Forty Years of Marburg Virus, by Werner Slenczka and Hans Di

Re: Forty Years of Marburg Virus, by Werner Slenczka and Han

Postby admin » Fri Jan 08, 2016 2:03 am

Marburg hemorrhagic fever (Marburg HF)
by Centers for Disease Control and Prevention

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Marburg hemorrhagic fever (Marburg HF) is a rare but severe hemorrhagic fever which affects both humans and non-human primates. Marburg HF is caused by Marburg virus, a genetically unique zoonotic (or, animal-borne) RNA virus of the filovirus family. The five species of Ebola virus are the only other known members of the filovirus family.

Marburg virus was first recognized in 1967, when outbreaks of hemorrhagic fever occurred simultaneously in laboratories in Marburg and Frankfurt, Germany and in Belgrade, Yugoslavia (now Serbia). Thirty-one people became ill, initially laboratory workers followed by several medical personnel and family members who had cared for them. Seven deaths were reported. The first people infected had been exposed to imported African green monkeys or their tissues while conducting research. One additional case was diagnosed retrospectively.

The reservoir host of Marburg virus is the African fruit bat, Rousettus aegyptiacus. Fruit bats infected with Marburg virus do not to show obvious signs of illness. Primates (including humans) can become infected with Marburg virus, and may develop serious disease with high mortality. Further study is needed to determine if other species may also host the virus.

This Rousettus bat is a sighted, cave-dwelling bat widely distributed across Africa. Given the fruit bat's wide distribution, more areas are potentially at risk for outbreaks of Marburg HF than previously suspected. The virus is not known to be native to other continents, such as North America.

Marburg HF typically appears in sporadic outbreaks throughout Africa; laboratory confirmed cases have been reported in Uganda, Zimbabwe, the Democratic Republic of the Congo, Kenya, Angola, and South Africa. Many of the outbreaks started with male mine workers working in bat-infested mines. The virus is then transmitted within their communities through cultural practices, under-protected family care settings, and under-protected health care staff. It is possible that sporadic, isolated cases occur as well, but go unrecognized.

Cases of Marburg HF have occurred outside Africa, such as during the 1967 outbreak, but are infrequent. In 2008, a Dutch tourist developed Marburg HF after returning to the Netherlands from Uganda, and subsequently died. Also in 2008, an American traveler developed Marburg HF after returning to the US from Uganda and recovered. Both travelers had visited a well-known cave inhabited by fruit bats in a national park. See the History of Outbreaks table for a chronological list of known cases and outbreaks.

Chronology of Marburg Hemorrhagic Fever Outbreaks

Known Cases and Outbreaks of Marburg Hemorrhagic Fever, in Chronological Order

Year(s) / Country / Apparent or suspected origin / Reported number of human cases / Reported number (%) of deaths among cases / Situation

1967 / Germany and Yugoslavia / Uganda / 31 / 7 (23%) / Simultaneous outbreaks occurred in laboratory workers handling African green monkeys imported from Uganda 1 In addition to the 31 reported cases, an additional primary case was retrospectively serologically diagnosed. 2

1975 / Johannesburg, South Africa / Zimbabwe 3 / 1 (33%) / A man with a recent travel history to Zimbabwe was admitted to hospital in South Africa. Infection spread from the man to his traveling companion and a nurse at the hospital. The man died, but both women were given vigorous supportive treatment and eventually recovered. 3

1980 Kenya / Kenya / 2 / 1 (50%) / Recent travel history included a visit to Kitum Cave in Kenya's Mount Elgon National Park. Despite specialized care in Nairobi, the male patient died. A doctor who attempted resuscitation developed symptoms 9 days later but recovered. 4

1987 Kenya / Kenya / 1 / 1 (100%) / A 15-year-old Danish boy was hospitalized with a 3-day history of headache, malaise, fever, and vomiting. Nine days prior to symptom onset, he had visited Kitum Cave in Mount Elgon National Park. Despite aggressive supportive therapy, the patient died on the 11th day of illness. No further cases were detected. 5

1990 / Russia / Russia / 1 / 1 (100%) / Laboratory contamination. 6

1998-2000 / Democratic Republic of Congo (DRC) / Durba, DRC / 154 / 128 (83%) / Most cases occurred in young male workers at a gold mine in Durba, in the north-eastern part of the country, which proved to be the epicentre of the outbreak. Cases were subsequently detected in the neighboring village of Watsa. 7

2004-2005 / Angola / Uige Province, Angola / 252 / 227 (90%) / Outbreak believed to have begun in Uige Province in October 2004. Most cases detected in other provinces have been linked directly to the outbreak in Uige. 8

2007 / Uganda / Lead and gold mine in Kamwenge District, Uganda / 4 / 1 (25%) / Small outbreak, with 4 cases in young males working in a mine. To date, there have been no additional cases identified. 9

2008 / USA ex Uganda / Cave in Maramagambo forest in Uganda, at the southern edge of Queen Elizabeth National Park / 1 / 0 (0) / A U.S traveler returned from Uganda in January 2008. The patient developed illness 4 days after returning, was hospitalized, discharged and fully recovered. The patient was retrospectively diagnosed with Marburg virus infection. 10

2008 / Netherlands ex Uganda / Cave in Maramagambo forest in Uganda, at the southern edge of Queen Elizabeth National Park 1 / 1 (100%) / A 40-year-old Dutch woman with a recent history of travel to Uganda was admitted to hospital in the Netherlands. Three days prior to hospitalization, the first symptoms (fever, chills) occurred, followed by rapid clinical deterioration. The woman died on the 10th day of the illness. 11 12

2012 / Uganda / Kabale / 15 / 4 (27%) / Testing at CDC/UVRI identified a Marburg virus disease outbreak in the districts of Kabale, Ibanda, Mbarara, and Kampala over a 3 week time period. 13

2014 / Uganda / Kampala / 1* / 1 / Investigations are on-going 14. Testing conducted at CDC/UVRI

*Numbers reflect laboratory confirmed cases only.


References

1. Siegert R. Marburg Virus. In. Virology. New York: Springer-Verlag; 1972; pp. 98-153.
2. Feldmann H, Slenczka W, Klenk HD. Emerging and reemerging of filoviruses. Archives of Virology. 1996;11(Suppl.):77-100.
3. World Health Organization. Marburg virus disease - South Africa. Weekly Epidemiological Record . 1975;50(12):124-125..
4. Smith DH, Johnson BK, Isaacson M, et al. Marburg-virus disease in Kenya. Lancet. 1982; 1(8276):816-820.
5. Johnson ED, Johnson BK, Silverstein D, et al. Characterization of a new Marburg virus isolated from a 1987 fatal case in Kenya. Archives of Virology. 1996;11(Suppl):101-114.
6. Nikiforov VV, Turovskii IU, Kalinin PP, et al. A case of laboratory infection with Marburg fever. Zhurnal Mikrobiologii, Epidemiologii i Immunobiologii. 1994(3):104-106.
7. Bausch DG, Nichol ST, Muyembe-Tamfum JJ, et al. Marburg hemorrhagic fever associated with multiple genetic lineages of virus. New England Journal of Medicine. 2006;355:909-919.
8. Towner JS, Khristova ML, Sealy TK, et al. Marburgvirus genomics and association with a large hemorrhagic fever outbreak in Angola. Journal of Virology. 2006;80(13):6497-6516.
9. Adjemian J, Farnon EC, Tschioko F, et al. Outbreak of Marburg hemorrhagic fever among miners in Kamwenge and Ibanda districts, Uganda, 2007. Journal of Infectious Diseases. 2011; 204(Suppl 3):S796-S799.
10. Centers for Disease Control and Prevention. Imported case of Marburg hemorrhagic fever - Colorado, 2008. Morbidity and Mortality Weekly Report. 2009; 58(49):1377-1381.
11. World Health Organization. Case of Marburg Haemorrhagic Fever Imported into the Netherlands from Uganda . 10 July 2008.
12. Timen A, Koopmans M, Vossen A, et al. Response to Imported Case of Marburg Hemorrhagic Fever, the Netherlands. Emerging Infectious Diseases. 2009; 15(8):1171-1175.
13. Albarino CG, Shoemaker T, Khristova ML, et al. Genomic analysis of filoviruses associated with four viral hemorrhagic fever outbreaks in Uganda and the Democratic Republic of the Congo in 2012. Virology. 2013;442(2):97-100.
14. Uganda Ministry of Health. Press Release, Oct. 8, 2014.
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Re: Forty Years of Marburg Virus, by Werner Slenczka and Han

