Between August 2001 and June 2003, researchers noted that wildlife outbreaks occurred prior to 5 human outbreaks in the same relative locations. During this same period, 98 animal carcasses were discovered in the region straddling northeast Gabon and the northwest Republic of Congo. Of these carcasses, 21 gorilla, chimp, and duiker carcasses were tested for the Ebola virus, with 14 samples being found positive. In 11 cases, instances of human infection were directly linked to gorilla, chimpanzee, and duiker carcasses^ref

Genomics : it differs from Zaire species in that the overlap between the glycoprotein and VP30 genes is absent and an intergenic region separates them

Genomics :

In the past decade, the highly virulent Zaire strain of Ebola virus (ZEBOV) has repeatedly emerged into rural human populations in Gabon and Republic of Congo^{ref1, ref2}. Compelling genetic evidence^ref suggests that ZEBOV entered human populations when people handled infected carcasses of western gorillas (Gorilla gorilla) and common chimpanzees (Pan troglodytes) during massive ape die-offs^ref (Huijbregts B, De Wachter P, Obiang LSN, Akou ME (2003) Ebola and the decline of gorilla Gorilla gorilla and chimpanzee Pan troglodytes populations in Minkebe Forest, north-eastern Gabon. Oryx 37: 437–443). The risk of new ZEBOV outbreaks in the two countries poses a continuing threat to humans as well as to the largest remaining gorilla and chimpanzee populations in the world.

It seems highly unlikely that ZEBOV has caused similarly damaging ape die-offs in Gabon and Congo during the past century. Ape reproductive rates are so low that recovery from population reductions as dramatic as recently caused by ZEBOV would take 75 years or more^ref. Thus, if large ape die-offs had occurred in the past half-century, one would expect to find large zones of low ape density. Extensive surveys conducted during the 1980s and early 1990s (Tutin CEG, Fernandez M (1984) Nationwide census of gorilla (Gorilla gorilla) and chimpanzee (Pan troglodytes) populations in Gabon. Am J Primatol 6: 313–336; Carroll RW (1988) Relative density, range extension, and conservation potential of the lowland gorilla (Gorilla gorilla) in the Dzanga-Sangha region of southwestern Central African Republic. Mammalia 52: 309–323; Fay M, Agnagna M (1992) Census of gorillas in northern Republic of Congo. Am J Primatol 27: 275–284; Stromayer AK, Ekobo A (1992) The distribution and number of forest dwelling elephants in extreme southeastern Cameroon. Pachyderm 15: 9–14; Williamson E, Usongo L (1996) Gorilla survey in the Dja Reserve, Cameroun. Gor Conserv News 10: 11–14; Bermejo M (1999) Status and conservation of primates in Odzala National Park, Democratic Republic of Congo. Oryx 33: 323–331) showed no such evidence. This observation is consistent with the fact that no human outbreaks of ZEBOV were recognized in Gabon or Congo before 1994. The big question is, thus: Why has ZEBOV now emerged so explosively? There are two contrasting answers to this question. First, many authors have either assumed^{ref1, ref2, ref3, ref4}, or concluded^{ref1, ref2, ref3, ref4, ref5, ref6} that ZEBOV has long been present in the region and that its emergence is due to an increase in the rate at which human or non-human apes come into contact with some yet-to-be-identified reservoir host. Both habitat disturbance^{ref1, ref2} and climatic factors^{ref1, ref2} (Tucker CJ, Wilson JM, Mahoney R, Anyamba A, Linthicum K, et al. (2002) Climatic and ecological context of the 1994–1996 Ebola outbreaks. Photogramm Eng Rem S 68: 147–152) have been proposed as triggers for ZEBOV emergence. The alternative, which so far has received little attention, is that the virus we know as ZEBOV has actually spread only recently to each outbreak site. While the history of ZEBOV has so far remained elusive, examples from many other viruses show that the spatio-temporal dynamics of a virus are reflected in its phylogenetic structure^{ref1, ref2, ref3}. Viruses that have long been maintained within a single host population, for example, tend to have a high diversity of genetic lineages, especially if they are subject to little or no selection at the host population level. Given sufficient levels of dispersal, related genotypes may be widely distributed and will show little spatial clustering. Hepatitis C virus, for instance, is considered to have a long association with humans, but has a number of strains with worldwide distribution, probably due to inadvertent infections resulting from medical interventions at a global scale^ref. In contrast, restricted genetic diversity and rapid turnover of genotypes are hallmarks of viruses that are either spreading or are subject to continuous positive selection. Fox rabies and influenza A are typical examples, respectively^{ref1, ref2}. Although, in either case, phylogenies exhibit a characteristic ladder-like pattern, the underlying mechanism (genetic drift in the former, selection in the latter) is fundamentally different and should leave distinguishable signatures in the spatio-temporal distribution and genetic substitution patterns of the virus. Given these general principles, our aim for the present study was to use a combination of genetic, spatial, and temporal data to discriminate between the two hypotheses for ZEBOV emergence (i.e. long term, local persistence versus recent spread). Genetic information for testing these hypotheses is available from gene sequences sampled from human outbreaks. If ZEBOV has been persistent at localities across the region for hundreds or thousands of years, then the virus should have diverged into a number of distinct genetic lineages whose most recent common ancestor (MRCA) long pre-dates the first recognized ZEBOV outbreak in 1976 at Yambuku, Democratic Republic of Congo (DRC). Furthermore, outbreaks subsequent to Yambuku could equally have been caused by more ancestral or more recently derived lineages. On the other hand, if ZEBOV has spread through the region only recently, all viruses sampled should be descendants of the same genetic lineage with an MRCA close to the 1976 sequence from Yambuku. Along a given spatial trajectory, genotypes involved in more recent outbreaks should be more or less direct descendants of viruses found during previous outbreaks (creating the characteristic ladder-like pattern). Successive outbreaks should also be progressively more divergent from the MRCA. The only reasonable scenario under which such a pattern would be expected from a long-resident virus is one of continuous selection for new virus variants. However, identical selection pressures would have to apply over much of the geographic range of the virus, and movement throughout the range would have to be high for the same selected variants to occur in different localities (e.g., influenza^ref). A second source of information lies in the spatio-temporal pattern of ZEBOV emergence. Under either hypothesis, local transmission events during a given outbreak might result in new cases appearing further and further away from the outbreak origin. However, if outbreaks are truly independent emergences from a persistent, widely distributed ZEBOV population, no spatial trend should be apparent in the locations of different outbreaks over the entire period since 1976. In contrast, if ZEBOV has spread from a mid-1970s origin near Yambuku, then new outbreaks should move further and further away from Yambuku as time passes. If ZEBOV is transmitted through some sort of local contact process, then the rate of spread should be consistent across spatial scales. If the spreading wave has made changes in direction, then outbreak date should be correlated with geographic distance along the invasion corridor rather than simply with straight-line distance from Yambuku. A third class of information lies in the spatial structure of virus genotypes. Outbreaks at the front of a spreading wave should show a correlation between genetic and spatial distance that is detectable at different spatial scales. Changes in the direction of spread might weaken such isolation by distance at large scales, but correlation strength would remain high if spatial distance was measured from the origin of the wave and along the putative path of spread. The development of strong spatial structuring would also be possible in a long-resident virus, but not necessarily, as high levels of gene flow would tend to spatially randomize genotypes (Epperson BK (2003) Geographical genetics. Princeton (New Jersey): Princeton University Press. 376 p). Thus, the absence of spatial structuring would argue against spread, but not necessarily against local persistence. We tested this series of predictions regarding local persistence or recent spread of ZEBOV by analyzing data on the spatio-temporal pattern of outbreaks together with glycoprotein (GP) gene sequences collected from human outbreaks. We found the data to be inconsistent with the idea that the ZEBOV outbreaks of the past 30 years are caused by a virus that has been a long-term resident at each site. Instead, all our results are concordant with the hypothesis of a recent ZEBOV wave that spread through the area in a relatively consistent and predictable manner.

