Early onset of neurological features differentiates two outbreaks of Lassa fever in Ebonyi state, Nigeria during 2017-2018.

Lassa fever (LF) is an acute viral haemorrhagic illness with various non-specific clinical manifestations. Neurological symptoms are rare at the early stage of the disease, but may be seen in late stages, in severely ill patients.The aim of this study was to describe the epidemiological evolution, socio-demographic profiles, clinical characteristics, and outcomes of patients seen during two Lassa fever outbreaks in Ebonyi State, between December 2017 and December 2018. Routinely collected clinical data from all patients admitted to the Virology Centre of the hospital during the period were analysed retrospectively. Out of a total of 83 cases, 70(84.3%) were RT-PCR confirmed while 13 (15.7%) were probable cases. Sixty-nine (83.1%) patients were seen in outbreak 1 of whom 53.6% were urban residents, while 19%, 15%, and 10% were farmers, students and health workers respectively. There were 14 (16.8%) patients, seen in second outbreak with 92.9% rural residents. There were differences in clinical symptoms, signs and laboratory findings between the two outbreaks. The case fatality rates were 29.9% in outbreak 1 and 85.7% for outbreak 2. Neurological features and abnormal laboratory test results were associated with higher mortality rate, seen in outbreak 2. This study revealed significant differences between the two outbreaks. Of particular concern was the higher case fatality during the outbreak 2 which may be from a more virulent strain of the Lassa virus. This has important public health implications and further molecular studies are needed to better define its characteristics.

Lassa fever outcomes and prognostic factors in Nigeria (LASCOPE): a prospective cohort study.

BACKGROUND: Lassa fever is a viral haemorrhagic fever endemic in parts of west Africa. New treatments are needed to decrease mortality, but pretrial reference data on the disease characteristics are scarce. We aimed to document baseline characteristics and outcomes for patients hospitalised with Lassa fever in Nigeria. METHODS: We did a prospective cohort study (LASCOPE) at the Federal Medical Centre in Owo, Nigeria. All patients admitted with confirmed Lassa fever were invited to participate and asked to give informed consent. Patients of all ages, including newborn infants, were eligible for inclusion, as were pregnant women. All participants received standard supportive care and intravenous ribavirin according to Nigeria Centre for Disease Control guidelines and underwent systematic biological monitoring for 30 days. Patients' characteristics, care received, mortality, and associated factors were recorded using standard WHO forms. We used univariable and multivariable logistic regression models to investigate an association between baseline characteristics and mortality at day 30. FINDINGS: Between April 5, 2018, and March 15, 2020, 534 patients with confirmed Lassa fever were admitted to hospital, of whom 510 (96%) gave consent and were included in the analysis. The cohort included 258 (51%) male patients, 252 (49%) female patients, 426 (84%) adults, and 84 (16%) children (younger than 18 years). The median time between first symptoms and hospital admission was 8 days (IQR 7-13). At baseline, 176 (38%) of 466 patients had a Lassa fever RT-PCR cycle threshold (Ct) lower than 30. From admission to end of follow-up, 120 (25%) of 484 reached a National Early Warning Score (second version; NEWS2) of 7 or higher, 67 (14%) of 495 reached a Kidney Disease-Improving Global Outcome (KDIGO) stage of 2 or higher, and 41 (8%) of 510 underwent dialysis. All patients received ribavirin for a median of 10 days (IQR 9-13). 62 (12%) patients died (57 [13%] adults and five [6%] children). The median time to death was 3 days (1-6). The baseline factors independently associated with mortality were the following: age 45 years or older (adjusted odds ratio 16·30, 95% CI 5·31-50·30), NEWS2 of 7 or higher (4·79, 1·75-13·10), KDIGO grade 2 or higher (7·52, 2·66-21·20), plasma alanine aminotransferase 3 or more times the upper limit of normal (4·96, 1·69-14·60), and Lassa fever RT-PCR Ct value lower than 30 (4·65, 1·50-14·50). INTERPRETATION: Our findings comprehensively document clinical and biological characteristics of patients with Lassa fever and their relationship with mortality, providing prospective estimates that could be useful for designing future therapeutic trials. Such trials comparing new Lassa fever treatments to a standard of care should take no more than 15% as the reference mortality rate and consider adopting a combination of mortality and need for dialysis as the primary endpoint. FUNDING: Institut National de la Santé et de la Recherche Médicale, University of Oxford, EU, UK Department for International Development, Wellcome Trust, French Ministry of Foreign Affairs, Agence Nationale de Recherches sur le SIDA et les hépatites virales, French National Research Institute for Sustainable Development.