Postby admin » Fri Jan 08, 2016 2:26 am

Introduction to Marburg virus: history of outbreaks
Posted by Tara C. Smith
August 30, 2007

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As I’ve noted before, filoviruses are some of my favorite pathogens. I don’t work on them myself–though in the pre-children era I certainly thought about it–but I find them absolutely fascinating to read about and follow the literature. Mostly, I think, this is because after knowing about them for so many years (Marburg was discovered in 1967), and so much research (over 1500 papers in Pubmed, or roughly a paper for every person these viruses have killed), we still know relatively little about the most basic questions–such as where there viruses are maintained in nature, and how they enter the human (or non-human primate) population, and why they’re so deadly.

Anyway, after a long time in Ebola’s shadow, Marburg virus has been in the news recently. As such, this is part one in a mini-series on Marburg virus, with an introduction to the virus and its history after the jump.

[See also Marburg in bats: has the elusive reservoir species been found?]

The year was 1967. Several laboratory workers, all from the same lab in Marburg, Germany, were hospitalized with a severe and strange disease. The physicians on staff realized the workers were all suffering from the same ailment, with symptoms that included fever, diarrhea, vomiting, massive bleeding from many different organs, shock, and eventually circulatory system collapse. An investigation began in an attempt to uncover the source of the outbreak. This led to the identification of the source of the virus in Germany: a species of African green monkeys, imported from Uganda, which were being used by the scientists for polio vaccine research. The virus was isolated, and found to exhibit a unique morphology, leading to the designation of a new group: the Filoviridae In that outbreak, a total of 31 human cases were observed, and the disease presented with a 23% mortality rate (7 deaths occurred out of 31 total infections).

After this episode, the virus went back into hiding for almost a decade, not surfacing again until 1975 in South Africa. The origin of this outbreak is unknown, although based on epidemiological studies it is assumed that the index case (the first person known to have been infected), a young man hitchhiking through Africa, acquired the disease in Zimbabwe, and then infected 2 others in South Africa when he arrived there. Only the index case died from the disease; the secondary cases (those infected due to contact with the index case) survived.

Marburg again disappeared until one case was reported in Kenya in 1980, and again in 1987 in the same area. In the first outbreak, again only the index case died, while a second patient survived. Only one infection was noted in the 1987 outbreak, resulting in death of the patient. Both of these Kenyan outbreaks occurred in the vicinity of a volcano named Mount Elgon, and there is evidence both index cases had spent time in a cave inside the mountain. This has led to (unconfirmed) speculation that bats may be a reservoir for filoviruses; a hypothesis which will be discussed in more detail in later posts.

Between 1987 and 1998, the only cases of Marburg were due to laboratory accidents, both in the former Soviet Union. One of these cases was fatal. However, in 1998, the largest natural outbreak of Marburg virus disease to date began in northeastern Democratic Republic of the Congo (DRC). This time, the focus of the outbreak was a town called Durba (population 16,000). A large number of men in this region work for the Kilo Moto Mining Company, which runs a number of illegal gold mines in the area. Working in this area is precarious; civil war broke out in 1996, and the socio-economic situation has deteriorated since then. Infectious diseases of all types are common, as vaccinations and medication are in short supply. The Marburg outbreak is thought to have started in November of 1998, although it was not reported to any international agencies until late April of 1999, following the death of the chief medical officer in the area from the disease.

Image

At this time, local officials contacted the Medecins sans Frontieres (Doctors without Borders) in Belgium regarding the ongoing hemorrhagic fever epidemic. Both this group and their sister group in Holland sent officers to investigate and to stem the spread of the epidemic. Patient samples were immediately sent to the National Institute of Virology in Johannesburg, South Africa; a diagnosis of Marburg virus as the cause of the illness was made on May 6th. Barrier nursing procedures were instituted, and isolation wards were fashioned at the hospital. Over the course of the epidemic, 149 cases with 123 deaths were recorded (83% fatality rate). Miners were found to be at a significantly higher risk of contracting Marburg than the general population of this area, suggesting they may be more frequently exposed to the natural reservoir of Marburg virus.

This outbreak was eclipsed beginning in 2004, when Marburg hit Angola in the fall. Due to civil war in the country and a non-existent public health infrastructure, the outbreak wasn’t identified until almost 6 months later, in March of 2005. 252 people were infected, with 227 deaths (90% fatality rate). The outbreak was declared over in November of 2005, after no additional cases had been reported since summer.

This brings us to present-day Uganda, where another Marburg outbreak is ongoing, again associated with miners. So far, this one has been minor, and seems to be contained; meanwhile, back in the DRC, there comes a report of another outbreak of hemorrhagic fever. It’s not known at the time what the causative agent is, but a hundred deaths have already been reported:

The deaths began following the funeral of 2 village chiefs. “Everyone that attended those funerals is now dead,” said the medical inspector. According to Kanow, haemorrhagic fever is transmitted by contact with an infected person.

In all, 4 villages are affected and 217 people have come down with the illness.


Is this the next Marburg or Ebola outbreak? A number of other pathogens can cause hemorrhagic fever, but the geography and epidemiology do point to either Marburg or Ebola as likely agents. It appears that Marburg infection is endemic in this area of the DRC; thus, sporadic cases are expected. The instability and conflict in this region makes it difficult to supply regular international aid; thus, it is likely that we will see future outbreaks in this region. More information on the reservoir and transmission of this virus would go a long way toward controlling both Marburg and Ebola virus infections in this region.

Images from http://www.who.int/features/marburg/02.jpg

Additional references and resources:

Bausch DG, et al. 2003. Risk factors for Marburg hemorrhagic fever, Democratic Republic of the Congo. Emerg. Infect. Dis. 9:1531-1537.

Beer B, Kurth R. 1999. Characteristics of Filoviridae: Marburg and Ebola Viruses. Naturwissenschaften, 86:8-17.

