked high-level viremia. We identify the IFN-γ-iNOS axis as an essential and potentially druggable molecular pathway to AHF-induced shock.

External quality assessment study for ebolavirus PCR-diagnostic promotes international preparedness during the 2014 - 2016 Ebola outbreak in West Africa.

During the recent Ebola outbreak in West Africa several international mobile laboratories were deployed to the mainly affected countries Guinea, Sierra Leone and Liberia to provide ebolavirus diagnostic capacity. Additionally, imported cases and small outbreaks in other countries required global preparedness for Ebola diagnostics. Detection of viral RNA by reverse transcription polymerase chain reaction has proven effective for diagnosis of ebolavirus disease and several assays are available. However, reliability of these assays is largely unknown and requires serious evaluation. Therefore, a proficiency test panel of 11 samples was generated and distributed on a global scale. Panels were analyzed by 83 expert laboratories and 106 data sets were returned. From these 78 results were rated optimal and 3 acceptable, 25 indicated need for improvement. While performance of the laboratories deployed to West Africa was superior to the overall performance there was no significant difference between the different assays applied.

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 fluid, 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 fluid 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 immunodeficient mice to test for infectivity. We used a linear mixed-effect model to analyse the dynamics of virus persistence in seminal fluid over time. FINDINGS: We enrolled 26 participants and tested 130 seminal fluid 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 fluid 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 fluid 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 affected 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 fluid 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. FUNDING: This study was funded by European Union's Horizon 2020 research and innovation programme, Directorate-General for International Cooperation and Development of the European Commission, Institut national de la santé et de la recherche médicale (INSERM), German Research Foundation (DFG), and Innovative Medicines Initiative 2 Joint Undertaking.

Evaluation of RealStar Reverse Transcription-Polymerase Chain Reaction Kits for Filovirus Detection in the Laboratory and Field.

BACKGROUND: Diagnosis of Ebola virus (EBOV) disease (EVD) requires laboratory testing. METHODS: The RealStar Filovirus Screen reverse transcription-polymerase chain reaction (RT-PCR) kit and the derived RealStar Zaire Ebolavirus RT-PCR kit were validated using in vitro transcripts, supernatant of infected cell cultures, and clinical specimens from patients with EVD. RESULTS: The Filovirus Screen kit detected EBOV, Sudan virus, Taï Forest virus, Bundibugyo virus, Reston virus, and Marburg virus and differentiated between the genera Ebolavirus and Marburgvirus The amount of filovirus RNA that could be detected with a probability of 95% ranged from 11 to 67 RNA copies/reaction on a LightCycler 480 II. The Zaire Ebolavirus kit is based on the Filovirus Screen kit but was optimized for detection of EBOV. It has an improved signal-to-noise ratio at low EBOV RNA concentrations and is somewhat more sensitive than the Filovirus kit. Both kits show significantly lower analytical sensitivity on a SmartCycler II. Clinical evaluation revealed that the SmartCycler II, compared with other real-time PCR platforms, decreases the clinical sensitivity of the Filovirus Screen kit to diagnose EVD at an early stage. CONCLUSIONS: The Filovirus Screen kit detects all human-pathogenic filoviruses with good analytical sensitivity if performed on an appropriate real-time PCR platform. High analytical sensitivity is important for early diagnosis of EVD.