Factors associated with progression to death in patients with Lassa fever in Nigeria: an observational study.

BACKGROUND: Lassa fever is endemic in several west African countries. Case-fatality rates ranging from 21% to 69% have been reported. The pathophysiology of the disease in humans and determinants of mortality remain poorly understood. We aimed to determine host protein biomarkers capable of determining disease outcome. METHODS: In this observational study, we analysed left-over blood samples from patients who tested positive for Lassa fever at Irrua Specialist Teaching Hospital, Nigeria, between January, 2014, and April, 2017. We measured viral load, concentrations of clinical chemistry parameters, and levels of 62 circulating proteins involved in inflammation, immune response, and haemostasis. Patients with a known outcome (survival or death) and at least 200 µL of good-quality diagnostic sample were included in logistic regression modelling to assess the correlation of parameters with Lassa fever outcome. Individuals who gave consent could further be enrolled into a longitudinal analysis to assess the association of parameters with Lassa fever outcome over time. Participants were divided into two datasets for the statistical analysis: a primary dataset (samples taken between Jan 1, 2014, and April 1, 2016), and a secondary dataset (samples taken between April 1, 2016, and April 1, 2017). Biomarkers were ranked by area under the receiver operating characteristic curve (AUC) from highest (most predictive) to lowest (least predictive). FINDINGS: Of 554 patients who tested positive for Lassa fever during the study period, 201 (131 in the primary dataset and 70 in the secondary dataset) were included in the biomarker analysis, of whom 74 (49 in the primary dataset and 25 in the secondary dataset) had died and 127 (82 in the primary dataset and 45 in the secondary dataset) had survived. Cycle threshold values (indicating viral load) and levels of 18 host proteins at the time of admission to hospital were significantly correlated with fatal outcome. The best predictors of outcome in both datasets were plasminogen activator inhibitor-1 (PAI-1; AUC 0·878 in the primary dataset and 0·876 in the secondary dataset), soluble thrombomodulin (TM; 0·839 in the primary dataset and 0·875 in the secondary dataset), and soluble tumour necrosis factor receptor superfamily member 1A (TNF-R1; 0·807 in the primary dataset and 0·851 in the secondary dataset), all of which had higher prediction accuracy than viral load (0·774 in the primary dataset and 0·837 in the secondary dataset). Longitudinal analysis (150 patients, of whom 36 died) showed that of the biomarkers that were predictive at admission, PAI-1 levels consistently decreased to normal levels in survivors but not in those who died. INTERPRETATION: The identification of PAI-1 and soluble TM as markers of fatal Lassa fever at admission, and of PAI-1 as a marker of fatal Lassa fever over time, suggests that dysregulated coagulation and fibrinolysis and endothelial damage have roles in the pathophysiology of Lassa fever, providing a mechanistic explanation for the association of Lassa fever with oedema and bleeding. These novel markers might aid in clinical risk stratification and disease monitoring. FUNDING: German Research Foundation, Leibniz Association, and US National Institutes of Health.

Monitoring of Lassa virus infection in suspected and confirmed cases in Ondo State, Nigeria.

INTRODUCTION: Lassa virus (LASV), the causative agent of Lassa fever (LF), an endemic acute viral haemorrhagic illness in Nigeria, is transmitted by direct contact with the rodent, contaminated food or household items. Person-to-person transmission also occurs and sexual transmission has been reported. Thus, this study investigated the presence of LASV in body fluids of suspected and confirmed cases. METHODS: this was a cross-sectional study between March 2018 and April 2019 involving 112 consenting suspected and post ribavirin confirmed cases attending the Lassa fever treatment center in Ondo State. Whole blood was collected from 57 suspected and 29 confirmed cases. Other samples from confirmed cases were 5 each of High Vaginal Swab (HVS) and seminal fluid; 12 breast milk and 4 urine. All samples were analyzed using reverse transcription-PCR (RT-PCR) targeting the S-gene of LASV. RESULTS: analysis of whole blood by RT-PCR showed that 1/57 (1.8%) suspected and 1/29 (3.4%) confirmed post ribavirin treated cases were positive. While LASV was detected in 2/5 (40%) post ribavirin treated seminal fluids and 1/11 (8.3%) breast milk. However, LASV was not detected in any of the HVS and urine samples. CONCLUSION: the detection of LASV in seminal fluid and breast milk of discharged post ribavirin treated cases suggests its persistence in these fluids of recovering Nigerians. The role of postnatal and sexual transmissions in the perennial outbreak of LF needs to be further evaluated.