Burton, A. Marburg Miner Mystery. The Lancet Infections Diseases, Vol. 4, 2004

Preston, R. 1994. The Hot Zone. New York: Random House.
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Re: Forty Years of Marburg Virus, by Werner Slenczka and Han

Postby admin » Fri Jan 08, 2016 2:36 am

Response to Imported Case of Marburg Hemorrhagic Fever, the Netherlands
by Aura Timen, Marion P.G. Koopmans, Ann C.T.M. Vossen, Gerard J.J. van Doornum, Stephan Günther, Franchette van den Berkmortel, Kees M. Verduin, Sabine Dittrich, Petra Emmerich, Albert D.M.E. Osterhaus, Jaap T. van Dissel, and Roel A. Coutinho1
Volume 15, Number 8—August 2009

National Institute for Public Health and the Environment, Bilthoven, the Netherlands (A. Timen, M.P.G. Koopmans, S. Dittrich, R.A Coutinho)
Leiden University Medical Center, Leiden, the Netherlands (M.P.G. Koopmans, A.C.T.M. Vossen, J.T. van Dissel)
University Medical Center, Rotterdam, the Netherlands (M.P.G. Koopmans, G.J.J. van Doornum, A.D.M.E. Osterhaus)
Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany (S. Gunther, P. Emmerich)
Elkerliek Hospital, Helmond, the Netherlands (F. van den Berkmortel, K.M. Verduin)
St. P.A.M.M., Veldhoven, the Netherlands (K.M. Verduin)
Academic Medical Center, Amsterdam, the Netherlands (R.A. Coutinho)
European Centre for Disease Prevention and Control, Stockholm, Sweden (S. Dittrich)

1 On behalf of the national response team.

Address for correspondence: Aura Timen, PO Box 1, 3720 BA Bilthoven, the Netherlands; e-mail: ln.mvir@nemit.arua

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Abstract

On July 10, 2008, Marburg hemorrhagic fever was confirmed in a Dutch patient who had vacationed recently in Uganda. Exposure most likely occurred in the Python Cave (Maramagambo Forest), which harbors bat species that elsewhere in Africa have been found positive for Marburg virus. A multidisciplinary response team was convened to perform a structured risk assessment, perform risk classification of contacts, issue guidelines for follow-up, provide information, and monitor the crisis response. In total, 130 contacts were identified (66 classified as high risk and 64 as low risk) and monitored for 21 days after their last possible exposure. The case raised questions specific to international travel, postexposure prophylaxis for Marburg virus, and laboratory testing of contacts with fever. We present lessons learned and results of the follow-up serosurvey of contacts and focus on factors that prevented overreaction during an event with a high public health impact.

Keywords: Marburg virus diseases, hemorrhagic fever, exposure, contacts, temperature monitoring, filovirus, viruses, perspective

In Western countries, Marburg hemorrhagic fever (MHF) is an imported disease with a low risk of occurrence, but it has a high profile in the public mind (1) because it can be transmitted from person to person, the course is fatal in up to 80% of cases, and the reservoir is uncertain (2,3). The infection is caused by the Marburg virus (MARV), an enveloped, nonsegmented, negative-stranded RNA virus belonging, with the Ebola virus, to the family Filoviridae. Although the main transmission route is direct contact with blood or other infected body fluids, transmission by droplets and aerosols cannot be ruled out and has been demonstrated in animal models (4).

MARV was identified in 1967 in Marburg, Germany, during a laboratory outbreak caused by handling tissues of African green monkeys (5,6). From 1975 through 1987, sporadic cases occurred in South Africa (1975, when the index case, a person exposed in Zimbabwe, was diagnosed in South Africa) (7) and in Kenya (1980, 1987) (8–10). Outbreaks were reported from the Democratic Republic of Congo in 1998–2000 (11,12), Angola in 2004–2005 (2) and Uganda in 2007 (13). Nonhuman primates and bats are suspected as sources of infection, but their role in the natural reservoir for MARV and transmission to humans is unclear (14).

In July 2008, an imported case of MHF was diagnosed in the Netherlands. We describe the public health response involving the management of 130 contacts at risk of acquiring the disease.

The Case

On July 5, 2008, a 41-year-old woman was referred by her general practitioner to the Elkerliek Hospital because of fever (39°C) and chills of 3 days’ duration after returning from a June 5–28 holiday in Uganda. She was placed in a hospital room with 3 other patients. Malaria was ruled out by 3 negative blood films. Routine bacteriologic tests were performed, and empiric treatment with ceftriaxone, 2 g/day, was started. On July 7, hemorrhagic fever was included among other infectious causes in the differential diagnosis because of rapid clinical deterioration and impending liver failure. An ambulance stripped of all unnecessary devices and equipped in accordance with strict isolation protocols transferred the patient to a single room with negative air pressure ventilation and anteroom in the Leiden University Medical Centre (LUMC).

After admission, rash, conjunctivitis, diarrhea, liver and kidney failure, and finally, hemorrhaging developed in the patient. Extensive bacteriologic and virologic analyses were conducted, and plasma samples were sent to Dutch national laboratories and to the Bernhard-Nocht-Institute for Tropical Medicine (BNI) in Hamburg, Germany, for testing to detect antibodies to and RNA from filoviruses. Initial laboratory results from the Dutch national reference laboratory were ambiguous for hemorrhagic fever. On July 10, BNI reported a positive reverse transcription–PCR result for MARV (15), which was confirmed by sequence analysis of the polymerase gene. The strain was related to, but distinct from, known isolates. MARV was confirmed by PCR by the Department of Virology at Erasmus Medical College (Rotterdam, the Netherlands). On July 11, the patient died of consequences of cerebral edema.

Travel History and Hypotheses for the Source of Infection

The patient’s travel group consisted of 7 Dutch tourists and 2 guides. Three of the tourists, including the patient, and 1 guide visited an empty cave on June 16 in Fort Portal and the Python Cave in the Maramagambo Forest on June 19. The patient’s partner recalled bats flying around in the latter cave, bumping against the visitors, and large amounts of droppings on the ground. She incurred no bite wounds, and no preexisting wounds were exposed to bats. On July 23, the travel group came within 5 m of gorillas in the wild and visited a village inhabited by pygmies, where they saw an elderly sick woman lying under a blanket.

We postulated that the most probable source of MARV infection was the visit to the Python Cave, known for its colony of Egyptian fruit-eating bats (Rousettus aegyptiacus). The party had photographed these bats, and this species of bat has been shown to carry filoviruses, including MARV (16,17) in other sub-Saharan locations. We estimated the incubation period of the infection to be 13 days.

Organization of Public Health Response

On July 8, the attending physician at the LUMC notified the Dutch public health authorities about the case. A national outbreak response team was formed of clinicians, medical microbiologists and virologists, public health specialists, staff members from the national response unit, and a press officer. This team convened a nearly daily teleconference to 1) to perform a structured assessment of the public health risks in the 2 hospitals and in the community, 2) perform risk classification of contacts, 3) issue guidelines for follow-up, 4) provide information to professionals and media, and 5) monitor progression of crisis response.

Immediately after the diagnosis was confirmed, on July 10, a press conference was held. Various press statements emphasizing the control measures designed to prevent secondary transmission followed the press conference. The World Health Organization was notified according to the International Health Regulations by the National Focal Point, and international warnings were issued through the Early Warning and Response System and through ProMED.

Management of Contacts

Although MARV infectivity is highest in the last stage of the disease, when severe bleeding coincides with high viral load, we considered the onset of fever (July 2) as the starting point for contact monitoring. Follow-up measures tailored to the risk group were undertaken during the 21 days after last possible exposure (14,18,19). The high-risk group comprised anyone with unprotected exposure of skin or mucosa to blood or other body fluids of the index patient. It included the other 3 patients in the patient’s room at Elkerliek and personnel who handled her specimens without protection. The low-risk contacts were LUMC and ambulance personnel who had employed the appropriate personal protective measures while caring for the patient or diagnostic samples. Persons who had been near the patient during her holiday, the return flight, and stay in the Netherlands until Elkerliek admission but who were not exposed to her body fluids during her febrile illness and personnel from reference laboratories who worked under BioSafety Level 3 conditions were categorized as casual contacts.

