Kikwit Outbreak Research Articles

Neurological, Cognitive, and Psychological Findings Among Survivors of Ebola Virus Disease From the 1995 Ebola Outbreak in Kikwit, Democratic Republic of Congo: A Cross-sectional Study

Clinical sequelae of Ebola virus disease (EVD) have not been described more than 3 years postoutbreak. We examined survivors and close contacts from the 1995 Ebola outbreak in Kikwit, Democratic Republic of Congo (DRC), and determined prevalence of abnormal neurological, cognitive, and psychological findings and their association with EVD survivorship. From August to September 2017, we conducted a cross-sectional study in Kikwit, DRC. Over 2 decades after the EVD outbreak, we recruited EVD survivors and close contacts from the outbreak to undergo physical examination and culturally adapted versions of the Folstein mini-mental status exam (MMSE) and Goldberg anxiety and depression scale (GADS). We estimated the strength of relationships between EVD survivorship and health outcomes using linear regression models by comparing survivors versus close contacts, adjusting for age, sex, educational level, marital status, and healthcare worker status. We enrolled 20 EVD survivors and 187 close contacts. Among the 20 EVD survivors, 4 (20%) reported at least 1 abnormal neurological symptom, and 3 (15%) had an abnormal neurological examination. Among the 187 close contacts, 14 (11%) reported at least 1 abnormal neurologic symptom, and 9 (5%) had an abnormal neurological examination. EVD survivors had lower mean MMSE and higher mean GADS scores as compared to close contacts (MMSE: adjusted coefficient: -1.85; 95% confidence interval [CI]: -3.63, -0.07; GADS: adjusted coefficient: 3.91; 95% CI: 1.76, 6.04). EVD survivors can have lower cognitive scores and more symptoms of depression and anxiety than close contacts more than 2 decades after Ebola virus outbreaks.

Antibodies block Ebola virus entry

Ebola Virus The recent Ebola virus outbreak in West Africa illustrates the need for both an effective vaccine and therapies to treat infected individuals. Corti et al. isolated two monoclonal antibodies from a survivor of the 1995 Kikwit outbreak and demonstrated their therapeutic efficacy in Ebola virus–infected macaques. In fact, one antibody protected macaques when it was given up to 5 days after infection. Misasi et al. solved the crystal structures of fragments of the two antibodies bound to the Ebola virus glycoprotein (GP), which mediates viral cell entry. The two antibodies targeted different regions of GP, but in both cases blocked steps required for viral entry. Science , this issue pp. [1339][1] & [1343][2] [1]: /lookup/doi/10.1126/science.aad5224 [2]: /lookup/doi/10.1126/science.aad6117

Assessing the Effectiveness of Ebola Control Measures in Kikwit, DRC 1995

We develop mathematical models to replicate the Ebola infection dynamics in Kikwit Districtof Congo DRC 1995. The models are used to assess various control measures undertaken during the outbreak and determine the type of control measures that were most effective in controlling the outbreak. Two models were designed for two distinct periods of the Ebola outbreak, that is the period before Ebola was declared an epidemic in Kikwit (January 1995 toApril 1995) and the period after Ebola was declared an outbreak in Kikwit (May 1995 to August1995. We also carry out cost benefit analysis for each intervention method. The aim of this study is to assess and suggest the best possible measures for effectively combating Ebola infection.

Characterizing alpha helical properties of Ebola viral proteins as potential targets for inhibition of alpha-helix mediated protein-protein interactions.