Phylogenetic Structure and Selection Patterns : maximum likelihood (ML) and Bayesian phylogenetic estimation approaches produced highly similar phylogenetic trees in which only one major lineage could be distinguished. All of the major structural features showed high statistical support. Both approaches placed the earliest outbreak (Yambuku,1976) very near the tree root (which estimates the MRCA), implying that ZEBOV sequences obtained at all other localities evolved from a virus very similar to Yambuku sometime after 1976. ML tree of full-length (> 2,000 bp) ZEBOV-GP sequences :

Tree was found in Paup* and rooted in a separate analysis using ICEBOV as an out-group. The latter analysis excluded a 576-bp variable region for which alignment with ZEBOV was uncertain. Numbers next to branches indicate percent support based on 1,000 bootstrap replicates and posterior probabilities obtained in a molecular clock-based analysis in program BEAST (only values > 70% are shown). Both trees also exhibited a series of ancestor–descendant relationships between outbreak localities (i.e., Yambuku?Mayibout, Mayibout?Booué, Booué?Mendemba) that closely mirrored the time sequence of ZEBOV outbreaks, with the most recent outbreaks falling furthest from the tree root. The tendency for new outbreaks to be directly descendent from immediately preceding outbreaks implies that outbreaks have occurred only in newly infected areas: either at the front of a narrow, advancing wave or through a series of long jumps in which each outbreak was seeded by the previous one. The rate of nucleotide substitution thereby remains fairly constant through time, as a molecular clock model could not be rejected. Because the observed rapid turnover of viral genotypes could potentially be the consequence of selection for new variants, we tested for positive selection in two ways. First, we examined the ratio of non-synonymous to synonymous substitutions (dN/dS) along tree branches. A model distinguishing different dN/dS for internal and tip branches did not fit any better than a model with one ratio applied to the entire tree (p = 0.395), indicating that there was no relative increase in dN associated with branches that gave rise to future lineages. Such an increase would be expected if positive selection were a major driving force behind the rapid turnover of virus genotypes^ref. But, given the small overall number of mutations on the tree, our statistical power to detect such an effect was low, especially if only a small number of sites were subject to positive selection. In our second analysis, we tested whether individual sites showed evidence of selection. A model of nearly neutral evolution was rejected in favor of a model accounting for sites under positive selection (p = 0.009). However, only one codon site (amino acid position 370) had a posterior probability greater than 95% of being under positive selection in a subsequent Bayesian assignment. This site appeared to have undergone three changes back and forth between isoleucine and methionine. A number of sites experienced a single amino acid change, many of which fell on internal branches. While these results are consistent with positive selection playing a role in ZEBOV evolution, they are inconclusive as to whether the specific amino acid replacements we observed are truly due to Darwinian selection or merely represent neutral evolution.