A total of 130 at-risk contacts were identified, 64 at high risk and 66 at low risk (Table). High-risk contacts were required to record their temperature 2×/day, report to the local health authorities 1×/day, and postpone any travel abroad. The low-risk contacts were asked to record their temperature 2×/day and to report to local health authorities if it was>38°C. No limits were imposed on the casual contacts.

Image
Control measures targeting contacts with risk for exposure to Marburg virus, the Netherlands, 2008*

Because asymptomatic MARV infection is rare (20,21) and thus unlikely to play a role in spreading the infection, we restricted further clinical and laboratory evaluation to persons with a temperature >38°C, measured at 2 points 12 hours apart. Every case of fever was to be assessed on an individual basis by the response team. Three academic hospitals provided stand-by isolation facilities for admission of contacts.

On August 1, the temperature monitoring of contacts ended. Fever of at least 12 hours’ duration or clinical signs of MHF did not develop in any of the contacts. Fever within 21 days did not develop in any of the travel companions and local guide who joined the patient in the bat cave. Because sustained fever did not develop in any of the high-risk or low-risk contacts during the surveillance period, no clinical or laboratory follow-up for MARV was needed. The Technical Appendix summarizes other findings during the monitoring period, dilemmas encountered with respect to travel restrictions, postexposure options in case of a high-risk accident, and laboratory diagnosis in the early stage of infection. The Technical Appendix also describes laboratory procedures used.

Serologic Follow-up

To identify asymptomatic seroconversion, a serosurvey was undertaken of 85/130 (65%) contact persons who participated in the study. They represented 78% (50/64) of high-risk contacts and 53% (35/66) of low-risk contacts and included the Dutch visitors to the bat cave. Blood samples were collected from December 2008 through February 2009, 5–7 months after possible exposure. The laboratory testing was performed at the BNI in Hamburg by using an immunofluorescent antibody (IFA) assay.

The IFA slides were prepared using the MARV strain of the index patient. Details about the laboratory testing are given in the Technical Appendix. In 2 initial evaluations, all but 2 samples were negative for antibodies against MARV. Additional screening found that all serum samples tested negative for immunoglobulin (Ig) G and IgM to MARV.

Discussion

We have described the public health response to the case of MHF in a Dutch woman returning from travel abroad, who was most likely exposed to MARV by visiting a bat cave. Outbreaks caused by filoviruses constitute a serious public health threat in sub-Saharan countries and have disruptive consequences at the individual and societal level. In countries in which these viruses are not endemic, imported cases occur only sporadically and are associated with little or no secondary transmission (22). Our patient represents a rare case of MARV infection imported to a Western country, and her case is unusual in that her only likely exposure was visiting a bat cave while traveling in Uganda. Insectivorous bats may have been the source of sporadic cases in Zimbabwe in 1975 (23) and Kenya in 1980 and 1987 (8,9). Furthermore, epidemiologic evidence linked a large outbreak of MHF in Durba (Democratic Republic of Congo) to a mine containing a large population of fruit-eating bats (24). Although the source of infection in our case is not certain, circumstantial evidence points to transmission in the Python Cave. Ecological surveys to assess the presence of infected bats in that cave are ongoing (P. Rollin, pers. comm.).

Our case shows that unnoticed exposure to an unknown reservoir in a country with no apparent cases of MHF can lead to infection. In countries with previous cases of MHF, entry into bat caves should certainly be avoided until we know the role of bats as reservoir for MARV. The importance of MHF for western countries may be increasing, with more persons traveling to high-risk regions and incurring exposure by intrusion into unaccustomed ecological niches. Hospital staff in low-risk countries must be alert to this possibility. In most travelers returning from tropical destinations, fevers are caused by common pathogens or by malaria. However, fever together with rapid clinical deterioration and hemorrhaging in a patient returned from a suspect region should suggest viral hemorrhagic fevers, especially if exposure to a possible reservoir could have occurred.

Inclusion of MHF in the differential diagnosis of a patient triggers an immediate public health response. This response aims primarily at reducing the chance of secondary transmission by identifying contact persons at risk. Person-to-person transmission occurs in countries to which MARV is endemic (22) but only once has been reported elsewhere (23). In this case we identified 130 contacts with possible risk. Two hospitals, 2 public health departments, and 3 laboratories were involved. We decided to trace all people who were in contact with the index patient after her fever developed and to assess their risk for exposure on a case-by-case basis. All contacts complied with temperature monitoring and daily reporting. All but 2 high-risk contacts postponed further travel until the theoretical incubation period of 21 days had elapsed.

In the Netherlands, statutory power to prevent a healthy person from traveling abroad is limited, but the Public Health Law is being revised, and emergency legal provisions are being considered. Despite various recommendations (14,18,25–27), no evidence-based, widely accepted international protocol is available to guide contact classification and monitoring in the case of MHF. Legislation on containment of dangerous pathogens (1) and measures applied to contacts differ among countries, sometimes with extreme consequences. These differences, together with privacy issues, make international exchange of information difficult.

The serosurvey of the contacts of this patient confirm that no secondary transmission took place between her and any contact who provided a blood sample. Our results are consistent with those of Borchert et al. (21), who found no serologic evidence for asymptomatic or mild MARV infection in a serosurvey of household contacts.

The present case was an exceptional situation in which visiting a tourist attraction led to MHF, a disease with a high potential for overreaction. Given this potential, a rational response must be built on a thorough and evidence-based risk assessment (1). The response in the Netherlands was low profile and did not lead to overreacting or public alarm. Its key factors were a coordinated risk assessment and contact monitoring, together with factual updates for health professionals and the public. MHF may be more often encountered in industrialized countries in the future due to adventure travel to regions endemic for MHF.

Supplementary Material

Technical Appendix:

Response to Imported Case of Marburg Hemorrhagic Fever, the Netherlands

Technical Appendix

Clinical Findings and Dilemmas during Contact Monitoring

Clinical Findings


In 1 high-risk contact, the body temperature once exceeded 38°C, but 12 hours later, the temperature had normalized. Another high-risk contact who shared the patient’s room at Elkerliek Hospital was readmitted to that hospital because of heart failure, pulmonary congestion, and subfebrile temperature. His first admission had ended days before his readmission; his condition did not differ between stays and could be attributed to the underlying end-stage heart disease. Several other contacts showed nonspecific symptoms such as nausea and headache, but without fever, and specific follow-up was deemed unnecessary.

The monitoring period led to emotional problems, mostly in high-risk contacts, due mainly to the restrictive measures on daily life and the relatively long period of uncertainty about their prognosis and possible transmission to family members. Psychological support was made available on a case-by-case basis by the occupational health department of the 2 hospitals.

Dilemmas in the Management of Contacts

Problems arose regarding international travel, testing of contacts, and postexposure prophylaxis. By the time Marburg hemorrhagic fever was diagnosed in the index patient, 2 contacts had left for holidays in Italy and the United States, respectively, where they remained for most of the monitoring period. The national authorities of both countries were contacted, and the protocols for temperature monitoring were conveyed with follow-up information on the health status of the 2 persons.

