Martin Gabriel

Emergence of Zaire Ebola Virus Disease in Guinea

In March 2014, the World Health Organization was notified of an outbreak of a communicable disease characterized by fever, severe diarrhea, vomiting, and a high fatality rate in Guinea. Virologic investigation identified Zaire ebolavirus (EBOV) as the causative agent. Full-length genome sequencing and phylogenetic analysis showed that EBOV from Guinea forms a separate clade in relationship to the known EBOV strains from the Democratic Republic of Congo and Gabon. Epidemiologic investigation linked the laboratory-confirmed cases with the presumed first fatality of the outbreak in December 2013. This study demonstrates the emergence of a new EBOV strain in Guinea.

First case of laboratory-confirmed Zika virus infection imported into Europe, November 2013.

In November 2013, an acute Zika virus (ZIKV) infection was diagnosed in a German traveller returning from Thailand. The patient reported a clinical picture resembling dengue fever. Serological investigations revealed anti-ZIKV-IgM and -IgG, as well as ZIKV-specific neutralising antibodies in the patients blood. In Europe, viraemic travellers may become a source of local transmission of ZIKV, because Aedes albopictus (Skuse) and Ae. aegypti (Linnaeus) are invasive mosquitoes and competent vectors for ZIKV. .

Epizootic emergence of Usutu virus in wild and captive birds in Germany

This study aimed to identify the causative agent of mass mortality in wild and captive birds in southwest Germany and to gather insights into the phylogenetic relationship and spatial distribution of the pathogen. Since June 2011, 223 dead birds were collected and tested for the presence of viral pathogens. Usutu virus (USUV) RNA was detected by real-time RT-PCR in 86 birds representing 6 species. The virus was isolated in cell culture from the heart of 18 Blackbirds (Turdus merula). USUV-specific antigen was demonstrated by immunohistochemistry in brain, heart, liver, and lung of infected Blackbirds. The complete polyprotein coding sequence was obtained by deep sequencing of liver and spleen samples of a dead Blackbird from Mannheim (BH65/11-02-03). Phylogenetic analysis of the German USUV strain BH65/11-02-03 revealed a close relationship with strain Vienna that caused mass mortality among birds in Austria in 2001. Wild birds from lowland river valleys in southwest Germany were mainly affected by USUV, but also birds kept in aviaries. Our data suggest that after the initial detection of USUV in German mosquitoes in 2010, the virus spread in 2011 and caused epizootics among wild and captive birds in southwest Germany. The data also indicate an increased risk of USUV infections in humans in Germany.

Nomenclature- and database-compatible names for the two Ebola virus variants that emerged in Guinea and the Democratic Republic of the Congo in 2014.

In 2014, Ebola virus (EBOV) was identified as the etiological agent of a large and still expanding outbreak of Ebola virus disease (EVD) in West Africa and a much more confined EVD outbreak in Middle Africa. Epidemiological and evolutionary analyses confirmed that all cases of both outbreaks are connected to a single introduction each of EBOV into human populations and that both outbreaks are not directly connected. Coding-complete genomic sequence analyses of isolates revealed that the two outbreaks were caused by two novel EBOV variants, and initial clinical observations suggest that neither of them should be considered strains. Here we present consensus decisions on naming for both variants (West Africa: “Makona”, Middle Africa: “Lomela”) and provide database-compatible full, shortened, and abbreviated names that are in line with recently established filovirus sub-species nomenclatures.

Unique human immune signature of Ebola virus disease in Guinea

Despite the magnitude of the Ebola virus disease (EVD) outbreak in West Africa, there is still a fundamental lack of knowledge about the pathophysiology of EVD. In particular, very little is known about human immune responses to Ebola virus. Here we evaluate the physiology of the human T cell immune response in EVD patients at the time of admission to the Ebola Treatment Center in Guinea, and longitudinally until discharge or death. Through the use of multiparametric flow cytometry established by the European Mobile Laboratory in the field, we identify an immune signature that is unique in EVD fatalities. Fatal EVD was characterized by a high percentage of CD4+ and CD8+ T cells expressing the inhibitory molecules CTLA-4 and PD-1, which correlated with elevated inflammatory markers and high virus load. Conversely, surviving individuals showed significantly lower expression of CTLA-4 and PD-1 as well as lower inflammation, despite comparable overall T cell activation. Concomitant with virus clearance, survivors mounted a robust Ebola-virus-specific T cell response. Our findings suggest that dysregulation of the T cell response is a key component of EVD pathophysiology.