Spatio-temporal structure of outbreaks : ZEBOV outbreaks showed a distinct spatio-temporal pattern, both over the entire period since 1976 and during shorter time intervals. For example, between 2001–2004 in the Gabon-Congo border area, both human outbreaks and animal carcasses that tested positive for Ebola independently showed statistically significant patterns of eastward spread. Furthermore, the eastward spread rate estimated for the 2001–2004 period (46.1 km per year) changed little if the 1996 human outbreak at Booué and a nearby Ebola-positive chimpanzee carcass were added to the analysis (47.6 km per year). This rate consistency over a large temporal interval is concordant with a single spread process, from Booué eastward through the Gabon-Congo border area. Spatial spread of ZEBOV :

The pattern of spread from west to east does not continue with the outbreaks preceding Booué. However, the ancestor–descendant relationships in the ZEBOV phylogeny suggest a coherent spread pattern in the period preceding the Booué outbreak. The position of Yambuku near the tree root suggests that the ZEBOV spread originated somewhere near Yambuku in about 1976 and continued both south to Kikwit and west to Booué. This hypothesis was supported by a ML search for the epizootic origin that maximized the correlation between geographic distance from the origin and time after the origin. This search chose a February 1973 origin just northwest of Yambuku. The phylogenetic position of the Booué sequence as a direct ancestor to all of the 2001–2003 outbreaks suggests that the western front then turned eastward toward the Gabon-Congo border. This abrupt change of direction may have been caused by natural features such as rivers, as frequently observed in spreading pathogens^{ref1, ref2, ref3}, and was suspected prior to any genetic data becoming available. The hypothesis of a pivot point at Booué was strongly supported by the observed relationship between geographic distance from the putative origin at Yambuku and time after Yambuku. If geographic distances from all other outbreaks to Yambuku were measured in a straight line, then the relationship was significant but relatively weak. However, if geographic distances to the Gabon-Congo border outbreaks were routed through Booué, correlation strength increased dramatically. The great strength of this relationship was not due to a single outlier point, as all of the major legs of the putative epizootic path showed similar spread rates (Yambuku => Kikwit = 51.7 km per year, Yambuku => Mekouka = 56.9 km per year, Booué => Mendemba = 48.5 km per year, Mendemba => Iboundji = 47.9 km per year).

Spatial structure of genotypes : at the local as well as the regional scale, spatial structure was evident in the distribution of ZEBOV genotypes. For instance, the 2001–2003 outbreaks on the Gabon-Congo border showed a clear pattern of decreasing genetic similarity with increasing geographic distances. The tight spatial structuring of genotypes at this relatively small scale fits the notion that ZEBOV transmission is a local contact process involving short movements of a few kilometers or less (Epperson BK (2003) Geographical genetics. Princeton (New Jersey): Princeton University Press. 376 p), a conclusion concordant with the observed consistency in the time rate of epizootic spread.

Correlation between geographic distance and patristic genetic distance :

Geographic structuring of genotypes was also evident at higher spatial scales. The correlation between geographic distance to Yambuku and genetic divergence from Yambuku was only weak. However, as with the spatio-temporal analysis, a striking improvement in model fit was observed when geographic distances were routed through Booué. A ML search for an epizootic pivot point that maximized the correlation between geographic distance and genetic divergences chose a pivot point just west of Booué.

Epizootic Pivot Point

Shading of each grid cell indicates the strength of correlation (R²) between geographic distance and patristic genetic distance when that grid cell (rather than Booué) is used as the epizootic pivot point. The position of the best fitting pivot point (shown with a white X) along the Ogooue River (blue line) is consistent with a river crossing near Booué, with subsequent movement east toward the Mendemba area. It is not consistent with gene flow directly between the other mid-1990s outbreak localities (Mekouka and Mayibout) and Mendemba.