Fatal case of newborn Lassa fever virus infection mimicking late onset neonatal sepsis: a case report from northern Nigeria.

BACKGROUND: Lassa fever is a zoonotic viral infection endemic to the West Africa countries. It is highly fatal during pregnancy and as such reports of neonatal onset Lassa fever infections are rare in scientific literature. We report a fatal case of Lassa fever in a 26-day-old neonate mimicking the diagnosis of late-onset neonatal sepsis. CASE PRESENTATION: The patient is a 26-day-old neonate who was admitted with a day history of fever, poor feeding, pre-auricular lymphadenopathy and sudden parental death. He was initially evaluated for late onset neonatal sepsis. He later developed abnormal bleeding and multiple convulsions while on admission, prompting the need to evaluate for Lassa fever using reverse transcription polymerase chain reaction (RT-PCR). He died 31 h into admission and RT-PCR result was positive for Lassa fever. CONCLUSIONS: Neonatal Lassa fever infection is highly fatal and can mimic neonatal sepsis. High index of suspicion is needed particularly for atypical presentations of neonatal sepsis in Lassa fever endemic areas.

Field evaluation of a Pan-Lassa rapid diagnostic test during the 2018 Nigerian Lassa fever outbreak.

Lassa virus (LASV) is the causative agent of Lassa fever (LF), an often-fatal hemorrhagic disease. LF is endemic in Nigeria, Sierra Leone and other West African countries. Diagnosis of LASV infection is challenged by the genetic diversity of the virus, which is greatest in Nigeria. The ReLASV Pan-Lassa Antigen Rapid Test (Pan-Lassa RDT) is a point-of-care, in vitro diagnostic test that utilizes a mixture of polyclonal antibodies raised against recombinant nucleoproteins of representative strains from the three most prevalent LASV lineages (II, III and IV). We compared the performance of the Pan-LASV RDT to available quantitative PCR (qPCR) assays during the 2018 LF outbreak in Nigeria. For patients with acute LF (RDT positive, IgG/IgM negative) during initial screening, RDT performance was 83.3% sensitivity and 92.8% specificity when compared to composite results of two qPCR assays. 100% of samples that gave Ct values below 22 on both qPCR assays were positive on the Pan-Lassa RDT. There were significantly elevated case fatality rates and elevated liver transaminase levels in subjects whose samples were RDT positive compared to RDT negative.

Public health response to two imported, epidemiologically related cases of Lassa fever in the Netherlands (ex Sierra Leone), November 2019.

On 20 November 2019, Lassa fever was diagnosed in a physician repatriated from Sierra Leone to the Netherlands. A second physician with suspected Lassa fever, repatriated a few days later from the same healthcare facility, was confirmed infected with Lassa virus on 21 November. Comprehensive contact monitoring involving high- and low-risk contacts proved to be feasible and follow-up of the contacts did not reveal any case of secondary transmission in the Netherlands.

Adaptive genetic diversification of Lassa virus associated with the epidemic split of north-central Nigerian and non-Nigerian lineages.

Lassa fever (LF) is a viral hemorrhagic fever that causes high morbidity and severe mortality annually. The disease is endemic to two geographically separate areas within tropical West Africa, one in Nigeria and the second predominantly in Sierra Leone-Guinea-Liberia-Mali. Lassa virus (LASV), the causative agent of the disease, exhibits clear delineation of phylogeography between the endemic areas. In order to characterize the genetic nature of Nigerian-non-Nigerian epidemic split, we performed molecular epidemiological analyses on non-Nigerian isolates (lineage IV as well as lineage V) and their sister group from north-central Nigeria (lineage III). The results showed that adaptive genetic diversification has occurred between these currently circulating clusters in the spread process, and a number of replacement divergences have been fixed between these clusters on the viral RNA-dependent RNA polymerase (L protein). This study highlights the viral L protein could be a determinant factor for the epidemic split.

Lassa virus RNA detection from suspected cases in Nigeria, 2011-2017.

