Marburg Virus, a close relative of the better-known Ebola Virus, is the founding member of the family Filoviridae, and is known to cause sporadic outbreaks of severe, often fatal disease in Humans. There have been 12 known Marburg Virus disease outbreaks, most recently in 2017 in Uganda. The largest Marburg Virus disease outbreak on record occurred in Uige, Angola, in 2005, with 227 deaths out of 252 known cases. This was the highest case-fatality ratio (90%) recorded for any large Filovirus outbreak, including the 2013–2016 Ebola Virus outbreak in West Africa (41%). A direct link to Marburg Virus spillover from Bats was not made during the event in Angola. Consistent with the high case-fatality ratio during the outbreak in Angola, the Marburg Virus Angola strain appears to be significantly more virulent than all other Marburg Virus strains (Musoke, Ravn, and Ozolin) in experimentally infected non-Human Primates. The Angola outbreak was the only Marburg Virus outbreak to originate outside of East Africa; all previous Marburg Virus disease outbreaks occurred in, or originated from, Uganda, Kenya, Democratic Republic of the Congo, or South Africa and Zimbabwe. Other Filoviruses circulating in Africa include the Marburgvirus, Ravn Virus, as well as five Ebolaviruses, Sudan Virus, Tai Forest Virus, Bundibugyo Virus, and the recently discovered Bombali Virus.
Extensive field studies in Uganda, Democratic Republic of the Congo, Kenya, South Africa, Gabon, and Zambia, as well as experimental infection studies in captive bats in the United States, have shown that the cave-dwelling Egyptian Rousette Bat, Rousettus aegyptiacus, is a primary natural reservoir of Marburg Virus. This discovery is consistent with the origins of Marburg Virus diseas outbreaks that, when known, have been linked to caves or mines, with Marburg Virus most often having spilled over to miners who work underground in known Egyptian Rousette Bat roosting sites, and occasionally to tourists who viewed Egyptian Rousette Bats too closely. Infected Egyptian Rousette Bats shed Marburg Virus in saliva and urine, and the Virus can persist for weeks in various tissues, particularly liver, spleen, and lymph nodes. Under experimental conditions, Marburg Virus can be transmitted directly between Egyptian Rousette Bats in the absence of Arthropod vectors. Furthermore, some infected Bats appear to be supershedders, capable of shedding a disproportionate amount of Virus, leading to increased Bat-Bat transmission in accordance with the Pareto principle. To date, Arthropod vectors do not appear to contribute to natural enzootic transmission of Marburg Virus among Egyptian Rousette Bats.
In equatorial Africa, Egyptian Rousette Bats live in very large, dense colonies sometimes numbering over 100 000 Bats. They can breed twice a year, producing thousands of susceptible juvenile Bats every six months in a single Egyptian Rousette Bats roost. Field studies in Uganda showed that 2–3% of all Egyptian Rousette Bats are actively infected with Marburgviruses (Marburg Virus and Ravn Virus) at any one time and that infection levels spike biannually, up to 12% on average, in juvenile Bats. Importantly, these seasonal spikes appear to be associated with increased risk of Human exposure, as they coincide with over 84% of known Marburg Virus spillover events to Humans. Despite the linkage of Egyptian Rousette Bats to Human Marburg Virus outbreaks, attempts to mitigate risk through Bat extermination were counterproductive and led to increased levels of active Marburg Virus infection in the recolonising Bat population.
Since 2007, over 80 distinct Marburg Virus genomic sequences and 21 Virus isolates have been obtained from tissues of infected wild-caught Egyptian Rousette Bats, representing every major Marburg Virus strain found in Marburg Virus disease outbreaks since 1967, with the exception of the Angola strain. In Gabon, South Africa and Zambia, Marburg Virus was detected in Egyptian Rousette Bats despite no known associated Human Marburg Virus disease outbreaks in the country.
In a paper published in the journal Nature Communications on 24 January 2020, a team of scientists led by Brian Amman of the Viral Special Pathogens Branch of the Centers for Disease Control and Prevention report the presence of Marburg Virus, including an Angola-like Marburg Virus, in Egyptian Rousette Bats in Sierra Leone, West Africa.
Importantly, no Marburg Virus disease outbreaks have been reported in Sierra Leone despite the presence of Marburg Virus. Amman et al.'s findings highlight the value of engaging with all stakeholders with appropriate messaging that identify and mitigate pathogens of public health concern before recognized spillovers occur. This is in consonant with measures that ensure Animal and environmental health. Moreover, it underpins the One Health surveillance approach that recognizes the interconnected relationship between people and other organisms (Plants and Animals) in a shared environment.