Our results clearly challenge the belief that ZEBOV has been persistently present for a long time at the outbreak sites in Gabon and Congo. First, our phylogenetic results imply that all known ZEBOV emergences occurring after Yambuku in 1976 were caused by direct and closely related descendents of a Yambuku-like virus. The descent of all known ZEBOV viruses from a very recent common ancestor is clearly inconsistent with the notion that they have long been evolving independently and in situ. Second, a similar ancestor–descendent relationship connects the outbreaks of the mid-1990s to those of 2001–2004. This replacement of virus over time and space by closely related but progressively more divergent genotypes is typically observed under spatial spread or continuous positive selection. Although our analyses were consistent with some codon sites being under positive selection, statistical power was too weak to reliably identify such sites. Fit of the molecular clock suggests that many of the observed substitutions are effectively neutral, so that the number of positively selected sites may be small. More importantly, spread and adaptation are not mutually exclusive. In fact, the very recent descent of all ZEBOV variants from a Yambuku-like common ancestor necessarily implies relatively rapid spread of variants across a large range. Thus, whether or not the ladder-like structure of the ZEBOV tree bears a signature of positive selection, it is much more consistent with recent spread than with independent evolution at each outbreak locality. Third, we found a general correlation between when new ZEBOV cases were observed and their geographical distance to previous cases. Importantly, this relationship and the corresponding rate of spread of about 50 km per year remained consistent over multiple spatial scales. The low p values in our correlation analyses indicate that observing such a pattern by chance, as the hypothesis of long-term presence of ZEBOV in the area would require, would be highly unlikely. A recent ZEBOV outbreak in May 2005 at Etoumbi village, which occurred after this paper was submitted for review, further provided an opportunity to test our model. Reassuringly, the spread rate from 2001–2003 did an excellent job of predicting the Etoumbi outbreak, given its distance from the 2001 outbreak at Mendemba. Spatial Spread from 2001–2005 :

Distance of each human outbreak site from the initial outbreak at Mendemba village, Gabon, plotted as a function of time after the Mendemba outbreak. Dashed regression line uses only outbreaks from 2001–2003 (R² = 0.43, p = 0.04). Solid regression line includes May 2005 outbreak at Etoumbi village (R² = 0.73, p < 0.001). Fourth, we identified a pattern of constantly increasing genetic divergence among virus genotypes with increasing geographic distance. As pointed out initially, a roughly linear relationship between genetic divergence and geographic separation is an expected outcome under spatial spread, particularly if spread has occurred along a relatively narrow front. Although isolation by distance itself could also be found among locally resident viruses, such a scenario would be inconsistent with any of the previous results, including the dramatically improved fit of the spatial-genetic correlation when routing distances through Booué. Taken together, our results clearly point to the conclusion that ZEBOV has gradually spread across central Africa from an origin near Yambuku in the mid-1970s. Under this scenario, the distinct phylogenetic tree structure, the strong correlation between outbreak date and distance from Yambuku, and the correlation between genetic and geographic distances can be interpreted as the outcome of a consistently moving wave of ZEBOV infection. The large-scale spatial correlations we identified were particularly strong under the assumption that the ZEBOV wave changed direction at Booué. This hypothesis may seem ad hoc but was actually posed by one of the authors (PDW) in a paper published a year before genetic data from the Gabon-Congo border region became available^ref. Transect surveys and numerous reports from local villagers had suggested that the second largest river system in equatorial Africa (the Ogooue-Ivindo-Ayina) had largely contained the 1994–1996 outbreaks in the Minkebe region of northern Gabon^ref (Huijbregts B, De Wachter P, Obiang LSN, Akou ME (2003) Ebola and the decline of gorilla Gorilla gorilla and chimpanzee Pan troglodytes populations in Minkebe Forest, north-eastern Gabon. Oryx 37: 437–443). An Ebola-positive chimpanzee was then found south of the river near Booué in 1996^ref, and subsequent surveys revealed suspiciously low ape densities southeast of Booué. Thus, all of the genetic correlation analyses we report here represent independent confirmation of an a priori hypothesis of spread from Booué, posed before genetic data were available. Likewise, the phylogenetic analysis, which identified the Booué virus sequence as the direct ancestor of all viruses observed later near the Gabon-Congo border, also indicates that Booué forms an epidemiologic link between previous and subsequent outbreaks. The effect of major rivers in channeling spread is well documented for other diseases in natural populations^{ref1, ref2, ref3}. Whether ZEBOV was resident (but undetected) in the central African forest block before the mid-1970s, or is an invader from outside the region remains unclear. Blood samples taken from both human^ref and non-human primates^ref suggest that some filovirus was already present in western equatorial Africa before the mid-1990s ape die-offs. Unfortunately, the serological tests employed were not specific to ZEBOV^ref. Therefore, it is impossible to tell whether these positive results were caused by a virus with a very recent common ancestor of the lineage we know as ZEBOV or by some more distantly related virus that is cross-reactive. The co-occurrence of both moderately high seropositivity and high ape densities at some sampling localities argues that the assayed virus was not highly virulent. ZEBOV has caused such high mortality rates in recent ape outbreaks that by the time these populations recover to high density (if they recover), individuals born after the outbreaks will greatly outnumber seropositive survivors (if any are still alive). Thus, moderate to high levels of seropositivity in ape populations are not consistent with high virulence. The absence of large human outbreaks in western equatorial Africa before the mid-1990s is consistent with a non-virulent virus, although the possibility that smaller outbreaks occurred but were not recognized cannot be excluded. The high rate of positive results in past serological surveys may explain why previous authors appear not to have seriously considered the possibility of recent ZEBOV spread. Apart from the serological results, the other major argument for long-term persistence at each locality has involved the mutational stability of ZEBOV-GP. The absence of mutations within several closely monitored human transmission chains has been used to argue that ZEBOV-GP evolves too slowly for a wildlife epizootic lasting only a few years to have generated the sequence variation observed in the recent Gabon-Congo border outbreaks^{ref1, ref2}. However, a formal statistical power analysis shows that the number of human cases involved in the cited transmission chains was far too small to reach this conclusion. In fact, our molecular clock analyses showed that ZEBOV evolves at a rate comparable to other RNA viruses, about 8 × 10^-4 substitutions per site per year and that the MRCA of the Gabon-Congo border outbreaks occurred in 1999 (CI = 1998–2000), well after the 1996 Booué outbreak. Thus, the genetic stability noted between ZEBOV outbreaks appears to be the consequence of short time separation rather than slow evolution. Although our results strongly support the hypothesis that ZEBOV spread recently to the outbreak sites in Gabon and Congo, it is still unclear through which reservoir host(s) ZEBOV spread occurred. Spread might have taken place through transmission within some wildlife reservoir endemic to the region or through the wave-like invasion of an infected reservoir. Whatever the reservoir species or group of species, the striking constancy in the rates of ZEBOV spread and evolution suggests either that its distribution and abundance are fairly uniform throughout the affected area, or that its range has been expanding at a uniform rate. At the same time, we found that the large-scale pattern of spread is well represented as one-dimensional, which contradicts expectations for a radiating wave in a uniformly distributed host. As pointed out before, we suspect that the channeling effect of rivers may be responsible for this pattern. The large time gaps between human outbreaks may simply reflect the fact that much of the proposed epizootic path is very lightly inhabited by humans. For example, the 1996 outbreak site at Booué and the 2001 outbreak site at Mendemba are separated by 250 km of forest crossed by a single road, along which lie only a handful of small villages. The conclusion that ZEBOV has recently spread further begs the question of whether this spread was triggered by some ecological change (perhaps anthropogenic in origin), by some change in the virus itself (for example, a mutation to higher virulence), or simply by some stochastic event. Answering these questions remains a challenge for future research. To what extent ZEBOV transmission between apes plays a role in either ZEBOV spatial spread or ape die-offs also remain open questions. Our results also warrant a re-evaluation of the potential for Ebola control. The consistent rate of Ebola spread suggests that control efforts may not need to encompass the entire region, but could be concentrated directly ahead of the advancing wave. Knowledge of the future path of spread could be used to strategically allocate the delivery of an Ebola vaccine^{ref1, ref2, ref3} (cf. rabies^ref) when a successful vaccine is developed. If the past spread rate of about 50 km per year continues in the current direction, Ebola Zaire should hit the populated areas north and east of Odzala National Park within the next one to two years. Most of the handful of parks still containing populations of gorillas large enough to be viable in the long term might be reached within three to six years. Saving these viable ape populations should be a top priority.