A third contact departed for Poland 3 days before completing the monitoring, after being instructed to carry on the monitoring and stay in daily contact with the Dutch authorities. The Polish authorities were informed because there were doubts about his compliance. Another person left for Morocco 1 day before the end of the monitoring period, but he kept in touch with the Dutch authorities.

To anticipate possible needlestick accidents or gross breaches of isolation measures by healthcare workers, use of experimental vaccines were assessed in a teleconference with international experts. They favored the vaccine in which attenuated recombinant vesicular stomatitis virus vector expresses the Marburg virus (MARV) glycoprotein (1–3) and developed protocols for its use, including regulatory aspects and measures to contain environmental shedding of VSV.

Laboratory Diagnosis in the Early Stage of Infection

Transportation and Processing of Samples


Transport of samples must be organized before sample collection to avoid bottlenecks. We therefore arranged for certified couriers to link hospitals quarantine facilities to laboratories, including the nearest reference laboratory in Germany.

Protocols were designed to encompass essential laboratory testing of severely ill patients, including chemical and bacteriologic diagnostic techniques, biosafety considerations, and methods for decontamination of equipment. No existing preparedness protocols included these considerations. We decided that diagnostic work-ups would be limited to contacts in whom fever developed. In that case, essential equipment for blood chemistry analyses would be placed inside the Intensive Care isolation facility.

Laboratory Assessment of Febrile Contacts: Differential Diagnosis

Protocols were developed for diagnosis of the most probable causes of illness, given the seasonal patters, in which prodromal symptoms resemble those seen in patients with a filovirus infection. These include fever, myalgia, and diarrhoea. Data from physician-based studies of respiratory diseases and gastroenteritis were used as a reference (4,5). Contacts with such symptoms would be tested for a range of pathogens to provide an alternative diagnosis. However, their removal from isolation would not be based solely on this testing because common pathogens are often detected in healthy controls.

Filovirus Evaluation in Contact Monitoring

Acute viremia develops in persons infected with Ebola virus, and viral antigens and RNA are detectable in serum, plasma, saliva, and occasionally other secreta (6,7). In early stages of infection, results of PCR-based assays have been positive 24–48 earlier than antigen-capture assays, making the PCR the method of choice. Although viral loads in severely ill patients are high, in the early course of illness, viral loads may be barely detectable (8). Therefore, proper evaluation of PCR-based methods, with particular emphasis on detection limits, is crucial for reliance on these diagnostics during monitoring. The filovirus diagnostics would therefore be conducted simultaneously in at least 2 laboratories. The Bernhard-Nocht-Institute for Tropical Medicine (BNI) in Hamburg, Germany, provided protocols for PCR-based detection of MARVs. They had been validated in a joint study between P4 laboratories, using all MARV isolates available in these laboratories as reference material (8).

Sequence analysis of the patient’s MARV strain showed it was most closely related to the first-identified Marburg virus isolate from Uganda, the Popp strain. Therefore, we assumed that detection limits reported for the Popp strain would apply to this strain as well. Reagent kits based on the Panning protocol were assembled at our request and kindly provided within a few days (Thomas Laue; QIAGEN, Hamburg, Germany). Evaluation of this kit, using extracts from patient serum and other possible sample types (throat swab, plasma, serum, feces), provided reliable results. Additionally, strain specific Taqman PCR was designed at the Department of Virology at the Erasmus University Hospital, with detection limits similar to those of the Panning protocol.

Laboratory Procedures Used in the Follow-up Survey

After inactivation and fixation on immunofluorescent antibody assay slides, the samples were stored at –20°C outside the high-containment laboratory, and further investigations using the inactivated virus were performed under BioSafety Level 2 conditions. Testing was performed using 1:10 and 1:40 dilutions in 1× phosphate-buffered saline of the contact sera, with positive (mouse monoclonal antibody against MARV) and negative (MARV-negative mouse sera) controls on every slide. In the initial screening, the presence of immunoglobulin (Ig) G and IgM was investigated by using IgM and anti-IgG secondary antibodies conjugated with fluorescein isothiocyanate (FITC).

After inconclusive results in the first screening, procedures were repeated using dilutions 1:20, 1:40, 1:80, 1:160, and 1:320 (plus negative and positive control) to enable identification of a potentially higher antibody titer. Inconclusive samples were double-stained with mouse monoclonal antibody and antibodies from the contact sera. The double fluorescence was detected by using 2 differently conjugated secondary antibodies: anti-mouse IgG-rhodamine and anti-human IgG-FITC to differentiate staining between virus particles.

In the initial evaluation of the slides, performed by 2 of the authors (P.E., S.D.), 2 samples could not clearly be identified as negative because they lacked the characteristic round virus inclusions in the cells. However, when virus particles in the infected cells were visualized using monoclonal antibody and overlaying it with the fluorescence of the human antibodies, all activity could be attributed to nonspecific background binding. No overlapping fluorescence of human antibody and mouse monoclonal antibody against Marburg hemorrhagic fever could be observed. Therefore, all tested sera were considered negative for IgG and IgM antibodies to MARV.

References

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2. Daddario-DiCaprio KM, Geisbert TW, Stroher U, Geisbert JB, Grolla A, Fritz EA, et al. Postexposure protection against Marburg haemorrhagic fever with recombinant vesicular stomatitis virus vectors in non-human primates: an efficacy assessment. Lancet. 2006;367:1399–404. PubMed DOI: 10.1016/S0140-6736(06)68546-2

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7. Towner JS, Rollin PE, Bausch DG, Sanchez A, Crary SM, Vincent M, et al. Rapid diagnosis of Ebola hemorrhagic fever by reverse transcription-PCR in an outbreak setting and assessment of patient viral load as a predictor of outcome. J Virol. 2004;78:4330–41. PubMed DOI: 10.1128/JVI.78.8.4330-4341.2004
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Acknowledgments

This article was written on behalf of the members of the national response team, which included the authors and L. Isken, W. Ransz, and A. Jacobi, B. van der Walle and P. Willemse, R. Daemen and D. van Oudheusden, A. Brouwer and C. Bleeker, and T. Schmitt.

We acknowledge the invaluable support from our colleagues across the world, particularly H. Feldmann, R. Swanepoel, Matthias Niedrig, Thomas Laue, John Towner, E. Leroy, E. Gavrilin, R. Andraghetti, F. Plummer, T. Geisbert, G. Nabel, C.J. Peters, and B. Graham. We thank M. van der Lubben, H. Vennema, B. Wilbrink, G.J. Godeke, B. van der Veer, M. Timmer, B. Niemeijer, C. Burghoorn-Maas, T. Mes, G. van Willigen, E. Kuijper, M. Feltkamp, J. van Pelt, and M. Wulff for their assistance; Jim van Steenbergen for critical comments on the manuscript; and Lucy Phillips for editing.

Parts of this study were supported by a grant from the Dutch Research Foundation (ZonMw).

Biography

• Dr Timen is a senior consultant in communicable disease control at the Centre for Infectious Diseases of the National Institute of Public Health and the Environment, the Netherlands. Her main research interest is the public health response to outbreaks and threats.