Molecular Diagnostics for Lassa Fever at Irrua Specialist Teaching Hospital, Nigeria: Lessons Learnt from Two Years of Laboratory Operation

Background Lassa fever is a viral hemorrhagic fever endemic in West Africa. However, none of the hospitals in the endemic areas of Nigeria has the capacity to perform Lassa virus diagnostics. Case identification and management solely relies on non-specific clinical criteria. The Irrua Specialist Teaching Hospital (ISTH) in the central senatorial district of Edo State struggled with this challenge for many years. Methodology/Principal Findings A laboratory for molecular diagnosis of Lassa fever, complying with basic standards of diagnostic PCR facilities, was established at ISTH in 2008. During 2009 through 2010, samples of 1,650 suspected cases were processed, of which 198 (12%) tested positive by Lassa virus RT-PCR. No remarkable demographic differences were observed between PCR-positive and negative patients. The case fatality rate for Lassa fever was 31%. Nearly two thirds of confirmed cases attended the emergency departments of ISTH. The time window for therapeutic intervention was extremely short, as 50% of the fatal cases died within 2 days of hospitalization—often before ribavirin treatment could be commenced. Fatal Lassa fever cases were older (p = 0.005), had lower body temperature (p<0.0001), and had higher creatinine (p<0.0001) and blood urea levels (p<0.0001) than survivors. Lassa fever incidence in the hospital followed a seasonal pattern with a peak between November and March. Lassa virus sequences obtained from the patients originating from Edo State formed—within lineage II—a separate clade that could be further subdivided into three clusters. Conclusions/Significance Lassa fever case management was improved at a tertiary health institution in Nigeria through establishment of a laboratory for routine diagnostics of Lassa virus. Data collected in two years of operation demonstrate that Lassa fever is a serious public health problem in Edo State and reveal new insights into the disease in hospitalized patients.

Structure of the Lassa virus nucleoprotein revealed by X-ray crystallography, small-angle X-ray scattering, and electron microscopy.

Background: Nucleoprotein (NP) is an essential component of the virus replication complex. Results: The crystal and quaternary structures of Lassa virus NP were determined. Conclusion: Lassa virus NP assembles into a symmetric trimer in solution. Significance: The trimeric complex may have a biological function in the virus life cycle, and its assembly could be a target for antivirals. The nucleoprotein (NP) of Lassa virus (LASV) strain AV was expressed in a recombinant baculovirus system. The crystal structure of full-length NP was solved at a resolution of 2.45 Å. The overall fold corresponds to that of NP of LASV strain Josiah (Qi, X., Lan, S., Wang, W., Schelde, L. M., Dong, H., Wallat, G. D., Ly, H., Liang, Y., and Dong, C. (2010) Nature 468, 779–783) with a root mean square deviation of 0.67 Å for all atoms (6.3% difference in primary sequence). As the packing in the crystal offers two different trimer architectures for the biological assembly, the quaternary structure of NP in solution was determined by small-angle x-ray scattering and EM. After classification and averaging of >6000 EM raw images, trimeric centrosymmetric structures were obtained, which correspond in size and shape to one trimer in the crystal structure formed around a crystallographic 3-fold rotation axis (symmetric trimer). The symmetric trimer is also a good model for the small-angle x-ray scattering data and could be well embedded into the ab initio model. The N-terminal domain of NP contains a deep nucleotide-binding cavity that has been proposed to bind cellular cap structures for priming viral mRNA synthesis. All residues implicated in m7GpppN binding were exchanged, and the transcription/replication phenotype of the NP mutant was tested using a LASV replicon system. None of the mutants showed a specific defect in mRNA expression; most were globally defective in RNA synthesis. In conclusion, we describe the full-length crystal structure and the quaternary structure in solution of LASV NP. The nucleotide-binding pocket of NP could not be assigned a specific role in viral mRNA synthesis.

Rapid outbreak sequencing of Ebola virus in Sierra Leone identifies transmission chains linked to sporadic cases

Abstract To end the largest known outbreak of Ebola virus disease (EVD) in West Africa and to prevent new transmissions, rapid epidemiological tracing of cases and contacts was required. The ability to quickly identify unknown sources and chains of transmission is key to ending the EVD epidemic and of even greater importance in the context of recent reports of Ebola virus (EBOV) persistence in survivors. Phylogenetic analysis of complete EBOV genomes can provide important information on the source of any new infection. A local deep sequencing facility was established at the Mateneh Ebola Treatment Centre in central Sierra Leone. The facility included all wetlab and computational resources to rapidly process EBOV diagnostic samples into full genome sequences. We produced 554 EBOV genomes from EVD cases across Sierra Leone. These genomes provided a detailed description of EBOV evolution and facilitated phylogenetic tracking of new EVD cases. Importantly, we show that linked genomic and epidemiological data can not only support contact tracing but also identify unconventional transmission chains involving body fluids, including semen. Rapid EBOV genome sequencing, when linked to epidemiological information and a comprehensive database of virus sequences across the outbreak, provided a powerful tool for public health epidemic control efforts.

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 Unions 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.