Laboratory accidents :

• in 1990 a worker at the Vektor State Scientific Centre of Virology and Biotechnology contracted Ebola virus and survived
• in a 1996 incident at another Russian biological research centre, the Defence Ministry's Virology Center in Sergiyev Posad near Moscow, a worker accidentally contracted the Ebola virus infection and died
• on Wed 10 Feb 2004 a civilian scientist at Fort Detrick remained free of Ebola symptoms after accidentally grazing her hand with a needle while injecting mice infected with a weakened form of the deadly virus. After being placed in a biosafety containment care 2-bed suite rated BSL-4 at the U.S. Army Institute of Infectious Diseases at Fort Detrick, about 45 miles northwest of Washington DC, did not develop symptoms, and survived unscathed, either because of the attenuated nature of the virus or because of the lowness of the dose. The patient was doing postdoctoral virology work at USAMRIID, where she has worked as a National Research Council fellow since June 2002. She grazed her hand on 11 Feb 2004 while studying potential treatments for Ebola. The mice she was treating were infected 2 days earlier with a low dose of the weakened virus : the woman slept at home the night of the accident (due to the incubation period, there was no risk to the community) and entered the suite on 12 Feb 2004 for a stay of up to 30 days. The suite is called the slammer because the metal door to a shower that must be used before leaving the area makes a loud noise when it's closed : the woman wears scrubs inside the chamber and is tended by nurses wearing gowns, gloves, surgical masks, and face shields. Her food goes in on trays. The containment consists of 2 hospital-like rooms each 180 square feet and a 300-square-foot treatment room. The air is exchanged up to 15 times an hour and is filtered coming in and going out, leaving it cleaner than when it entered. The suite was last used for patient care in 1985, for observation of a lab worker after a possible finger puncture while working with Junin virus : no illness resulted.
• on May 5, 2004, while working with guinea-pigs infected with Ebola virus, a female member of staff of the Vektor State Scientific Centre of Virology and Biotechnology (set up in 1974) was accidentally injected. This accident occurred in the course of a scientific experiment in the Department of Dangerous viral Pathogens of the Molecular Biology Scientific Research Institute. The woman scientist was hospitalized in the dangerous infections department of a special hospital with the greatest possible level of biological defence. There were constant consultations with Health Ministry specialists regarding treatment. A doctor who has often been involved in treating patients with Ebola hemorrhagic fever in Africa was consulted. In spite of the treatment, the scientist died on Wed 19 May. According to the Vector press office, an analysis of clinical materials is currently underway. The staff who were involved in the treatment and investigation of the patient will be kept under observation for 21 days: they will undergo a daily medical examination and twice-daily temperature measurement.

