Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Studies of Reservoir Hosts for Marburg Virus

Studies of Reservoir Hosts for Marburg Virus Studies of Reservoir Hosts for Marburg Virus Robert Swanepoel,* Sheilagh B. Smit,* Pierre E. Rollin,† Pierre Formenty,‡ Patricia A. Leman,* Alan Kemp,* Felicity J. Burt,§ Antoinette A. Grobbelaar,* Janice Croft,* Daniel G. Bausch,¶ Hervé Zeller,# Herwig Leirs,** †† L.E.O. Braack,‡‡ Modeste L. Libande,§§ Sherif Zaki,† Stuart T. Nichol,† Thomas G. Ksiazek,† and Janusz T. Paweska,* on behalf of the International Scientifi c and Technical Committee for Marburg Hemorrhagic Fever Control in the Democratic Republic of the Congo To determine reservoir hosts for Marburg virus (MARV), 1998 through September 2000. The outbreak involved 154 we examined the fauna of a mine in northeastern Demo- patients (48 confi rmed and 106 suspected cases); the case- cratic Republic of the Congo. The mine was associated with fatality ratio was 83% (1). Primary cases occurred in young a protracted outbreak of Marburg hemorrhagic fever during male miners and spread as secondary cases to family mem- 1998–2000. We found MARV nucleic acid in 12 bats, com- bers and, less frequently, to healthcare workers and others prising 3.0%–3.6% of 2 species of insectivorous bat and 1 in the community. Most cases occurred in Durba, but a few species of fruit bat. We found antibody to the virus in the se- secondary cases occurred elsewhere, including nosocomial rum of 9.7% of 1 of the insectivorous species and in 20.5% infections in nearby Watsa village, where severely ill pa- of the fruit bat species, but attempts to isolate virus were tients sought care. The occurrence of sporadic cases and unsuccessful. short chains of human-to-human transmission suggested that infection had been repeatedly introduced into the hu- arburg virus (MARV) and Ebola virus, members of man population; this suggestion was substantiated by the Mthe family Filoviridae, cause outbreaks of severe detection of at least 9 genetically distinct viruses circulat- hemorrhagic fever in Africa. Although humans have on ing during the outbreak. Identical sequences of MARV occasion acquired infection from contact with tissues of were found in patients within but not across clusters of diseased nonhuman primates and other mammals, the res- epidemiologically linked cases, although viruses with the ervoir hosts of the viruses in nature remain unknown. same sequences reappeared at irregular intervals during the An outbreak of Marburg hemorrhagic fever ran a pro- outbreak. Most (94%) affected miners worked underground tracted course in the gold-mining village of Durba, north- in Goroumbwa Mine, rather than in the 7 opencast mines eastern Democratic Republic of the Congo, from October in the village. Cessation of the outbreak coincided with the fl ooding of Goroumbwa Mine. Interviews with long-term *National Institute for Communicable Diseases, Sandringham, Re- residents and healthcare workers and review of hospital public of South Africa; †Centers for Disease Control and Preven- records showed that a syndrome hémorragique de Durba tion, Atlanta, Georgia, USA; ‡World Health Organization, Geneva, [hemorrhagic syndrome of Durba] had been associated Switzerland; §University of the Free State, Bloemfontein, South Af- with the mine since at least 1987, and a survivor of a 1994 rica; ¶Tulane School of Public Health and Tropical Medicine, New outbreak was found to have antibodies against MARV. The Orleans, Louisiana, USA; #Institut Pasteur, Lyon, France; **Uni- fauna of Goroumbwa Mine included bats, rodents, shrews, versity of Antwerp, Antwerp, Belgium; ††University of Aarhus, Kon- frogs, snakes, cockroaches, crickets, spiders, wasps, and gens Lyngby, Denmark; ‡‡Conservation International, Cape Town, moth fl ies (1). We present the results of virus reservoir host South Africa; and §§Department of Health, Watsa, Democratic Re- studies conducted during the outbreak. public of the Congo Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007 1847 RESEARCH serum sample confi rmed positive for MARV and used as an Methods internal control. Cutoff values for recording positive results In parallel with human epidemiologic studies, visits were deliberately selected to be stringent at 3 × (mean + were made to Durba in May and October 1999 to collect 