INTRODUCTION: The diagnosis of Lassa fever is crucial to confirm cases, as well as to control/prevent nosocomial and community-based transmission and initiation of treatment, which is still limited in the country. Thus, we aimed at providing some information on the laboratory detection of Lassa from suspected cases in Nigeria. METHODS: This was a retrospective study of seasonal Lassa fever outbreaks data from 1,263 samples analyzed using Reverse Transcription-Polymerase Chain Reaction (RT-PCR) at the Virology Research Laboratory, College of Medicine, University of Lagos/Lagos University Teaching Hospital between year 2011 and 2017. Data were analyzed using the 21st edition of SPSS statistical software (2015). RESULTS: The RT-PCR test confirmed the presence of Lassa in 112 (8.9%) comprising 61 (54.4%) males, 48 (42.9%) females and 3 (2.7%) individuals without gender information. Those aged between 18 and 49 years were mostly affected. There was a decline in the detection of Lassa from 4.7% in 2011/2012 to less than 1% by the 2014/2015. However, during the 2015/2016 and 2016/2017 seasons the detection rates increased to 10.4% and 15.1% respectively. The Northern region of Nigeria reported high confirmed cases of Lassa. The South Western region also witnessed an increased Lassa fever positivity rate of 13.4% of which Lagos and Ogun states being the focal state of Lassa activity in the region. CONCLUSION: These established the need for heightening the continued surveillance for Lassa as well as the establishment of other testing facilities within these endemic regions for prompt diagnosis of Lassa fever.

Lethal Infection of Lassa Virus Isolated from a Human Clinical Sample in Outbred Guinea Pigs without Adaptation.

Lassa virus (LASV), a member of the family Arenaviridae, is the causative agent of Lassa fever. Lassa virus is endemic in West African countries, such as Nigeria, Guinea, Liberia, and Sierra Leone, and causes outbreaks annually. Lassa fever onset begins with "flu-like" symptoms and may develop into lethal hemorrhagic disease in severe cases. Although Lassa virus is one of the most alarming pathogens from a public health perspective, there are few licensed vaccines or therapeutics against Lassa fever. The fact that animal models are limited and the fact that mostly laboratory-derived viruses are used for studies limit the successful development of countermeasures. In this study, we demonstrated that the LASV isolate LF2384-NS-DIA-1 (LF2384), which was directly isolated from a serum sample from a fatal human Lassa fever case in the 2012 Sierra Leone outbreak, causes uniformly lethal infection in outbred Hartley guinea pigs without virus-host adaptation. This is the first report of a clinically isolated strain of LASV causing lethal infection in outbred guinea pigs. This novel guinea pig model of Lassa fever may contribute to Lassa fever research and the development of vaccines and therapeutics.IMPORTANCE Lassa virus, the causative agent of Lassa fever, is a zoonotic pathogen causing annual outbreaks in West African countries. Human patients can develop lethal hemorrhagic fever in severe cases. Although Lassa virus is one of the most alarming pathogens from a public health perspective, there are few available countermeasures, such as antiviral drugs or vaccines. Moreover, the fact that animal models are not readily accessible and the fact that mostly laboratory viruses, which have been passaged many times after isolation, are used for studies further limits the successful development of countermeasures. In this study, we demonstrate that a human isolate of Lassa virus causes lethal infection uniformly in Hartley guinea pigs. This novel animal model of Lassa fever may contribute to Lassa fever research and the development of vaccines and therapeutics.

Fatal Case of Lassa Fever, Bangolo District, Côte d'Ivoire, 2015.

Lassa fever has not been reported in Côte d'Ivoire. We performed a retrospective analysis of human serum samples collected in Côte d'Ivoire in the dry seasons (January-April) during 2015-2018. We identified a fatal human case of Lassa fever in the Bangolo District of western Côte d'Ivoire during 2015.

The Role of Reference Materials in the Research and Development of Diagnostic Tools and Treatments for Haemorrhagic Fever Viruses.

Following the Ebola outbreak in Western Africa in 2013-16, a global effort has taken place for preparedness for future outbreaks. As part of this response, the development of vaccines, treatments and diagnostic tools has been accelerated, especially towards pathogens listed as likely to cause an epidemic and for which there are no current treatments. Several of the priority pathogens identified by the World Health Organisation are haemorrhagic fever viruses. This review provides information on the role of reference materials as an enabling tool for the development and evaluation of assays, and ultimately vaccines and treatments. The types of standards available are described, along with how they can be applied for assay harmonisation through calibration as a relative potency to a common arbitrary unitage system (WHO International Unit). This assures that assay metrology is accurate and robust. We describe reference materials that have been or are being developed for haemorrhagic fever viruses and consider the issues surrounding their production, particularly that of biosafety where the viruses require specialised containment facilities. Finally, we advocate the use of reference materials at early stages, including research and development, as this helps produce reliable assays and can smooth the path to regulatory approval.