Epidemiology :

• 1967 (the first filovirus discovered) : Marburg and Frankfurt, West Germany (31 cases and 7 deaths), and Belgrade, Yugoslavia (6 cases) subsequently linked to contact with infected Cercopithecus aethiops imported from Uganda. Human infection occurred primarily in those harvesting organs for use in vaccine production, a process certainly parallel to butchering for consumption.
• 1975, in Johannesburg, South Africa (an Australian hitchhiker who may have contracted the disease while traveling through Rhodesia (now Zimbabwe) and who died subsequently. The infection was passed to his traveling companion and a nurse, both of whom recovered)
• 1980, in Kenya (2 cases, one of which fatal : a Frenchman was infected in Kitum Cave in the Mt. Elgon area, not far from the Uganda region where the monkeys had been trapped, and died. He spread the virus to his attending physician, who survived. When medical researchers put

animals in the cave, no virus was traced, and the animals remained healthy)
• 1982 (a young man who had recently come from Zimbabwe to South Africa, on the basis of transient antibody activity, but it was subsequently stated that this diagnosis should be regarded as unsubstantiated)
• 1987, in Kenya (a fatal case in a Danish boy)
• 1988 : Nikolai Ustinov, a 44-years old researcher at Vector State Scientific Centre of Virology and Biotechnology, accidentally self-injected Marburg virus and died on Apr 30 (hence the "Variant U" biowarfare Marburg virus)^ref
• 1990 : another workerat Vector State Scientific Centre of Virology and Biotechnology contracted the virus and survived^ref
• late 1998 to late 2000 in the Democratic Republic of Congo : the largest outbreak on record, which involved 154 cases (48 laboratory-confirmed and 106 suspected), of which 123 were fatal (CFR = 83%). The outbreak was initially concentrated in workers at a gold mine in Durba. Genetic evidence of the virus was found in cave-dwelling bats in the mine where primary human cases arose, but we were not able to isolate live viruses from bats, and the outbreak stopped when the mine flooded. Evidence indicated multiple introductions into the human population from a reservoir which was linked to an abandoned mine. Cases were associated with at least 9 different genetic lineages of Marburg virus, representing the entire genetic repertoire of previously characterized East African isolates of the Lake Victoria Marburg virus species^{ref1, ref2}. These multiple introductions were thought to be a factor in the unusually high number of cases reported, as past outbreaks identified as having one point source introduction resulted in a limited number of human-to-human transmission cycles. A serosurvey of health care workers showed no transmission, and one conclusion^ref was that there was minimal secondary transmission of Marburg virus during this outbreak. A serosurvey conducted in the Central African Republic^ref demonstrated 3.2% of the population (137/4295) had positive antibodies against Marburg virus. There was a report in the literature of a cluster of cases in the DRC outbreak that involved an infant : there were 6 cases, 3 of which had a mild clinical presentation, and the infant was believed to be infected during "nurturing" (breast feeding)^ref. The mean age was 28 years (range: 7 days to 72 years). Only 12 (8%) of the cases were under age 5, and the case fatality for this group was the same.  Most cases were in young males exposed in nearby gold mines. Secondary transmission in the household appeared to be limited. There was evidence of transmission of Marburg virus in the region prior to 1998, but that did not result in a high prevalence of antibody in the population^{ref1, ref2, ref3}. Although precise calculations are close to impossible, age-stratified attack rates appear to vary significantly between filovirus outbreaks. The relatively low incidence of pediatric cases of Ebola-Zaire during the 1995 outbreak in Kikwit, DRC, prompted theories of natural immunity in children. These theories were scrapped after the 2000 Ebola-Sudan outbreak in Gulu, Uganda, where fatal infection in children was common.
• October 13 2004-27 Jul 2005 : it infected 374 cases (252 laboratory confirmed; 2 previous cases proved negative) and killed 329 (227 laboratory confirmed; including 16 nurses, 2 doctors, and 5 healers) people (CFR among known cases = 89.5%) in Angola^{ref1, ref2}
• 99% (368 cases and 323 deaths) from the northern Uije / Uige province (a city of 500,000 inhabitants, about 225 km (140 miles) north of Luanda) : 21 contacts are being followed in Uige municipality and 111 in other municipalities
• 197 cases and 183 deaths occurred in Uige municipality
• other municipalities in Uige Province account for an additional 56 cases and 50 deaths.
52 contacts in the municipality of Songo, Uige Province, are followed up as Aug 24 2005. The virus was identified by CDC and WHO labs on Tue 22 Mar 2005 in samples from 9 of 12 fatal cases : while children under the age of 5 years (thus not in school and generally carried on the backs of their mothers) initially accounted for around 75% of cases, recent cases are including an increasing number of adults. There is probably a local factor which has not been highlighted yet. Several assumptions can be considered: viral variation, a local activity responsible for the spread, or infection of children admitted to the hospitals of Uige for another infection (measles, malaria, and typhoid have all been reported from the adjacent Democratic Republic of Congo). More surprising is the fact that mothers are little affected despite very close contact with their infants. that the high mortality