Ebola, considered till recently as a rare and endemic disease, has dramatically transformed into a potentially global humanitarian crisis. The genome of Ebola, a member of the Filoviridae family, encodes seven proteins. Based on the recently implemented software (PAGAL) for analyzing the hydrophobicity and amphipathicity properties of alpha helices (AH) in proteins, we characterize the helices in the Ebola proteome. We demonstrate that AHs with characteristically unique features are involved in critical interactions with the host proteins. For example, the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain has an AH with a large hydrophobic moment. The neutralizing antibody (KZ52) derived from a human survivor of the 1995 Kikwit outbreak recognizes a protein epitope on this AH, emphasizing the critical nature of this secondary structure in the virulence of the Ebola virus. Our method ensures a comprehensive list of such `hotspots'. These helices probably are or can be the target of molecules designed to inhibit AH mediated protein-protein interactions. Further, by comparing the AHs in proteins of the related Marburg viruses, we are able to elicit subtle changes in the proteins that might render them ineffective to previously successful drugs. Such differences are difficult to identify by a simple sequence or structural alignment. Thus, analyzing AHs in the small Ebola proteome can aid rational design aimed at countering the `largest Ebola epidemic, affecting multiple countries in West Africa' ( http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/index.html).

Ebola Virus Disease in the Democratic Republic of Congo

BackgroundThe 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.MethodsWe 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.ResultsThe 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.ConclusionsThe 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.)

Characterizing alpha helical properties of Ebola viral proteins as potential targets for inhibition of alpha-helix mediated protein-protein interactions

Ebola, considered till recently as a rare and endemic disease, has dramatically transformed into a potentially global humanitarian crisis. The genome of Ebola, a member of the Filoviridae family, encodes seven proteins. Based on the recently implemented software (PAGAL) for analyzing the hydrophobicity and amphipathicity properties of alpha helices (AH) in proteins, we characterize the helices in the Ebola proteome. We demonstrate that AHs with characteristically unique features are involved in critical interactions with the host proteins. For example, the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain has an AH with a large hydrophobic moment. The neutralizing antibody (KZ52) derived from a human survivor of the 1995 Kikwit outbreak recognizes a protein epitope on this AH, emphasizing the critical nature of this secondary structure in the virulence of the Ebola virus. Our method ensures a comprehensive list of such `hotspots'. These helices probably are or can be the target of molecules designed to inhibit AH mediated protein-protein interactions. Further, by comparing the AHs in proteins of the related Marburg viruses, we are able to elicit subtle changes in the proteins that might render them ineffective to previously successful drugs. Such differences are difficult to identify by a simple sequence or structural alignment. Thus, analyzing AHs in the small Ebola proteome can aid rational design aimed at countering the `largest Ebola epidemic, affecting multiple countries in West Africa' (http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/index.html).

Characterizing alpha helical properties of Ebola viral proteins as potential targets for inhibition of alpha-helix mediated protein-protein interactions

Ebola, considered till recently as a rare and endemic disease, has dramatically transformed into a potentially global humanitarian crisis. The genome of Ebola, a member of the Filoviridae family, encodes seven proteins. Based on the recently implemented software (PAGAL) for analyzing the hydrophobicity and amphipathicity properties of alpha helices (AH) in proteins, we characterize the helices in the Ebola proteome. We demonstrate that AHs with characteristically unique features are involved in critical interactions with the host proteins. For example, the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain has an AH with a large hydrophobic moment. The neutralizing antibody (KZ52) derived from a human survivor of the 1995 Kikwit outbreak recognizes a protein epitope on this AH, emphasizing the critical nature of this secondary structure in the virulence of the Ebola virus. Our method ensures a comprehensive list of such `hotspots'. These helices probably are or can be the target of molecules designed to inhibit AH mediated protein-protein interactions. Further, by comparing the AHs in proteins of the related Marburg viruses, we are able to elicit subtle changes in the proteins that might render them ineffective to previously successful drugs. Such differences are difficult to identify by a simple sequence or structural alignment. Thus, analyzing AHs in the small Ebola proteome can aid rational design aimed at countering the `largest Ebola epidemic, affecting multiple countries in West Africa' ( http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/index.html).