Lassa fever in pregnancy with a positive maternal and fetal outcome: A case report.

BACKGROUND: The signs and symptoms of Lassa fever are initially indistinguishable from other febrile illnesses common in the tropics and complications of pregnancy. Surviving Lassa fever during pregnancy is rare. Only few cases have been documented. The antiviral drug of choice is ribavirin. CASE DESCRIPTION: A 25-year-old multigravida farmer with fever who was initially thought to have malaria in pregnancy at 29 weeks gestation. Further changes in her clinical state and laboratory tests led to a confirmation of Lassa fever. The Liver enzymes were markedly deranged and the packed cell volume was 27%. She commenced on ribavirin and subsequently was delivered of a live male neonate who was RT PCR negative for Lassa fever virus. Her clinical state improved, repeat RT PCR on day 15 was negative and she made full recovery. DISCUSSION: The case reported had similar clinical features of fever and abdominal pain and resulted in the initial diagnoses of Malaria in pregnancy. When she failed to respond to antimalarial and antibiotics treatments, a strong suspicion of viral hemorrhagic fever was made. At this time the patient was in advanced stage of the disease with bleeding from vagina and puncture sites. On the third day of admission she was delivered of a live male neonate who remained negative after 2 consecutive RT PCR tests for Lassa fever virus. Lassa fever carries a high risk of death to the fetus throughout pregnancy and to the mother in the third trimester. Mothers with Lassa fever improved rapidly after evacuation of the uterus by spontaneous abortion, or normal delivery. She was clinically stable following delivery. Her laboratory investigations were essentially normal. Throughout her management transmission based precautions were observed. None of the six close contacts developed symptoms after been followed up for 21 days. CONCLUSION: This report adds to the body of literature that individuals can survive Lassa fever during pregnancy with good maternal and fetal outcome.

Development and Evaluation of a One-Step Quantitative RT-PCR Assay for Detection of Lassa Virus.

Lassa fever is a severe viral hemorrhagic illness caused by Lassa virus. Based on estimates, the number of LASV infections ranges from 300,000 to 500,000 cases in endemic areas with a fatality rate of 1%. Development of fast and sensitive tools for the control and prevention of Lassa virus infection as well as for clinical diagnostics of Lassa fever are crucial. Here we reported development and evaluation of a one-step quantitative RT-qPCR assay for the Lassa virus detection - LASV-Fl. This assay is suitable for the detection of lineages I-IV of Lassa virus. The limit of detection of the assay ranged from 103 copies/ml to 105 copies/ml and has 96.4% diagnostic sensitivity, whereas analytical and diagnostic specificities both were 100%. Serum, whole blood and tissue are suitable for use with the assay. The assay contains all the necessary components to perform the analysis, including an armored positive control (ARC+) and an armored internal control (IC). The study was done during the mission of specialized anti-epidemic team of the Russian Federation (SAET) in the Republic of Guinea in 2015-2018. Based on sequencing data, LASV-specific assay was developed using synthetic MS2-phage-based armored RNA particles, RNA from Lassa virus strain Josiah, and further, evaluated in field conditions using samples from patients and Mastomys natalensis rodents.

Protection From Lethal Lassa Disease Can Be Achieved Both Before and After Virus Exposure by Administration of Single-Cycle Replicating Lassa Virus Replicon Particles.

Lassa fever is a frequently severe human disease that is endemic to several countries in West Africa. To date, no licensed vaccines are available to prevent Lassa virus (LASV) infection, even though Lassa fever is thought to be an important disease contributing to mortality and both acute and chronic morbidity. We have previously described a vaccine candidate composed of single-cycle LASV replicon particles (VRPs) and a stable cell line for their production. Here, we refine the genetic composition of the VRPs and demonstrate the ability to reproducibly purify them with high yields. Studies in the guinea pig model confirm efficacy of the vaccine candidate, demonstrate that single-cycle replication is necessary for complete protection by the VRP vaccine, and show that postexposure vaccination can confer protection from lethal outcome.