compared to the sporadic outbreaks (25%) and the epidemic of Durba (82%) are explainable by the fact that the population initially consults traditional practitioners and "kimbandeiros," since the disease is locally ascribed to fate. They go to hospital only after failure of these treatments and often arrive moribund. The sparse resources available to the centers of provincial care are also in question as well as contaminations by re-used needles. This is the 1st epidemic caused by a filovirus which has developed in an urban environment, whereas previously they have been rural zoonoses : this outbreak -- like prior outbreaks of viral hemorrhagic fevers -- began outside the urban environment but transmission was potentiated in the hospital setting. The index case may well have been an adult male whose wife was infected after caring for him, and who then infected their child(ren). An infected child was then hospitalized in an environment where infectious disease isolation is not routinely practiced, and where supplies are limited so that needles and syringes (or other medical devices) may not be adequately sterilized between uses. Beds may have become contaminated and although washed between patients, perhaps not sufficiently to eliminate the virus. All of the above were described as sources of transmission in prior viral hemorrhagic fever outbreaks in which the hospital served as a potentiator for transmission of the virus. The outbreak then took hold in the pediatric ward, among patients that were already admitted for other illnesses not related to Marburg. Because these were hospitalized children, the direct contact with their families was a bit more limited than if they had stayed home. But I suspect that there was more transmission to the family members who then stayed at home and were ill (and died) at home, away from the attention of the formal health sector. That would explain the early apparent waves in the pediatric population, but now the shifting age distribution involving the hospital staff, and probably family members of the children. I suspect the final epidemiologic reports will reflect this. With the above theory, the index case probably had the more "traditional route" (whatever that may be) of primary infection -- exposure to bat droppings (as postulated from an isolated Ebola case related to spelunking in Kenya, and for the outbreak in miners in the Democratic Republic of Congo), or exposure to an infected primate through skinning and preparing for food (I do not know if monkeys are eaten in Angola, but I suspect they are, especially in a zone where food resources are limited). I suspect that the observed high mortality rate may be a function of "in-hospital" mortality rate, and the true rate may be closer to that seen in the Durba outbreak. The disparity from earlier reported mortalities with Marburg may be related to either a different strain, or more likely to level of medical care available (earlier outbreaks were in more highly developed areas).  It will be interesting to see if an analysis of CFR by age group indicates a differential based on age.  A true CFR would require doing a baseline serosurvey in the community to establish the denominator of infected individuals. It is possible that there are milder cases that are not within the radarscope of the health sector. Given the high emotional state in the area, I suspect that such a serosurvey will have to wait until after things quiet down and "return to normal". If this outbreaks was due to a single point source introduction and human-to-human spread, it would be noteworthy, as previous Marburg virus outbreaks have been limited when one point source introduction occurred. Whether this is a property of the virus or the state of health care and socio-economic status in the affected areas would be an interesting consideration in the study of the current outbreak. In the present outbreak we do not yet know the true age, as there may have been a significant proportion of mild cases that did not come to the attention of the health sector in the early reports of the outbreak. Another explanation is that pre-existing immunity in the older population from earlier outbreaks would leave the younger people susceptible. And of course a 3rd explanation is that there is a common exposure for the younger age group that has been placing it at higher risk (less likely, as the younger age groups tend to stay more within their family compounds and do not tend to congregate in the pre-school age group). Sloppy injection practices explain why most victims are below age 5 : doctors often give medicine to young children by injection rather than by mouth. It should be remembered that Angola underwent a prolonged civil war from 1972 to 2002, during which the basic infrastructure -- such as health care -- was limited, with limited communications as well. It is possible that Marburg or other hemorrhagic virus activity in the country did not come to the attention of the international health sector. There were previous occasional individual case reports of hemorrhagic fever-like illness in 2001 and 1995. Since the start of the outbreak, the monthly number of cases has progressively increased, but this increase could be the result of intensified surveillance. Cases in adults include 12 health care workers : most deaths have occurred between 3 to 7 days following the onset of symptoms. 