Characterizing alpha helical properties of Ebola viral proteins as potential targets for inhibition of alpha-helix mediated protein-protein interactions

Ebola, considered till recently as a rare and endemic disease, has dramatically transformed into a potentially global humanitarian crisis. The genome of Ebola, a member of the Filoviridae family, encodes seven proteins. Based on the recently implemented software (PAGAL) for analyzing the hydrophobicity and amphipathicity properties of alpha helices (AH) in proteins, we characterize the helices in the Ebola proteome. We demonstrate that AHs with characteristically unique features are involved in critical interactions with the host proteins. For example, the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain has an AH with a large hydrophobic moment. The ability of this AH to bind to other host proteins is disrupted by a neutralizing antibody derived from a human survivor of the 1995 Kikwit outbreak, emphasizing the critical nature of this secondary structure in the virulence of the Ebola virus. Our method ensures a comprehensive list of such `hotspots'. These helices probably are or can be the target of molecules designed to inhibit AH mediated protein-protein interactions. Further, by comparing the AHs in proteins of the related Marburg viruses, we are able to elicit subtle changes in the proteins that might render them ineffective to previously successful drugs. Such differences are difficult to identify by a simple sequence or structural alignment. Thus, analyzing AHs in the small Ebola proteome can aid rational design aimed at countering the `largest Ebola epidemic, affecting multiple countries in West Africa' (http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/index.html).

Courage is not the absence of fear: responding to the Ebola outbreak in Liberia.

Courage is not the absence of fear: responding to the Ebola outbreak in Liberia.

Diagnostic schemes for reducing epidemic size of African viral hemorrhagic fever outbreaks.

Viral hemorrhagic fever (VHF) outbreaks, with high mortality rates, have often been amplified in African health institutions due to person-to-person transmission via infected body fluids. By collating and analyzing epidemiological data from documented outbreaks, we observed that diagnostic delay contributes to epidemic size for Ebola and Marburg hemorrhagic fever outbreaks. We used a susceptible-exposed-infectious-removed (SEIR) model and data from the 1995 outbreak in Kikwit, Democratic Republic of Congo, to simulate Ebola hemorrhagic fever epidemics. Our model allows us to describe the dynamics for hospital staff separately from that for the general population, and to implement health worker-specific interventions. The model illustrates that implementing World Health Organization/US Centers for Disease Control and Prevention guidelines of isolating patients who do not respond to antimalarial and antibacterial chemotherapy reduces total outbreak size, from a median of 236, by 90% or more. Routinely employing diagnostic testing in post-mortems of patients that died of refractory fevers reduces the median outbreak size by a further 60%. Even greater reductions in outbreak size were seen when all febrile patients were tested for endemic infections or when febrile health-care workers were tested. The effect of testing strategies was not impaired by the 1-3 day delay that would occur if testing were performed by a reference laboratory. In addition to improving the quality of care for common causes of febrile infections, increased and strategic use of laboratory diagnostics for fever could reduce the chance of hospital amplification of VHFs in resource-limited African health systems.

Ebolavirus Glycoprotein Structure and Mechanism of Entry

Ebolavirus (EBOV) is a highly virulent pathogen capable of causing a severe hemorrhagic fever with 50-90% lethality. The EBOV glycoprotein (GP) is the only virally expressed protein on the virion surface and is critical for attachment to host cells and catalysis of membrane fusion. Hence, the EBOV GP is a critical component of vaccines as well as a target of neutralizing antibodies and inhibitors of attachment and fusion. The crystal structure of the Zaire ebolavirus GP in its trimeric, prefusion conformation (3 GP(1) plus 3 GP(2)) in complex with a neutralizing antibody fragment, derived from a human survivor of the 1995 Kikwit outbreak, was recently determined. This is the first near-complete structure of any filovirus glycoprotein. The overall molecular architecture of the Zaire ebolavirus GP and its role in viral entry and membrane fusion are discussed in this article.

Techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein.