A total of 1755 Bats from 42 species were captured and sampled from 4 districts in Sierra Leone: Moyamba (Kasewe Cave), Kailahun (Tailu Village), Koinadugu (Kakoya Cave), and Kono (Koema Cave). All Bat samples were tested for 5 Filoviruses (Ebola Virus, Tai Forest Virus, Bundibugyo, Marburg Virus, and Ravn Virus). Of these, 435 Bats were identified as Egyptian Rousette Bats (186 from Kasewe Cave; 7 from Tailu Village; 131 from Kakoya Cave; and 111 from Koema Cave), from which 11 bats (2.5%) tested positive for active Marburg Virus infection by Virus-specific real-time reverse transcription polymerase chain reaction or consensus reverse transcription polymerase chain reaction. Marburg Virus-positive samples included six liver/spleen, five lymph nodes, two oral swabs, one salivary gland, and one whole blood sample. Marburg Virus isolation was attempted on all polymerase chain reaction positive tissues (13) and swabs (2), and from those, four Virus isolates were obtained from three Egyptian Rousette Bats caught at Kasewe Cave. Two Marburg Virus isolates were obtained from one Bat (no. 960), one from the liver/spleen and the other from lymph node, while one isolate was obtained from two other Bats (nos. 968 and 1000), each from liver/spleen. Owing to a non-destructive sampling protocol, tissue specimens from Egyptian Rousette Bats captured at Kakoya and Koema Caves were not available for similar analysis.
Marburg Virus sequences from small diagnostic NP and VP35 gene fragments were determined from 10 of the 11 polymerase chain reaction-positive bats using an array of sequencing approaches, depending on the institution performing the surveillance and sequence analysis. These Marburg Virus sequences were then compared by maximum-likelihood phylogenetic analysis to 128 NP and/or VP35 sequence fragments obtained previously from Egyptian Rousette Bats or Humans in Uganda, Democratic Republic of the Congo, Angola, Gabon, and Kenya. The phylogenetic analysis shows that the Sierra Leone-derived Marburg Virus sequences are most closely related to sequences obtained in Gabon and Angola. In addition, Marburg Virus full-length genome sequences were determined by genome walking of Marburg Virus RNA extracted from oral swabs and whole blood (2), one of which was phylogenetically similar to the Angola-like Marburg Virus isolates (4). Unexpectedly, Marburg Virus isolate sequences from Bats nos. 960 (2), 968, and 1000 were 100% identical across the full-length Virus genome. To rule out cross-contamination during Virus isolation, RNA was extracted directly from Egyptian Rousette Bats tissues and approximately 5 kb of Marburg Virus RNA was sequenced using an Angola strain-specific tiling and amplification approach. As with the Marburg Virus isolate sequences, all tissue-derived Marburg Virus sequences from those three Bats were 100% identical.
Among the 193 Egyptian Rousette Bats captured at Kasewe Cave and Tailu Village, 140 (72.5%) were juveniles (forearm length under 90 mm), and 53 (27.5%) were adults. All of the Marburg Virus polymerase chain reaction-positive Kasewe Cave Egyptian Rousette Bats (9/186) were classified as juveniles (4.8%). A total of 242 Egyptian Rousette Bats were sampled at Kakoya and Koema Caves. Of these, 87 (36%) were juveniles and 155 (64%) were adults. Like the Kasewe Cave and Tailu Village sites, all Marburg Virus polymerase chain reaction positive Egyptian Rousette Bats (2/242; 0.8%) were juveniles. A significant age bias was detected among Marburg Virus-positive Bats; all 11 polymerase chain reaction-positive Bats were juveniles. No sexual bias with respect to Marburg Virus active infection was detected between male (6) and female (5) polymerase chain reaction-positive Bats.
Marburg Virus-specific IgG antibody was detected in 24/140 (17.1%) Egyptian Rousette Bats captured at Kasewe Cave (136 serum tested) and Tailu Village (4 serum tested). Notably, two of these Marburg Virus IgG antibody-positive Bats were also positive by real-time quantitative reverse transcription polymerase chain reaction. No sexual bias was observed in Marburg Virus-specific IgG antibody-positive Egyptian Rousette Bats (5/49, or10.2%, female; 19/91, or 20.9%, male). Consistent with previous studies of wild-caught Egyptian Rousette Bats in Uganda, there was a significant age bias, as 32.4% of adults (12/37) were antibody-reactive to Marburg Virus compared to 11.7% of juveniles (12/103). Sera from Egyptian Rousette Bats captured at Kakoya and Koema Caves were not available for IgG analysis.