Development of an RT-LAMP assay for the detection of Lassa viruses in southeast and south-central Nigeria.

Lassa virus (LASV) causes Lassa fever (LF), a viral hemorrhagic fever endemic in West Africa. LASV strains are clustered into six lineages according to their geographic location. To confirm a diagnosis of LF, a laboratory test is required. Here, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using a portable device for the detection of LASV in southeast and south-central Nigeria using three primer sets specific for strains clustered in lineage II was developed. The assay detected in vitro transcribed LASV RNAs within 23 min and was further evaluated for detection in 73 plasma collected from suspected LF patients admitted into two health settings in southern Nigeria. The clinical evaluation using the conventional RT-PCR as the reference test revealed a sensitivity of 50% in general with 100% for samples with a viral titer of 9500 genome equivalent copies (geq)/mL and higher. The detection limit was estimated to be 4214 geq/mL. The assay showed 98% specificity with no cross-reactivity to other viruses which cause similar symptoms. These results suggest that this RT-LAMP assay is a useful molecular diagnostic test for LF during the acute phase, contributing to early patient management, while using a convenient device for field deployment and in resource-poor settings.

A medical records and data capture and management system for Lassa fever in Sierra Leone: Approach, implementation, and challenges.

Situated in southeastern Sierra Leone, Kenema Government Hospital (KGH) maintains one of the world's only Lassa fever isolation wards and was a strategic Ebola virus disease (EVD) treatment facility during the 2014 EVD outbreak. Since 2006, the Viral Hemorrhagic Fever Consortium (VHFC) has carried out research activities at KGH, capturing clinical and laboratory data for suspected cases of Lassa fever. Here we describe the approach, progress, and challenges in designing and maintaining a data capture and management system (DCMS) at KGH to assist infectious disease researchers in building and sustaining DCMS in low-resource environments. Results on screening patterns and case-fatality rates are provided to illustrate the context and scope of the DCMS covered in this study. A medical records system and DCMS was designed and implemented between 2010 and 2016 linking historical and prospective Lassa fever data sources across KGH Lassa fever units and its peripheral health units. Data were captured using a case report form (CRF) system, enzyme-linked immunosorbent assay (ELISA) plate readers, polymerase chain reaction (PCR) machines, blood chemistry analyzers, and data auditing procedures. Between 2008 and 2016, blood samples for 4,229 suspected Lassa fever cases were screened at KGH, ranging from 219 samples in 2008 to a peak of 760 samples in 2011. Lassa fever case-fatality rates before and following the Ebola outbreak were 65.5% (148/226) and 89.5% (17/19), respectively, suggesting that fewer, but more seriously ill subjects with Lassa fever presented to KGH following the 2014 EVD outbreak (p = .040). DCMS challenges included weak specificity of the Lassa fever suspected case definition, limited capture of patient survival outcome data, internet costs, lapses in internet connectivity, low bandwidth, equipment and software maintenance, lack of computer teaching laboratories, and workload fluctuations due to variable screening activity. DCMS are the backbone of international research efforts and additional literature is needed on the topic for establishing benchmarks and driving goal-based approaches for its advancement in developing countries.

Increase in Lassa Fever Cases in Nigeria, January-March 2018.

We reviewed data pertaining to the massive wave of Lassa fever cases that occurred in Nigeria in 2018. No new virus strains were detected, but in 2018, the outbreak response was intensified, additional diagnostic support was available, and surveillance sensitivity increased. These factors probably contributed to the high case count.

Standardized focus assay protocol for biosafety level four viruses.

Working in accordance with biosafety level four practices is highly complex and time-consuming. Therefore, the respective laboratory protocols should be as uniform as possible, simple to perform and straightforward in readout. Here we describe the successful application of a standardized 24-well plate focus assay protocol for the titration of Zaire ebolavirus (two isolates), Marburg virus (three isolates), Lassa virus (two isolates), Crimean Congo hemorrhagic fever virus (one isolate), and tick-borne encephalitis virus (two isolates). Viral titers are determined based on a simple visual readout. The protocol exhibits high precision, with coefficients of variation for interassay variability ranging between 0.05 and 0.21 and those for intraassay variability between 0.08 and 0.23. All reagents required for the test, including primary and secondary antibodies, are commercially available, facilitating the establishment of the protocol in other laboratories.