The trimeric membrane-anchored ebolavirus envelope glycoprotein (GP) is responsible for viral attachment, fusion and entry. Knowledge of its structure is important both for understanding ebolavirus entry and for the development of medical interventions. Crystal structures of viral glycoproteins, especially those in their metastable prefusion oligomeric states, can be difficult to achieve given the challenges in production, purification, crystallization and diffraction that are inherent in the heavily glycosylated flexible nature of these types of proteins. The crystal structure of ebolavirus GP in its trimeric prefusion conformation in complex with a human antibody derived from a survivor of the 1995 Kikwit outbreak has now been determined [Lee et al. (2008), Nature (London), 454, 177-182]. Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing are described. Glycoproteins were produced in human embryonic kidney 293T cells, which allowed rapid screening of constructs and expression of protein in milligram quantities. Complexes of GP with an antibody fragment (Fab) promoted crystallization and a series of deglycosylation strategies, including sugar mutants, enzymatic deglycosylation, insect-cell expression and glycan anabolic pathway inhibitors, were attempted to improve the weakly diffracting glycoprotein crystals. The signal-to-noise ratio of the search model for molecular replacement was improved by determining the structure of the uncomplexed Fab. Phase combination with Fab model phases and a selenium anomalous signal, followed by NCS-averaged density modification, resulted in a clear interpretable electron-density map. Model building was assisted by the use of B-value-sharpened electron-density maps and the proper sequence register was confirmed by building alternate sequences using N-linked glycan sites as anchors and secondary-structural predictions.

Immunoglobulin G in Ebola Outbreak Survivors, Gabon

To the Editor: Three well-documented outbreaks of Ebola hemorrhagic fever occurred from 1996 through 2001 in Gabon in central Africa (1). All were caused by the highly pathogenic species Zaire ebolavirus, which is associated with an ≈80% case-fatality rate. The first outbreak hit Mayibout, a village in northeast Gabon in January and February 1996, causing 31 cases and 21 deaths. The first victims were children who helped carry and butcher a chimpanzee carcass found in the forest. The second outbreak lasted from October 1996 through March 1997 and occurred in the Booue region, about 150 km southwest of Mayibout, Gabon. The outbreak area was located along a trunk road and railroad track, and the infection spread to several villages around Booue, then to Libreville, the capital of Gabon, where 15 cases were recorded. The third outbreak occurred October 2001 through May 2002 in the Mekambo area, about 150 km from Mayibout in the east (2). This outbreak consisted of several independent chains of human transmission arising from infected animal carcasses, mainly chimpanzees and gorillas. It caused 65 cases and 53 deaths and coincided with major outbreaks in great apes that decimated wild populations (3,4). A total of 207 human cases were recorded during these 3 outbreaks; 149 persons died. Of the fatal and nonfatal cases 31 and 24, respectively, were confirmed by real-time reverse transcription–PCR, antigen detection, and immunoglobulin (Ig) G ELISA at Centre International