Footnotes

Suggested citation for this article: Timen A, Koopmans MPG, Vossen ACTM, van Doornum GJJ, Günther S, van den Berkmortel F, et al. Response to imported case of Marburg hemorrhagic fever, the Netherlands. Emerg Infect Dis [serial on the Internet]. 2009 Aug [date cited]. Available fromhttp://www.cdc.gov/EID/content/15/8/1171.htm

References

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2. Centers for Disease Control and Prevention Outbreak of Marburg virus hemorrhagic fever—Angola, October 1, 2004–March 29, 2005. MMWR Morb Mortal Wkly Rep2005;54:308–9 [PubMed]

3. Borio L, Inglesby T, Peters CJ, Schmaljohn AL, Hughes JM, Jahrling PB, et al.Hemorrhagic fever viruses as biological weapons: medical and public health management. JAMA 2002;287:2391–405 10.1001/jama.287.18.2391 [PubMed][Cross Ref]

4. Leffel EK, Reed DS Marburg and Ebola viruses as aerosol threats. Biosecur Bioterror2004;2:186–91 10.1089/bsp.2004.2.186 [PubMed] [Cross Ref]

5. Peters CJ Marburg and Ebola virus hemorrhagic fevers. In: Mandell GL, Bennett JE, Dolin R, editors. Mandell, Douglas, and Bennett’s principles and practice of infectious diseases. Philadelphia: Elsevier Inc.; 2005. p. 2057–9.

6. Siegert R Marburg virus. Virology monograph. New York: Springer-Verlag; 1972. p. 98–153.

7. Gear JS, Cassel GA, Gear AJ, Trappler B, Clausen L, Meyers AM, et al. Outbreak of Marburg virus disease in Johannesburg. BMJ 1975;4:489–93 10.1136/bmj.4.5995.489[PMC free article] [PubMed] [Cross Ref]

8. Johnson ED, Johnson BK, Silverstein D, Tukei P, Geisbert TW, Sanchez AN, et al.Characterization of a new Marburg virus isolated from a 1987 fatal case in Kenya. Arch Virol Suppl 1996;11:101–14 [PubMed]

9. Smith DH, Johnson BK, Isaacson M, Swanapoel R, Johnson KM, Killey M, et al.Marburg-virus disease in Kenya. Lancet 1982;1:816–20 10.1016/S0140-6736(82)91871-2 [PubMed] [Cross Ref]

10. Khan AS, Sanchez A, Pflieger AK Filoviral haemorrhagic fevers. Br Med Bull1998;54:675–92 [PubMed]

11. World Health Organization Marburg fever, Democratic Republic of the Congo. Wkly Epidemiol Rec 1999;74:145. [PubMed]

12. World Health Organization Viral haemorrhagic fever/Marburg, Democratic Republic of the Congo. Wkly Epidemiol Rec 1999;74:157–8 [PubMed]

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14. European Network for Diagnostics of Imported Viral Diseases Management and control of viral hemorrhagic fevers [cited 2008 Aug 13]. Available fromhttp://www.enivd.de

15. Drosten C, Gottig S, Schilling S, Asper M, Panning M, Schmitz H, et al. Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR. J Clin Microbiol2002;40:2323–30 10.1128/JCM.40.7.2323-2330.2002 [PMC free article] [PubMed][Cross Ref]

16. Towner JS, Pourrut X, Albarino CG, Nkogue CN, Bird BH, Grard G, et al. Marburg virus infection detected in a common African bat. PLoS One 2007;2:e764 10.1371/journal.pone.0000764 [PMC free article] [PubMed] [Cross Ref]

17. Swanepoel R, Smit SB, Rollin PE, Formenty P, Leman PA, Kemp A, et al. Studies of reservoir hosts for Marburg virus. Emerg Infect Dis 2007;13:1847–51 [PMC free article][PubMed]

18. Filoviruses. In: Steenbergen JE, Timen A, Beaujean DJ, editors. LCI guidelines infectious disease control edition 2008 [in Dutch]. Bilthoven: National Institute for Public Health and the Environment; 2008. p. 478–84.

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Re: Forty Years of Marburg Virus, by Werner Slenczka and Han

Postby admin » Fri Jan 08, 2016 3:38 am

Marburg Virus Infection Detected in a Common African Bat
by Jonathan S. Towner,1 Xavier Pourrut,2,3 César G. Albariño,1 Chimène Nze Nkogue,2 Brian H. Bird,1,4 Gilda Grard,2 Thomas G. Ksiazek,1 Jean-Paul Gonzalez,5 Stuart T. Nichol,1 and Eric M. Leroy2,3,*
Published online 2007 Aug 22. doi: 10.1371/journal.pone.0000764
PMCID: PMC1942080

Philip Stevenson, Academic Editor

1Centers for Disease Control and Prevention, Special Pathogens Branch, Atlanta, Georgia, United States of America
2Centre International de Recherches Médicales de Franceville, Franceville, Gabon
3Institut de Recherche pour le Développement, UR178, Franceville, Gabon
4School of Veterinary Medicine, University of California at Davis, Davis, California, United States of America
5Institut de Recherche pour le Développement, UR178, Nakhonpathom, Thaïland
Cambridge University, United Kingdom
* To whom correspondence should be addressed. E-mail: rf.dri@yorel.cire

Conceived and designed the experiments: EL XP JT. Performed the experiments: EL XP JT GG CA CN. Analyzed the data: SN EL JT BB JG. Contributed reagents/materials/analysis tools: SN JT TK. Wrote the paper: SN EL JT.

NOTICE: THIS WORK MAY BE PROTECTED BY COPYRIGHT

YOU ARE REQUIRED TO READ THE COPYRIGHT NOTICE AT THIS LINK BEFORE YOU READ THE FOLLOWING WORK, THAT IS AVAILABLE SOLELY FOR PRIVATE STUDY, SCHOLARSHIP OR RESEARCH PURSUANT TO 17 U.S.C. SECTION 107 AND 108. IN THE EVENT THAT THE LIBRARY DETERMINES THAT UNLAWFUL COPYING OF THIS WORK HAS OCCURRED, THE LIBRARY HAS THE RIGHT TO BLOCK THE I.P. ADDRESS AT WHICH THE UNLAWFUL COPYING APPEARED TO HAVE OCCURRED. THANK YOU FOR RESPECTING THE RIGHTS OF COPYRIGHT OWNERS.


[Dr. Leonard George Horowitz] But the biggest best seller, "The Hot Zone," by author Richard Preston, was different. Extremely dishonest. Author Richard Preston was paid by the Alfred P. Sloan Foundation, among the cancer industry's largest benefactors, intimately tied to the Rockefeller family's influence, to present easily-proven deceptions. Preston reported that Ebola emerged from bat guano, at the bottom of a deep dark cave in Southern Sudan. The fact is, this is very close to where Litton Bionetics' labs operated, testing these precise agents on native Africans.

[BIONETICS RESEARCH LABORATORIES, INC. (NIH-71-2025)
Title: Investigations of Viral Carcinogenesis in Primates
Contractor's Project Directors: Dr. John Landon, Dr. David Valerio, Dr. Robert Ting
Project Officers (NCI): Dr. Roy Kinard, Dr. Jack Gruber, Dr. Robert Gallo
Objectives: (1) Evaluation of long-term oncogenic effects of human and animal viral inocula in primates of various species, especially newborn macaques, (2) maintenance of monkey breeding colonies and laboratories necessary for inoculation, care and monitoring of monkeys, and (3) biochemical studies of transfer RNA under conditions of neoplastic transformation and studies on the significance of RNA-dependent DNA polymerase in human leukemic tissues.
Major Findings: This contractor continues to produce over 300 excellent newborn monkeys per year. This is made possible by diligent attention to reproductive physiological states of female and male breeders. Semen evaluation, artificial insemination, vaginal cytology and ovulatory drugs are used or tried as needed.
Inoculated and control infants are hand-fed and kept in modified germ-free isolators. They are removed from isolators at about 8 weeks of age and placed in filtered air cages for months or years of observation. The holding ...
Date Contract Initiated: February 12, 1962]


Here, for close examination, is the exact experiment administered by Litton Bionetics in 1965 and ending in 1970, precisely timed for the first Marburg virus outbreak.

