Role of Wildlife in Emergence of Ebola Virus in Kaigbono (Likati), Democratic Republic of the Congo, 2017.

After the 2017 Ebola virus (EBOV) outbreak in Likati, a district in northern Democratic Republic of the Congo, we sampled small mammals from the location where the primary case-patient presumably acquired the infection. None tested positive for EBOV RNA or antibodies against EBOV, highlighting the ongoing challenge in detecting animal reservoirs for EBOV.

Accounting for population structure reveals ambiguity in the Zaire Ebolavirus reservoir dynamics.

Ebolaviruses pose a substantial threat to wildlife populations and to public health in Africa. Evolutionary analyses of virus genome sequences can contribute significantly to elucidate the origin of new outbreaks, which can help guide surveillance efforts. The reconstructed between-outbreak evolutionary history of Zaire ebolavirus so far has been highly consistent. By removing the confounding impact of population growth bursts during local outbreaks on the free mixing assumption that underlies coalescent-based demographic reconstructions, we find-contrary to what previous results indicated-that the circulation dynamics of Ebola virus in its animal reservoir are highly uncertain. Our findings also accentuate the need for a more fine-grained picture of the Ebola virus diversity in its reservoir to reliably infer the reservoir origin of outbreak lineages. In addition, the recent appearance of slower-evolving variants is in line with latency as a survival mechanism and with bats as the natural reservoir host.

Author Correction: Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance.

Metagenomic next-generation sequencing (mNGS), the shotgun sequencing of RNA and DNA from clinical samples, has proved useful for broad-spectrum pathogen detection and the genomic surveillance of viral outbreaks. An additional target enrichment step is generally needed for high-sensitivity pathogen identification in low-titre infections, yet available methods using PCR or capture probes can be limited by high cost, narrow scope of detection, lengthy protocols and/or cross-contamination. Here, we developed metagenomic sequencing with spiked primer enrichment (MSSPE), a method for enriching targeted RNA viral sequences while simultaneously retaining metagenomic sensitivity for other pathogens. We evaluated MSSPE for 14 different viruses, yielding a median tenfold enrichment and mean 47% (±16%) increase in the breadth of genome coverage over mNGS alone. Virus detection using MSSPE arboviral or haemorrhagic fever viral panels was comparable in sensitivity to specific PCR, demonstrating 95% accuracy for the detection of Zika, Ebola, dengue, chikungunya and yellow fever viruses in plasma samples from infected patients. Notably, sequences from re-emerging and/or co-infecting viruses that have not been specifically targeted a priori, including Powassan and Usutu, were successfully enriched using MSSPE. MSSPE is simple, low cost, fast and deployable on either benchtop or portable nanopore sequencers, making this method directly applicable for diagnostic laboratory and field use.

2017 Outbreak of Ebola Virus Disease in Northern Democratic Republic of Congo.

BACKGROUND: In 2017, the Democratic Republic of the Congo (DRC) recorded its eighth Ebola virus disease (EVD) outbreak, approximately 3 years after the previous outbreak. METHODS: Suspect cases of EVD were identified on the basis of clinical and epidemiological information. Reverse transcription-polymerase chain reaction (RT-PCR) analysis or serological testing was used to confirm Ebola virus infection in suspected cases. The causative virus was later sequenced from a RT-PCR-positive individual and assessed using phylogenetic analysis. RESULTS: Three probable and 5 laboratory-confirmed cases of EVD were recorded between 27 March and 1 July 2017 in the DRC. Fifty percent of cases died from the infection. EVD cases were detected in 4 separate areas, resulting in > 270 contacts monitored. The complete genome of the causative agent, a variant from the Zaireebolavirus species, denoted Ebola virus Muyembe, was obtained using next-generation sequencing. This variant is genetically closest, with 98.73% homology, to the Ebola virus Mayinga variant isolated from the first DRC outbreaks in 1976-1977. CONCLUSION: A single spillover event into the human population is responsible for this DRC outbreak. Human-to-human transmission resulted in limited dissemination of the causative agent, a novel Ebola virus variant closely related to the initial Mayinga variant isolated in 1976-1977 in the DRC.

Metagenomic Next-Generation Sequencing of the 2014 Ebola Virus Disease Outbreak in the Democratic Republic of the Congo.

We applied metagenomic next-generation sequencing (mNGS) to detect Zaire Ebola virus (EBOV) and other potential pathogens from whole-blood samples from 70 patients with suspected Ebola hemorrhagic fever during a 2014 outbreak in Boende, Democratic Republic of the Congo (DRC) and correlated these findings with clinical symptoms. Twenty of 31 patients (64.5%) tested in Kinshasa, DRC, were EBOV positive by quantitative reverse transcriptase PCR (qRT-PCR). Despite partial degradation of sample RNA during shipping and handling, mNGS followed by EBOV-specific capture probe enrichment in a U.S. genomics laboratory identified EBOV reads in 22 of 70 samples (31.4%) versus in 21 of 70 (30.0%) EBOV-positive samples by repeat qRT-PCR (overall concordance = 87.1%). Reads from Plasmodium falciparum (malaria) were detected in 21 patients, of which at least 9 (42.9%) were coinfected with EBOV. Other positive viral detections included hepatitis B virus (n = 2), human pegivirus 1 (n = 2), Epstein-Barr virus (n = 9), and Orungo virus (n = 1), a virus in the Reoviridae family. The patient with Orungo virus infection presented with an acute febrile illness and died rapidly from massive hemorrhage and dehydration. Although the patient's blood sample was negative by EBOV qRT-PCR testing, identification of viral reads by mNGS confirmed the presence of EBOV coinfection. In total, 9 new EBOV genomes (3 complete genomes, and an additional 6 ≥50% complete) were assembled. Relaxed molecular clock phylogenetic analysis demonstrated a molecular evolutionary rate for the Boende strain 4 to 10× slower than that of other Ebola lineages. These results demonstrate the utility of mNGS in broad-based pathogen detection and outbreak surveillance.