2 patients fled the hospital^{ref1, ref2, ref3}. The outbreak has spread to the capital Luanda (a city of nearly 3 million), killing on Mar 25 a 15-year-old boy at the Josina Machel hospital, a Vietnamese doctor, and a 51-years old Italian paediatrician working for Medici con Africa, Maria Bonino, in the Sagrada Esperanca clinic, who had all been in the northern Uige province to which the virus had previously been confined : at least 4 other people coming from Uije have been diagnosed with the virus in the capital. Another here is Matondo Alexandre, a UN child protection expert based in Angola. Together, the pair raised the alarm over Marburg earlier in 2005, broadcasting their warnings over the radio in February, when the government wasn't yet taking the outbreak seriously. The radio broadcast attracted the attention of Angola's health ministry and the WHO. But tragically, Dr Bonino contracted the disease and died, less than a week after a laboratory in Atlanta confirmed her suspicions. Mr Alexandre has been denounced as a witch, and his aunt was beaten by Uige residents. The virus had spread to a fourth province, Kwanza Norte province east of the capital : a death occurred in the town of Camabatela, which lies 85km south of Uige and 300km east of the coastal capital. A total of 5 nurses have died. Emergency efforts were under way, and on Thu 24 Mar 2005 the European Union pledged € 500,000 (US$ 650 000) to help fight the outbreak. The pandemic has spread through 8 of the 18 provinces: Luanda, Cabinda (the first death was a pregnant woman on Sat Mar 26, having travelled there from Uige), Kuanza Sul, Kuanza Norte, Malanje, Zaire, Bengo, and Uige, all concentrated in the northwestern part of the country. On Sat 9 Apr 2005, WHO vehicles carrying 18 staff were attacked and damaged in Uige by angry people, even though they were accompanied by government officials and police officers : health workers had been killed by residents who erroneously believed the workers were exposing them to the virus. Teams from the WHO halted operations in Angola's north eastern Uige province, where the outbreak began : local people have reportedly stoned their vehicles and threatened their safety. In Uige, mobile surveillance teams resumed operations on Sat 10 Apr 2005, following intensive campaigns to improve public understanding of the disease. Campaigns have benefited from support by the provincial governor and officials from the health department, who have made personal visits to affected communities. Religious leaders are also helping to sensitize the public. WHO launched an appeal Fri Apr 1 for $2.4 million to support the emergency response to the outbreak. MSF has urged the government to close the regional hospital here, at the center of the outbreak, saying the medical center itself is a source of the deadly infection : 2 other hospitals within 60 miles of Uige may also have to be shut down. But the virtual wipe out of hospital staff during the Ebola outbreak in Kikwit, Zaire, in 1995 led to ongoing transmission of disease in homes where family care-givers had absolutely zero protection against acquisition of disease once a single member became ill. It is a massive public health decision, and it must be taken by the government. The health-care workers are encapsulated in air-filled white suits, white aprons, green gloves, face masks and face guards. On their backs, they wear battery packs to keep the suits inflated. Some carry canisters of bleach or chlorine on their backs and hose sprays so they can disinfect the homes of the sick. A few have sprayed so much bleach that the buckles on their shoes are rusting. Medical workers warn visitors not to shake hands with anyone and not to stnd directly in front of residents when talking to them, for fear that a cough could release an infectious spray of spittle. Silo Margarita is one of the few nurses still working at the 500-bed regional hospital here, a sprawling collection of well-kept, one-story concrete buildings that appeared almost deserted on Sat 9 Apr 2005 afternoon. Wearing a surgical mask and plastic wrapped on her boots, she continued to care for 12 patients, despite the fact, she said, that as many as 15 of the hospital's nurses and 2 doctors have died from Marburg virus infection. 2 nurses died only last Thu 7 Apr 2005. The could check as many as a 100 samples a day. But because the medical teams were forced on Thu 7 Apr 2005 to suspend their community searches for new cases, he said he was now getting only 4 samples a day. When somebody dies, anthropologists explained to the neighbors how the "astronauts" are coming to take the body away. Panic has spread through the region, with some people hiding their infected relatives and dead bodies from health workers, compounding the problem. Officials also told reporters that a local tradition of delaying the burial of loved ones risked increasing the spread of the deadly virus. Health workers also said local residents lacked sufficient information about the disease and that hospitals failed to observe basic rules of hygiene that could help stem the epidemic. 2 unusual aspects of the current epidemic have been noted: its emergence outside the cave or mine environment where the disease has often originated in the past^{ref1, ref2, ref3}, and the large percentage of victims who were children. I would like to suggest an environmental factor that may have been one of many conditions contributing to the early stages of the epidemic in Uige Province, before person-to-person transmission became more prevalent: the floors on which the victims lived. The epidemic in Uige emerged during the rainy season, which has been as wet as or wetter than usual in northeastern Angola. The dwellings of many of the victims, particularly outside urban centers, were likely to have floors of packed earth, and were also likely to be dimly lit, at least during the daylight hours with the cloudy conditions of