[Plyctm: Polycythemia
PPLO: Mycoplasma
R: Rubella
Rau Vi: Rauscher virus
RCS: Reticulum cell sarcoma
Reo 1: Reovirus 1
Reo 3: Reovirus 3
Rhabd L: Rhabdomysarcoma + leukemia
Rhabdo: Rhabdomysarcoma
RTC: Rous transformed cells
S: Sarcoma
S20S40: SV-20 + SV-40
SA 7: Simian agent 7
SCL: Stem cell leukemia
Sq S: Squamous cell sarcoma
SV-5: Simian virus 5
SV-20: Simian virus 20
SV-40: Simian virus 40]


This NCI document refers to John Landon, Chief scientist at Litton Bionetics overseeing the experiment. Notice that at least half of the Rhabdo virus simian-infected monkeys died or were transferred. The ones that lived were transferred to the vaccine production labs for vaccine production.

This is how the first Marburg virus outbreaks occurred. Since that time, every single Marburg virus outbreak, as well as Ebola virus outbreak, has either been associated with a bioweapons lab accident, or an intentional release for terrorism's objectives: political or financial gain.

But not according to America's leading Outbreak journalist, Laurie Garrett. In her bestseller, "The Coming Plague," she neglects Litton's links and dismisses the CIA biological weapons contributions to these outbreaks as though she too were on the Rockefeller's payroll.

It should be noted that these unnaturally evolved, man-made viruses are far less genetically stable. They are more fragile. They mutate more rapidly than naturally evolved viruses. That's why when the second Marburg virus outbreak occurred in the southern part of Uganda in 1975, many scientists suspected foul play by the CIA since the genetic sequence of the 1975 strain was essentially identical to the 1968 strain. The only explanation for this fact remains: refrigeration.

-- In Lies We Trust: The CIA, Hollywood, & Bioterrorism, a documentary by Dr. Leonard George Horowitz


Abstract

Marburg and Ebola viruses can cause large hemorrhagic fever (HF) outbreaks with high case fatality (80–90%) in human and great apes. Identification of the natural reservoir of these viruses is one of the most important topics in this field and a fundamental key to understanding their natural history. Despite the discovery of this virus family almost 40 years ago, the search for the natural reservoir of these lethal pathogens remains an enigma despite numerous ecological studies. Here, we report the discovery of Marburg virus in a common species of fruit bat (Rousettus aegyptiacus) in Gabon as shown by finding virus-specific RNA and IgG antibody in individual bats. These Marburg virus positive bats represent the first naturally infected non-primate animals identified. Furthermore, this is the first report of Marburg virus being present in this area of Africa, thus extending the known range of the virus. These data imply that more areas are at risk for MHF outbreaks than previously realized and correspond well with a recently published report in which three species of fruit bats were demonstrated to be likely reservoirs for Ebola virus.

Introduction

Forty years after the discovery of Marburg virus as the cause of a hemorrhagic fever outbreak among laboratory workers in Germany, the natural reservoir for this highly pathogenic filovirus remains unknown [1]. Until 2000, the virus origins of all Marburg hemorrhagic fever (MHF) cases could be traced to eastern Africa. However, in 2005 the largest outbreak of MHF on record occurred in Uige, Angola, expanding the known range of the disease, and likely the natural reservoir, to the far western edge of the Congo basin [2], [3]. We hypothesized that Marburg virus is present in the rain forests of Gabon, based on ecologic similarities and relative proximity (<800 km) to northern Angola. Bats were the focus of this study based on the recent discovery of the related filovirus, Ebola virus, in fruit bats in Gabon and Republic of Congo [4] (RC), and epidemiologic linkage of MHF cases to a gold mine containing sizeable numbers of bats during a large MHF outbreak in Durba, Democratic Republic of Congo (DRC) in 2000 [5], [6]. Further evidence for a bat reservoir include the linkage of a MHF case in 1987 to Kitum cave at Mt. Elgon, Kenya [7], and transient viremias in asymptomatic bats experimentally infected with Ebola virus [8].

Here, we report testing of bats collected from Gabon and Republic of Congo and demonstrate Marburg virus infection in a common species of fruit bat (Rousettus aegyptiacus) as evidenced by the presence of virus specific RNA and antibody.

Materials and Methods

Marburg virus nucleic acid and IgG detection


For each bat, approximately 100 mg of tissue was incubated overnight at 4°C in 450 ul of cold 2X cellular lysis buffer (ABI) to inactivate virus. Each tissue was then diluted to 1X and homogenized for 2 minutes, at 1500 strokes/min using a ball-mill tissue grinder (Genogrinder 2000, Spex Centriprep). Total RNA was extracted from ∼150 ul of the homogenate [9] and tested for Marburg virus using slightly modified real-time [10] or nested RT-PCR assays. The nested VP35 RT-PCR assay is previously described [6], while the four primers used for the nested NP assay are (5′ to 3′) MBG704F1-GTAAAYTTGGTGACAGGTCATG, MBG719F2-GGTCATGATGCCTATGACAGTATCAT, MBG1248R1-TCTCGTTTCTGGCTGAGG, and MBG1230R2-ACGGCIAGTGTCTGACTGTGTG. The annealing conditions were 50° C for the first round and 55° C for the second round using high-fidelity RT-PCR reagents (Invitrogen). Primer concentrations and amplification conditions used were as described by the manufacturer. Samples 1448, 1519, 1631 and 2296 were independently assayed a minimum of three times in the real-time RT-PCR assay, each time using RNA extracted from newly cut tissue. Potential false positives due to PCR or sample cross-contamination could be ruled out due to the unique virus sequences obtained and the complete lack of any previous Marburg virus testing in the laboratory. The positive control RNA used for the PCR analysis was derived from the Ravn 1987 isolate and is >15% divergent from the known sequence obtained from the three nested RT-PCR-positive bats.

IgG was detected from bat sera diluted 1∶100 using a previously described protocol modified for Marburg virus [11]. Bats with corrected OD values >0.13 were additionally tested at 1∶400 and 1∶1600 dilutions. Bat IgG was detected using Protein A-G peroxidase or HRP-conjugated goat sera raised against a cocktail of IgG from six diverse bat species. Bats of the species Epomops franqueti (N = 47) were used as the negative control group.

Phylogenetic analyses

Bat derived sequence fragments (Genbank accession numbers EU068108-13) were concatenated then aligned with 18 MBG virus genomes (Genbank accession numbers DQ447649-60, AY358025, DQ217792, Z29337.) Maximum likelihood analyses (bootstrap 500 replicates) were completed (PAUP v4.0b10, Sinauer.). Genetic distances were calculated using the Wisconsin package of GCG version 10.3. (Accelrys Inc., San Diego, CA).

Collection of bat organs

Bat species were morphometrically determined in the field using the species identification key developed by Bergman [12]. In addition, bats were photographed and catalogued noting weight, sex, age (adult or juvenile) and forearm measurements. Bats were euthanized individually after which the organs were immediately harvested and frozen in liquid nitrogen. All organs were then stored under nitrogen vapor until placed in a −80 mechanical freezer for storage for subsequent analysis.