Amman et al. present evidence of active Marburg Virus circulation in West African Egyptian Rousette Bat based on polymerase chain reaction, antibody, and Virus isolation data and provide the first report of an Angola-like strain of Marburg Virus since it was first detected in humans in 2005. Importantly, this discovery occurred prior to any known Marburg Virus disease outbreak in Sierra Leone and was used to implement evidence-based public health messaging to at-risk communities about Marburg Virus spillover risk. To accomplish this, a comprehensive One Health communications approach leveraging the human, animal, and environmental and emergency health sectors within the Ministries of Health and Sanitation, and Agriculture and Forestry and Food Security along with other international partners was implemented across national, district, and local community levels. Through several engagement meetings with Ministry of Health and Sanitation and with several relevant ministries, departments and agencies, (Ministry of Agriculture Forestry and Food security, Ministry of Local Government, Ministry of Lands, Ministry of Mines and Mineral Resources, Environment and Protection Agency, Office of National Security) over a two-week period, briefing documents including Marburg factsheets, Marburg Virus disease preparedness, detection and response plans were developed and presented at a national conference. This resulted in recommendations for public health outreach, with a team comprised of key stakeholders (government health and agriculture units, universities, development partners and district and local authorities) across the capital city and three of the districts (Moyamba, Koinadugu and Kono). This outreach team conducted initial information sharing events in each community near the Egyptian Rousette Bat colonies followed by regular in-person meetings with traditional community leaders and other local stakeholders to provide key messages related to virus exposure risks and methods to reduce contact with Bats. Concerns raised by local communities where bushmeat consumption brings them in contact with Bats for livelihood were noted and discussed, and local perceptions about Bats were explored in developing options for minimizing exposure risks. As an additional national-level public preparedness measure, Marburg Virus disease has now been included in testing regimens at national laboratories in Sierra Leone.
Marburgviruses have been found in multiple Egyptian Rousette Bat populations across sub-Saharan and South Africa. Though fragmented, the geographic range for Egyptian Rousette Bats extends into West Africa, covering areas of Liberia, Sierra Leone, and Guinea that contain fruiting trees and caves. Therefore, finding 11 Marburg Virus positive Egyptian Rousette Bats from three separate districts (Moyamba, Koinadugu, and Kono) in Sierra Leone is not unexpected, and together with previous field studies supports the evidence that Egyptian Rousette Bats are the primary Marburg Virus natural reservoir. The finding of multiple and diverse Marburg Virus genetic lineages simultaneously circulating in geographically distinct locations in Sierra Leone suggests that Marburg Virus has been present in West Africa for an extended period of time and is not a recent introduction from other areas of Africa. Indirect fluorescent antibody data suggested that Human Marburg Virus infections may have occurred and gone unrecognised in Liberia in the late 1970s, yet due to specificity issues with the indirect immunofluorescence antibody test at that time, the significance of the findings were unclear. Nevertheless, the isolation of genetically identical viruses from 3/9 Bats caught at the same cave (Kasewe Cave) was surprising. Of note, all three infected juvenile Bats were caught at approximately the same time (within a day of each other). During similar Marburg Virus surveillance activities in Uganda from 2007–2012, 21 genetically distinct Marburgviruses, including Ravn Virus, were isolated directly from Egyptian Rousette Bats, but none were genetically identical to another. In Sierra Leone, we suspect that finding two or more Bats simultaneously infected with the same Marburg Virus lineage is a consequence of being infected from a single point source, perhaps a supershedder Egyptian Rousette Bat interacting with other Bats in a small colony. Moreover, juvenile Bats are known to roost together in caves, a behavior that may facilitate Bat-to-Bat transmission from infected to susceptible individuals. In addition, the field teams did not observe evidence of massive Egyptian Rousette Bat colonies at Kasewe, Kakoya, and Koema Caves like those seen in East Africa making multiple infections stemming from one source more likely. The determination that the Egyptian Rousette Bat colonies are comparatively small is based on the lack of widespread fecal deposits on vegetation near the colony entrances, unlike the copious amounts normally seen in East African Egyptian Rousette Bat populations. Future investigations will include mark-recapture studies to better estimate population sizes at these locations. Overall, the presence of the Egyptian Rousette Bat natural reservoir throughout portions of sub-Saharan Africa implies that Marburgviruses could be present in Bat populations in many localities with suitable habitat for this species even though no Marburg Virus disease outbreaks have yet been recorded.