3SD) PP values determined for stored bat (n = 188) and specimens for virus ecostudies. The ecostudies were ap- rodent (n = 360) serum samples that had been collected for proved by the International Scientifi c and Technical Com- unrelated purposes in Kruger National Park, South Africa, mittee for Marburg Hemorrhagic Fever Control, which was from 1984 through 1994, and tested at a dilution of 1:100. coordinated by the World Health Organization on behalf of The Kruger bat samples were collected from 3 species of the government of the Democratic Republic of the Congo. fruit bats (Megachiroptera) and 12 species of insectivorous In view of the epidemiologic fi ndings during the outbreak, bats (Microchiroptera), including samples from 56 Chae- emphasis was placed on the fauna of Goroumbwa Mine. rephon pumila, 32 Rousettus aegyptiacus, 27 Mops con- Bats were caught with mist nets at mine entrances; rodents dylurus, 16 Hipposideros caffer, plus 57 samples from 11 and shrews were caught live with Sherman traps within and other species. close to the mine; and arthropods (cockroaches, crickets, spiders, wasps, and moth fl ies, plus streblid, nycteribiid, Results and Discussion and mite parasites of bats) were collected by hand or with The numbers of specimens collected, plus the results sweepnets. Vertebrates were euthanized and dissected on of RT-PCR, nested PCR, attempts to isolate virus in cell site. Blood samples were collected; and samples of liver, culture, and ELISA antibody determinations, are summa- lung, spleen, kidney, testes, brain, salivary glands, and fe- rized in the Table. With the exception of a Nycteris hispida tuses of pregnant females were preserved along with the bat, which was caught near a house in Durba, all specimens arthropods in liquid nitrogen dry-shipping containers for were collected within Goroumbwa Mine or its immediate transport to the National Institute for Communicable Dis- surroundings. An estimated minimum of 10,000 Egyptian eases in South Africa. Extra liver samples were collected fruit bats (R. aegyptiacus) roosted in the mine, clustered for phylogenetic studies on bats and rodents, and formalin- within the upper galleries. Although the numbers of insec- fi xed tissue samples were kept for possible histopathologic tivorous bats were diffi cult to estimate because these bats and immunohistochemical examination. Carcasses were roosted mainly in the deeper recesses of the mine, the catch fi xed in formalin for α-taxonomy purposes. rates indicated substantial numbers of the eloquent horse- Vertebrate tissue and arthropod suspensions were shoe bat (Rhinolophus eloquens) and the greater long-fi n- processed and tested for fi lovirus nucleic acids by reverse gered bat (Miniopterus infl atus). Few microchiropterans transcription–PCR (RT-PCR) and nested PCR by us- were caught in May, but catch rates improved in October ing fi lovirus-specifi c large (L) protein gene primers and after adjustment of trapping hours and the gauge of mist nested MARV-specifi c viral protein 35 (VP35) primers nets used. Pregnancy was recorded in 12 (24%) of 50 R. as described for samples from human patients during the aegyptiacus females in May and in 2 (4.2%) of 47 females outbreak (1). Nucleotide sequencing of amplicons and se- in October; descended testes were found in 2 (6%) of 33 quence data analysis were also performed as described pre- males in May and 19 (25%) of 76 in October. The only in- viously (2), except that MEGA version 3.1 software was dication of breeding activity observed in microchiropterans used (3). Initial RT-PCR and nested PCR were performed was that 1/7 Rh. eloquens females was pregnant in May. with pooled tissue samples of individual vertebrates; when The L primer RT-PCR, which was applied to all speci- possible, for specimens that produced positive results, all mens, produced no positive result. In contrast, the nested tissues were retested separately. In attempts to isolate virus MARV VP35 PCR, which was applied only to specimens as detected by indirect immunofl uorescence, suspensions collected in October 1999, produced positive results on (≈10%) of vertebrate tissues pooled for individual animals specimens from 12 bats: 1 (3.0%) of 33 M. infl atus, 7 and arthropods pooled by species were subjected to 3 se- (3.6%) of 197 Rh. eloquens, and 4 (3.1%) of 127 R. ae- rial