Results and Discussion

We tested over 1100 bats representing 10 species (Table 1) collected from five locations throughout Gabon and northwest Republic of Congo (Figure 1), and show evidence of Marburg virus infection in only one species, Rousettus aegyptiacus. Homogenized liver and spleen samples of 1138 bats were first analyzed using a Marburg virus-specific real-time RT-PCR assay to the VP40 gene recently used for human diagnostic testing during the 2005 Angola MHF outbreak [10]. Four bats (sample numbers 1448, 1631, 2296 and 1519), all Rousettus aegyptiacus, were positive at low levels (cycle threshold values >33) in the real-time assay. All four of these bats were trapped near caves in 2005 and early 2006 at two geographic locations in Gabon 250 km apart and approximately 700 km north of Uige, Angola. These four samples were then subjected to further analysis by conventional nested RT-PCR targeting the virus VP35 and NP genes (Materials and Methods). Three bats tested positive in each of the nested assays while a fourth bat (sample 1519), though never found positive by conventional RT-PCR, tested positive five independent times in the real-time assay, each time using RNA extracted from newly cut tissue.

Image
Figure 1. Animal collection sites in Gabon and Republic of Congo.

Image
Table 1. Summary of bat species tested for Marburg virus specific RNA and/or antibody.

Sequence analysis of the purified PCR products identified unique sequences from each bat which together form a well supported single lineage distinct from all previously characterized Marburg viruses, including the Ravn strain used as a positive control in these PCR-based assays (Figure 2A). The Gabon bat Marburg virus sequences differed from those of the other western African Marburg virus lineage, Angola, by approximately 5% at the nucleotide level, far less than the 15% diversity observed among East African Marburg virus isolates (data not shown). An alignment of the NP and VP35 sequences (Figures 2B and C) shows the three bat sequences are divergent from each other at nine positions, and different from a consensus sequence of eight historical Marburg virus isolates at 14 positions. We suspect that the virus load of the fourth bat (positive in the real-time assay only) is just below the limit of detection by the nested assays and/or the sequence has mis-matches at critical PCR-priming positions in the NP and VP35 assays.

Image
Figure 2. Phylogenetic analyses and nucleotide sequence alignments of NP and VP35 sequences derived from bat tissues.

After screening the collection of bats by real-time RT-PCR, we tested for Marburg virus specific antibody in sera (if available) from all bats trapped at the two locations (438 bats) from which PCR positive bats were found (Table 1). Interestingly, sera from 29 bats had corrected OD values greater than 0.13, a threshold value that is three standard deviations from the average OD of the control group (Epomops franqueti) (Figure 3A). Moreover, all 29 of these bats were Rousettus aegyptiacus while none of the other species tested (N = 196) had corrected OD values greater than this threshold value. Sera from three of the 29 bats had OD values greater than 0.13 when diluted 1∶400 while another five bats, including 1448 and 2296, met the same criteria at dilutions of 1∶1600 (Figure 3B). Unfortunately, serum was unavailable from bat 1519 that had tested positive by real-time RT-PCR. Initial serologic testing was completed with a protein A/G conjugate. In the event that the protein A/G conjugate showed a species-specific preference for Rousettus aegyptiacus IgG, we re-tested those sera with OD values greater than 0.13 (in addition to 50 Marburg antibody “negative” bats) using a goat anti-bat conjugate made by immunization with IgG from multiple diverse bat species (both micro and mega-chiropterans). The results of this secondary testing were identical to the initial serological findings using protein A/G (data not shown). These data indicate a substantial fraction, almost 9%, of Rousettus aegyptiacus trapped at these locations may have low-level antibody to Marburg virus, while another 3% have more significant Marburg antibody titers. Among the R. aegyptiacus population tested for which age determinations could be made, evidence of Marburg infection in bats favored adults (24/138) over juveniles (4/86), 17.4% to 4.6% respectively (p = 0.005, Chi-Square test ). However, firm conclusions about the proportion of infected adult versus juvenile populations are difficult because the majority of ‘positive’ bats show only low titers of Marburg antibody while those bats with more conclusive evidence of Marburg infection, by being either PCR positive and/or having IgG titers greater than or equal to 1∶400 (N = 8), are more equivalently distributed (five adults and three juveniles). In addition, there could be residual maternal Marburg-specific antibody in the juvenile bat population. Fifteen of the bats, with OD values greater than 0.13, were males while among the three PCR-positive animals, two were adults (male and female) and one was a juvenile (male).

Image
Figure 3. Marburg antibody testing in bats collected at locations where PCR positive bats were found.

The serological data, combined with the PCR data, are suggestive that these bats may represent a bon-a-fide reservoir species. However, we cannot rule out periodic contact by the bats with an as yet unnamed reservoir. The presence of both virus RNA and IgG antibodies in three (or possibly four) of the animals is consistent with extended viremias, but may well represent late acute phase infections. While it is difficult to determine from these data if Marburg virus causes significant morbidity in R. aegyptiacus, it is worth noting that all of the animals caught appeared clinically healthy and were strong enough to leave their roost to forage for food. Virus isolation attempts and antigen detection tests on the same liver/spleen organ extracts were negative, which along with the quantitative PCR data, indicate low levels of Marburg virus in these organs.

Although inconclusive, several other lines of evidence are consistent with R. aegyptiacus representing a natural host for Marburg virus. Cave roosting is not generally observed with most fruit bats [13], including the three species thought to harbor Ebola virus [4]. However, R. aegyptiacus is known to roost in caves, a behavior that correlates well with the epidemiologic linkage of greater than 80% of human cases in the Durba MHF outbreak to mining activity in a gold mine [5] harboring large bat populations. Furthermore, the home range of R. aegyptiacus encompasses the geographic origin of all known sources of Marburg virus outbreaks (Figure 4) [13] as well as the locations from which the Marburg virus PCR and IgG positive bats were found. These Marburg virus positive bats represent the first naturally infected non-primate animals ever identified, and this is the first report of any Marburg virus activity in Gabon, a region of Africa recently hit by multiple outbreaks of the highly pathogenic Ebola virus (species Zaire) [14]. Together, these facts predict that 1) the potential for Marburg virus contact could be more wide-spread than previously recognized, 2) fruit bats from the sub-family Pteropodinae may serve as filovirus hosts and 3) the species of fruit bats that harbor Ebola and Marburg viruses are likely distinct, yet their home ranges may have large areas of overlap. Identification of the reservoir host should allow development of risk reduction measures to help mitigate the potential of future disease outbreaks.

Image
Figure 4. Geographic distribution (green shade) of Rousettus aegyptiacus in Africa.

Acknowledgments

We thank all those involved in the field collections. We also thank W. Karesh, from Wildlife Conservation Society, for his continuous support. Finally, we thank Drs. Brian Halloway and Pierre Rollin from Centers for Disease Control and Prevention, Atlanta, USA, for providing diagnostic reagents. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agencies.

Footnotes

Competing Interests: The authors have declared that no competing interests exist.

Funding: CDC is supported by the Government of the United States of America. CIRMF is supported by the Government of Gabon, Total-Fina-Elf Gabon, and the Ministère de la Coopération Française. This work was also supported by a Fonds de Solidarité Prioritaire grant from the Ministère des Affaires Etrangères de la France (FSP n° 2002005700).

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