The Marburg Virus infection data from the four capture sites indicates an age bias towards juvenile Egyptian Rousette Bats that is consistent with previous studies in Uganda and South Africa. Overall, more juveniles were actively infected with Marburg Virus, while more adults had antibody reactive to Marburg Virus. As with Uganda and South Africa, this is indicative of juveniles having maternal antibody for the first few months after birth, providing protection against Marburg Virus infection. That antibody eventually wanes, leaving the older (4-6 months) juvenile cohort susceptible to infection. As the Bats get older, the chances of having been infected with Marburg Virus increase, leading to the increased prevalence of Marburg Virus-specific antibody detected in the adults.
The Marburg Virus phylogeny shows that sequences obtained from Egyptian Rousette Bats in Sierra Leone align most closely with viruses previously found in Egyptian Rousette Bats in Gabon and Democratic Republic of the Congo from 2006–2009, and in Humans in Angola in 2005. The detection of an Angola-like strain is noteworthy because this is the first time it has been identified in Egyptian Rousette Bats even though all other major Marburgvirus lineages, including Ravn Virus, have been detected co-circulating in a single Egyptian Rousette Bat population in Uganda or Democratic Republic of the Congo. In that context, the overall genetic diversity detected to date in the West African Marburg Virus sequences is comparatively lower and may be a consequence of smaller colony sizes compounded by long-term immunity in previously infected bats. Experimental infection studies of captive Egyptian Rousette Bats have shown that bats retain immunity to Marburg Virus reinfection for up to two years despite diminished antibody levels, suggesting that reinfection is not a major driver of Virus persistence in the population. This type of infection dynamic in Egyptian Rousette Bats would further limit the number of susceptible Bat hosts within a colony, thereby potentially limiting the number of Virus strains that can co-circulate within an Egyptian Rousette Bat roost. The fact that the Marburg Virus strains detected in Sierra Leonean Egyptian Rousette Bats are most similar to those seen in other locations on the west coast of Africa (Gabon and Angola) may be reflective of restricted Egyptian Rousette Bat movement and consistent with isolation of Egyptian Rousette Bat populations in Sierra Leone from the larger metapopulation of Egyptian Rousette Bats across most of Central and East Africa. One reason for this isolation could be loss of contiguous habitat through degradation of forested lands that bridge the gap between the Congo Basin and West Africa.
The clear and unwavering recommendation by the Amman et al. is for individuals living and working in close proximity to caves and mines inhabited by Egyptian Rousette Bats to avoid these Bats. Extermination of a reservoir species as a means of zoonotic pathogen control has been shown to be ineffective and can result in higher ratios of active infection. In one recent example, a Ugandan gold mine was sealed and more than 100 000 Egyptian Rousette Bats destroyed. Over the course of several years, the Bats returned and the prevalence of Marburg Virus infection in the Bat population more than doubled. This recolonization was soon followed by the largest human Marburg Virus disease outbreak in Ugandan history, centered in a nearby town. These data show that culling Bat populations may lead to increased Human health risks and thus should be avoided as a pathogen control measure. Furthermore, as a frugivorous species, Egyptian Rousette Bats play an extremely important ecological role in forest regeneration by dispersing seeds and facilitating pollination of the fruiting trees they visit on a nightly basis. The ecological benefits of Bat activity are critical for the survival of the threatened environment in which they live. Tropical forests in Sierra Leone, Liberia, and Côte d’Ivoire were reported to be most at risk in terms of vulnerability, exposure, and pressure from agricultural expansion. Of those West African countries, Sierra Leone was identified as having the greatest pressure from population and income growth resulting in commodity crop expansion and foreign land investment. With reports of existing vegetative cover in the upper Guinean forests showing losses of nearly 80%, ecologically important species like Egyptian Rousette Bats are crucial to the health and longevity of this fragile ecosystem. Perhaps identifying Egyptian Rousette Bats as the source of Marburg Virus in West Africa can serve as a public deterrent and promote Bat avoidance instead of destruction.
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