de Recherches Medicales de Franceville (CIRMF) in Gabon. Because of the lack of available samples from survivors, little is known about the duration of IgG antibody response. However, studies of 20 survivors convalescing after the 1995 Kikwit outbreak in the Democratic Republic of the Congo (DRC) showed that Zaire ebolavirus IgG appeared 5 to 18 days after symptom onset and persisted at least 21 months (5,6). With the exception of 2 survivors sampled 10 years after the 1976 Yambuku outbreak in DRC (7), no data are available on Zaire ebolavirus IgG persistence beyond 21 months. Low seroprevalence rates of Ebola virus or Marburg virus found in surveys of patients in outbreak areas have been attributed to seroreversion (8–10). To investigate the persistence of Zaire ebolavirus IgG, we studied laboratory-confirmed survivors of the 3 outbreaks in Gabon. The study was approved by the Gabon Ministry of Health and by the traditional chief of each village, and written informed consent was obtained from each survivor. During 3 months of investigations in the different outbreak areas beginning in June 2007, we located 11, 3, and 6 survivors of the 2001 Mekambo, 1996 Booue, and 1996 Mayibout outbreaks, respectively. During home visits, the survivors underwent a brief medical consultation, malaria smears were taken, and basic medicines were provided to the villagers. We collected blood samples in EDTA tubes; plasma was separated by centrifugation in the field and stored in dry nitrogen until transfer to the CIRMF laboratory in Gabon, where it was stored at –80°C. ELISA was performed as previously described, using reagents provided by the Special Pathogens Branch, Centers for Disease Control and Prevention (Atlanta, GA, USA) (7). The optical density (OD) cut-off value (0.13) was calculated as the mean + 3 SD of adjusted OD values for 103 negative control serum samples obtained from Caucasian persons living in Europe. All 20 survivors had positive test results for Zaire ebolavirus IgG (Table). The adjusted OD values at a dilution of 1:1,600 ranged from 0.3 to 3.4 in the 9 survivors of the 1996 outbreaks and from 0.7 to 3.5 in the 11 survivors of the 2001 outbreak. Adjusted OD values determined during the symptomatic period and/or a few days to 1 month after recovery were available for some survivors (Table). Specific IgG appeared by day 5 after symptom onset, increased during the symptomatic period (as shown by higher titers on day 10), peaked by day 30 (2 weeks after recovery), then declined slowly over several years. Zaire ebolavirus IgG remained detectable, often at high levels, >11 years after the infection. Table Adjusted OD values in patients infected with Zaire ebolavirus during 3 outbreaks in Gabon, determined by testing at days 5, 10, and/or 30 after symptom onset and again in 2007 (7 or 11 years after recovery)* These long-lasting IgG antibody responses found in 20 survivors of 3 different Zaire ebolavirus outbreaks rule out the hypothesis that low Ebola virus (and Marburg virus) seroprevalence rates found in epidemic regions of Africa are due to rapid loss of specific IgG. Whether this immunity is sufficient to protect from recurrent infection remains undetermined. These findings show that IgG ELISA is suitable for epidemiologic and epizootiologic investigations of Ebola and that Zaire ebolavirus IgG is an excellent indicator of Zaire ebolavirus circulation in humans.

Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor

Ebola virus (EBOV) entry requires the surface glycoprotein (GP) to initiate attachment and fusion of viral and host membranes. Here we report the crystal structure of EBOV GP in its trimeric, pre-fusion conformation (GP1+GP2) bound to a neutralizing antibody, KZ52, derived from a human survivor of the 1995 Kikwit outbreak. Three GP1 viral attachment subunits assemble to form a chalice, cradled by the GP2 fusion subunits, while a novel glycan cap and projected mucin-like domain restrict access to the conserved receptor-binding site sequestered in the chalice bowl. The glycocalyx surrounding GP is likely central to immune evasion and may explain why survivors have insignificant neutralizing antibody titres. KZ52 recognizes a protein epitope at the chalice base where it clamps several regions of the pre-fusion GP2 to the amino terminus of GP1. This structure provides a template for unravelling the mechanism of EBOV GP-mediated fusion and for future immunotherapeutic development.

Openness is key in fight against disease outbreaks

When pneumonic plague struck Surat, Gujarat, India in 1994, thousands of people panicked and fled city. We were sitting in Ottawa watching CNN showing pictures of people fleeing, said Dr Ron St John, Director-General of Centre for Emergency Preparedness and Response at Public Health Agency of Canada, recalling how news media were first source of information on outbreak for public health officials, including those at WHO. The slow response to that outbreak and an Ebola outbreak in Kikwit in Democratic Republic of Congo following year led to panic and unnecessary deaths. We had no capacity to respond to outbreaks on our own, or even to deal with information coming in. All we had was a fax machine. The switchboards at WHO were completely overwhelmed, said Dr David Heymann, acting Assistant Director-General for Communicable Diseases and WHO Director-General's Representative for Pandemic Influenza. New techniques were clearly needed to respond to disease threats, such as pandemic influenza, that could kill millions. In 1996, then director-general, Dr Hiroshi Nakajima, asked Heymann to set up a new emerging infectious disease programme to deal with outbreaks. In age of real-time electronic media and television, journalists became a vital source of instant information that public health authorities could use to detect outbreaks, in addition to information from governments, nongovernmental organizations and health-care workers, said Dr Thomas Grein, Medical Officer, Alert and Response Operations at WHO. The question was: how to search through maze of thousands of reports filed by journalists every day? The answer came from Canada. St John linked up with Dr Rudi Nowak from Health Canada, Canadian health ministry, and they proposed development of a computerized system that would collect raw news feeds from international news agencies such as Agence France Presse, Associated Press and Reuters, and scan these feeds automatically to find news of disease outbreaks. Development work on software began in 1997 funded by a Canadian government grant of 800 000 Canadian dollars (US$ 500 000 at time). The news filtering system St John and Nowak developed, known as Global Public Health Intelligence Network (GPHIN, pronounced G-finn), went live in 1999 and scanned news feeds in English and French. We were astounded at how much information we could get, said St John, adding that system collects thousands of reports everyday. But information had to be verified, and incorrect information discarded. Nowak came to WHO in Geneva for two years to work with Heymann, Dr Guenael Rodier, Special Adviser for Communicable Diseases to Regional Director, WHO Regional Office for Europe, and Dr Mike Ryan, Director of Department of Epidemic and Pandemic Alert and Response, in establishing a team that would be responsible for verification. Every weekday morning at 9am about 20 members of that team meet in Strategic Health Operations Centre, nicknamed the SHOC room, at WHO's headquarters, to discuss outbreak reports that have come in and which ones need to be verified, by contacting WHO country offices, which, in turn, contact their host governments. The reports are also analysed by relevant crisis centres set up at WHO's regional offices. Ryan and his team set up Global Outbreak Alert and Response Network of 126 institutions across globe. Its experts can be despatched to field, where they work together with WHO staff from regional offices and headquarters to stem any outbreaks. Before WHO and other subscribers receive reports from GPHIN, these have been screened by a team of eight public health specialists in Canada. The SHOC was opened in May 2004 with funding from United States. After death of WHO's Director-General Dr LEE Jong-wook in May 2006, it was renamed JW Lee Centre for Strategic Health Operations. …

David Heymann—WHO's public health guru

David Heymann is the Representative of the Director General for polio eradication at the WHO. Before that he was Executive Director of WHO’s communicable diseases cluster which includes WHO’s programmes on infectious and tropical diseases and he led the public-health response to severe acute respiratory syndrome (SARS) in 2003. In the mid to late 1990s Heymann was Director of WHO’s programme on emerging and other communicable diseases and before that was the chief of research activities in WHO’s global programme on AIDS. Before joining WHO Heymann worked for 13 years as a medical epidemiologist in sub-Saharan Africa on an assignment from the US Centers for Disease Control and Prevention (CDC). He worked on a range of projects including improving control and surveillance of infectious diseases with special emphasis on the childhood immunisable diseases including measles and polio African haemorrhagic fevers poxviruses and malaria. While based in Africa Heymann investigated the first outbreak of Ebola in Yambuku (former Zaire) in 1976 then in the second outbreak of Ebola in 1977 in Tandala and in 1995 directed the international response to the Ebola outbreak in Kikwit. Before 1976 one of Heymann’s first public-health jobs was in India as a medical officer in the WHO smallpox eradication programme. (excerpt)

Ebola virus can be effectively neutralized by antibody produced in natural human infection.