Contribution of Environment Sample-Based Detection to Ebola Outbreak Management.

Detection of chains of transmission is critical to interrupt Ebola virus (EBOV) outbreaks. For >25 years, quantitative reverse transcription polymerase chain reaction performed on biological fluids has been the reference standard for EBOV detection and identification. In the current study, we investigated the use of environmental sampling to detect EBOV shed from probable case patients buried without the collection of bodily fluids. During the 2012 Bundibugyo virus (BDBV) outbreak in the Democratic Republic of the Congo, environmental samples were screened for BDBV RNA by means of real-time polymerase chain reaction. Low levels of BDBV genomic RNA were detected in a hospital and in a house. Detection of BDBV RNA in the house led to the identification of the last chain of transmission still active, which resulted in the safe burial of the person with the last laboratory-confirmed case of this outbreak. Overall, environmental sampling can fill specific gaps to help confirm EBOV positivity and therefore be of value in outbreak management.

Ebola Virus Disease, Democratic Republic of the Congo, 2014.

During July-November 2014, the Democratic Republic of the Congo underwent its seventh Ebola virus disease (EVD) outbreak. The etiologic agent was Zaire Ebola virus; 66 cases were reported (overall case-fatality rate 74.2%). Through a retrospective observational study of confirmed EVD in 25 patients admitted to either of 2 Ebola treatment centers, we described clinical features and investigated correlates associated with death. Clinical features were mainly generic. At admission, 76% of patients had >1 gastrointestinal symptom and 28% >1 hemorrhagic symptom. The case-fatality rate in this group was 48% and was higher for female patients (67%). Cox regression analysis correlated death with initial low cycle threshold, indicating high viral load. Cycle threshold was a robust predictor of death, as were fever, hiccups, diarrhea, dyspnea, dehydration, disorientation, hematemesis, bloody feces during hospitalization, and anorexia in recent medical history. Differences from other outbreaks could suggest guidance for optimizing clinical management and disease control.

Transmission of Ebola viruses: what we know and what we do not know.

Available evidence demonstrates that direct patient contact and contact with infectious body fluids are the primary modes for Ebola virus transmission, but this is based on a limited number of studies. Key areas requiring further study include (i) the role of aerosol transmission (either via large droplets or small particles in the vicinity of source patients), (ii) the role of environmental contamination and fomite transmission, (iii) the degree to which minimally or mildly ill persons transmit infection, (iv) how long clinically relevant infectiousness persists, (v) the role that "superspreading events" may play in driving transmission dynamics, (vi) whether strain differences or repeated serial passage in outbreak settings can impact virus transmission, and (vii) what role sylvatic or domestic animals could play in outbreak propagation, particularly during major epidemics such as the 2013-2015 West Africa situation. In this review, we address what we know and what we do not know about Ebola virus transmission. We also hypothesize that Ebola viruses have the potential to be respiratory pathogens with primary respiratory spread.

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.

Ebola virus disease in the Democratic Republic of Congo.

BACKGROUND: The seventh reported outbreak of Ebola virus disease (EVD) in the equatorial African country of the Democratic Republic of Congo (DRC) began on July 26, 2014, as another large EVD epidemic continued to spread in West Africa. Simultaneous reports of EVD in equatorial and West Africa raised the question of whether the two outbreaks were linked. METHODS: We obtained data from patients in the DRC, using the standard World Health Organization clinical-investigation form for viral hemorrhagic fevers. Patients were classified as having suspected, probable, or confirmed EVD or a non-EVD illness. Blood samples were obtained for polymerase-chain-reaction-based diagnosis, viral isolation, sequencing, and phylogenetic analysis. RESULTS: The outbreak began in Inkanamongo village in the vicinity of Boende town in Équateur province and has been confined to that province. A total of 69 suspected, probable, or confirmed cases were reported between July 26 and October 7, 2014, including 8 cases among health care workers, with 49 deaths. As of October 7, there have been approximately six generations of cases of EVD since the outbreak began. The reported weekly case incidence peaked in the weeks of August 17 and 24 and has since fallen sharply. Genome sequencing revealed Ebola virus (EBOV, Zaire species) as the cause of this outbreak. A coding-complete genome sequence of EBOV that was isolated during this outbreak showed 99.2% identity with the most closely related variant from the 1995 outbreak in Kikwit in the DRC and 96.8% identity to EBOV variants that are currently circulating in West Africa. CONCLUSIONS: The current EVD outbreak in the DRC has clinical and epidemiologic characteristics that are similar to those of previous EVD outbreaks in equatorial Africa. The causal agent is a local EBOV variant, and this outbreak has a zoonotic origin different from that in the 2014 epidemic in West Africa. (Funded by the Centre International de Recherches Médicales de Franceville and others.).