Mayaro virus infection in traveler returning from Amazon Basin, northern Peru

To the Editor: We report the case of a 27-year-old male Swiss tourist who spent 3.5 weeks (July 6–30, 2011) vacationing in the vicinity of Tarapoto, a small city located in the rainforests of the Amazon Basin in northern Peru. An acute febrile illness developed in the man during the second week of his stay. Signs and symptoms of illness were chills, malaise, frontal headache, generalized myalgia, a self-limiting painful cervical and inguinal lymphadenopathy (lasting ≈1 week), slowly progressive and pronounced polyarthralgic pains of the peripheral joints, and a transient nonpruritic maculopapular rash (starting at the forearms ≈1 week after onset of fever and spreading to the trunk and later to the neck and face before fading after 3 days). The traveler sought care at the local hospital, where physicians diagnosed suspected dengue fever on the basis of the clinical signs and symptoms, and received symptomatic treatment with paracetamol. The fever and other signs subsided within 1 week, except the arthralgia, which did not improve. The polyarthritis initially was accompanied by swelling of the affected joints and showed a symmetric pattern, mainly affecting the small joints of the hands and feet as well as the wrists, ankles, and knees. After returning home to Switzerland, the patient consulted his general practitioner (August 1, 2011) because of persisting, incapacitating joint pains. The patient reported stiffness of the affected joints, mainly in the morning and after immobility. Physical examination of the affected joints did not reveal visible clinical signs of inflammation (swelling, redness, effusion). Laboratory tests were performed for complete blood count, liver and kidney function, C-reactive protein, and serologic testing for dengue virus, chikungunya virus, Borrelia burgdorferi, Chlamydia trachomatis, Epstein-Barr virus, parvovirus B19, Salmonella Typhi, and S. Paratyphi, but none revealed a cause for the symptoms. Over almost 2 more months, the joint pains did not improve; thus, the patient was referred to a rheumatologic clinic and subsequently to the Swiss Tropical and Public Health Institute, Basel, Switzerland, for evaluation of a putative travel-related cause of the polyarthralgia. Because of the patient’s travel history, the course of the illness and clinical signs and symptoms experienced during the journey, and the evolution and characteristics of the persisting joint pains, we suspected an underlying Mayaro virus (MAYV) infection. Serologic testing (indirect immunofluorescence and virus neutralization assays) for several alphaviruses were performed as described (1), and the results (Table) confirmed our presumptive diagnosis. Table Results of serologic testing for a Mayaro virus–infected Swiss traveler returning from the Amazon Basin, northern Peru, 2011* Several viral infections (e.g., dengue, rubella, parvovirus B19, hepatitis B, hepatitis C, HIV, and human T-lymphotropic virus type 1) can be accompanied by arthralgia. However, the most prominent and long-lasting polyarthritic symptoms occur in patients infected by alphaviruses (family Togaviridae). Alphaviruses are arthropod-borne viruses (arboviruses) that circulate among a wide variety of wild animals in relative mosquito vector–specific and host-specific enzootic cycles; infection in humans (dead-end hosts) is almost exclusively incidental. Clinical cases and virus isolation have been reported only from northern South America, where MAYV circulates in an enzootic sylvatic cycle (similar to that for yellow fever) involving forest-dwelling Haemagogus spp. mosquitoes as vectors and nonhuman primates as natural hosts (2). Infections in humans mostly occur sporadically, are strongly associated with occupational or recreational exposure in rainforest environments, and are assumed to represent spillover from the enzootic cycle (2). MAYV was first isolated in Trinidad in 1954; since then, sporadic cases, clusters, outbreaks, and small epidemics of Mayaro fever have been reported from Brazil, Bolivia, Columbia, French Guiana, Guyana, Peru, Venezuela, and Surinam (3). In addition to the clinical cases and virus isolates reported from northern South America, serologic survey findings suggest the presence of MAYV in Costa Rica, Guatemala, and Panama (4) (Figure A1). Clinical signs of this acute, dengue-like, febrile illness last 3–7 days and typically include chills, headache, retro-orbital and epigastric pain, myalgia, arthralgia, nausea, vomiting, diarrhea, and a maculopapular rash (sometimes followed by desquamation) (7). However, as with other alphavirus infections (i.e., chikungunya [Africa, Asia], o’nyong-nyong [Africa], Ross River [Australia, Oceania], Barmah Forest [Australia], and Sindbis [Africa, Europe, Asia, Australia]), the hallmark of MAYV infection is the highly debilitating arthralgia. Permanent damage of the affected joints has not been reported. Hemorrhagic manifestations of MAYV infections are rare but have been described (3). Concerns over the potential emergence of urban transmission of MAYV were raised after a laboratory study showed vector competence of Aedes aegypti mosquitoes (8). International travelers are rarely given a diagnosis of MAYV infection (9,10). This might be attributed to the overall low frequency of the infection; the dengue-like signs and symptoms, which may lead to a misdiagnosis; and the fact that the disease is not well known outside MAYV-endemic regions. Physicians treating patients with signs and symptoms of a dengue-like illness and a recent history of travel to MAYV-endemic areas should consider MAYV infection in the differential diagnosis, especially if arthralgia is prominent and prolonged and dominates the clinical picture.