The activity of antibodies against filoviruses is poorly understood but has important consequences for vaccine design and passive prophylaxis. To investigate this activity, a panel of recombinant human monoclonal antibodies to Ebola virus antigens was isolated from phage display libraries constructed from RNA from donors who recovered from infection in the 1995 Ebola virus outbreak in Kikwit, Democratic Republic of Congo. Antibodies reactive with nucleoprotein (NP), envelope glycoprotein (GP), and secreted envelope glycoprotein (sGP) were characterized by immunofluorescence and radioimmunoprecipitation assays. Four antibodies reacting strongly with sGP and weakly with GP and two antibodies reacting with NP were not neutralizing. An antibody specific for GP neutralized Ebola virus to 50% at 0.4 microgram/ml as the recombinant Fab fragment and to 50% at 0.3 microgram/ml (90% at 2.6 microgram/ml) as the corresponding whole immunoglobulin G1 molecule. The studies indicate that neutralizing antibodies are produced in infection by Ebola virus although probably at a relatively low frequency. The neutralizing antibody may be useful in vaccine design and as a prophylactic agent against Ebola virus infection.

Persistence and genetic stability of Ebola virus during the outbreak in Kikwit, Democratic Republic of the Congo, 1995.

Ebola virus persistence was examined in body fluids from 12 convalescent patients by virus isolation and reverse transcription-polymerase chain reaction (RT-PCR) during the 1995 Ebola hemorrhagic fever outbreak in Kikwit, Democratic Republic of the Congo. Virus RNA could be detected for up to 33 days in vaginal, rectal, and conjunctival swabs of 1 patient and up to 101 days in the seminal fluid of 4 patients. Infectious virus was detected in 1 seminal fluid sample obtained 82 days after disease onset. Sequence analysis of an RT-PCR fragment of the most variable region of the glycoprotein gene amplified from 9 patients revealed no nucleotide changes. The patient samples were selected so that they would include some from a suspected line of transmission with at least three human-to-human passages, some from 5 survivors and 4 deceased patients, and 2 from patients who provided multiple samples through convalescence. There was no evidence of different virus variants cocirculating during the outbreak or of genetic variation accumulating during human-to-human passage or during prolonged persistence in individual patients.

Ebola outbreak in Kikwit, Democratic Republic of the Congo: discovery and control measures.

The Ebola epidemic in Kikwit, Democratic Republic of the Congo, was recognized because of a nosocomial outbreak in Kikwit General Hospital. Initially, a diagnosis of shigella infection was suspected because many patients presented with bloody diarrhea. On 4 May 1995, blood samples from 14 acutely ill patients were sent to the Centers for Disease Control and Prevention (Atlanta), and on 9 May, a diagnosis of Ebola hemorrhagic fever was confirmed. The major disease control measures that were undertaken were the isolation of patients in a quarantine ward at Kikwit General Hospital, the distribution of protective equipment to health care workers and family members caring for Ebola patients, the use of barrier nursing techniques, the distribution of health education material, active and passive case finding, and the burying of the deceased in plastic bags by a trained team of Red Cross volunteers who wore gloves and protective clothing.

Recombinant human monoclonal antibodies to Ebola virus.

Human Fab (IgG1kappa) phage display libraries were constructed from bone marrow RNA from 2 donors who recovered from infection with Ebola (EBO) virus during the 1995 outbreak in Kikwit, Democratic Republic of the Congo. The libraries were initially panned against a radiation-inactivated EBO virus-infected Vero cell lysate, but only weak binders were identified. In contrast, panning against secreted EBO glycoprotein (SGP) resulted in Fabs showing very strong reactivity with SGP in ELISA. These Fabs also reacted with a virion membrane preparation. The Fabs were strongly positive in IFAs with cells infected with EBO (subtype Zaire) virus but negative with uninfected cells, with a characteristic punctate staining pattern in the cytoplasm. The Fabs showed weak or no reactivity with the virus cell lysate although donor serum did react. The Fabs are now being characterized in structural and functional terms. Major interest will focus on the ability of antibodies to neutralize EBO virus and, later, to protect animals against infection.