passages in Vero 76 cell cultures. Serum samples from gyptiacus. Nested VP35 PCR on individual tissues of the bats and rodents were tested for antibody to MARV by positive bats produced positive results for liver, spleen, ELISA by using a modifi cation of the technique described kidney, lung, salivary gland (3/5 bats), and heart (2/5 bats). previously for human serum (1). ELISA antigen consisted Attempts to isolate virus in cell cultures from pooled or- of lysate of Vero cell cultures infected with the Musoke gans were uniformly negative. Applying an ELISA cutoff strain of MARV. Bat antibody was detected with antibat value of 16.4 PP, determined as 3 × (mean + 3 SD) of val- immunoglobulin–horseradish peroxidase conjugate (Beth- ues recorded for 188 bat serum samples from Kruger Na- yl, Montgomery, AL, USA) and rodent antibody with an- tional Park, antibody activity to MARV was detected by timouse immunoglobulin conjugate (Zymed Laboratories, ELISA in 20 (9.7%) of 206 Rh. eloquens and in 32 (20.5%) San Francisco, CA, USA). Net ELISA optical density val- of 156 R. aegyptiacus serum specimens from Durba (Table; ues were expressed as percent positivity (PP) of a human 1848 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007 Studies of Reservoir Hosts for Marburg Virus Table. Results from Marburg virus testing of specimens collected in Durba, northeastern Democratic Republic of the Congo, May and October 1999 Marburg ELISA Marburg nested VP35 Total no. antibody, no. Filovirus L RT-PCR RT-PCR, no. Species sampled positive/no. tested (%) and virus isolation positive/no. tested (%) Chiroptera: Microchiroptera Hipposideros caffer 13 0/10 0/13 0/7 H. commersoni 17 0/16 0/17 0/13 Miniopterus inflatus 38 0/34 0/38 1/33 (3.0) Nycteris hispida 1 0/1 0/1 0/1 Rhinolophus eloquens 222 20/206 (9.7) 0/222 7/197 (3.6) Rh. landeri 10/1 Chiroptera: Megachiroptera Lissonycteris angolensis 3 0/3 0/3 0/3 Rousettus aegyptiacus 230 32/156 (20.5) 0/230 4/127 (3.1) Rodentia Lemniscomys striatus 10 0/10 0/10 Lophuromys sikapusi 20/2 0/2 Mastomys natalensis 4 0/4 0/4 0/1 Mus (Nannomys) minutoides 11 0/11 0/11 0/2 Praomys delectorum 14 0/14 0/14 0/4 Taterillus emini 1 0/1 0/1 0/1 Rattus norvegicus 5 0/5 0/5 0/1 Insectivora: Sorcidae (Crocidura spp.) 3 0/3 0/3 Amphibia: Anura (unidentified frog) 1 0/1 0/1 Arthropoda: Crustacea (unidentified crab) 4 0/4 0/4 Arthropoda: Hexapoda, Arachnida* ≈2,000 0/22† *Cockroaches, crickets, spiders, wasps, moth flies, streblids, nycteribiids, mites. †Pooled specimens. Figure 1). Prevalence of nucleic acid or antibody did not VP35 gene fragments detected in the Durba patients and differ signifi cantly between male and female bats or adults bats are representative of the diversity of the complete and juveniles (determined on the basis of body mass) or MARV genome and encompass the entire genetic spectrum between bats collected in May and October. The only RT- of isolates obtained over the past 40 years (1,4). This fact PCR–positive bat that had antibody was a Rh. eloquens indicates that the virus evolves slowly and that any possible male collected in October. All other investigations pro- relationship with bats in the Goroumbwa Mine must have duced negative results. extended over a long period. The diversity of MARV se- Phylogenetic analysis of the sequences determined for quences detected suggests compartmentalized circulation of the twelve 302-nt MARV VP35 gene fragments amplifi ed from bat specimens (GenBank accession nos. EU11794– EU118805) showed that 6 corresponded to sequences pre- viously determined for virus isolates from humans during the epidemic (1), 1 corresponded to a 1975 human isolate from Zimbabwe, and the remaining 5 represented novel se- quences; these last 6 variants from bats, combined with the 9 variants from humans, make a total of 15 distinct MARV sequences found to have been in circulation during the Durba epidemic (Figure 2). Although the differences ob- served between MARV sequences during the 1999 Durba outbreak were minor, the sequences were consistent in se- quential isolates from individual patients and within groups of epidemiologically linked patients (e.g., intrafamilial transmission). In addition, phylogenetic analysis on L gene Figure 1. Marburg virus ELISA percent positivity (PP) values recorded on bat serum samples collected in 1999 in Durba, fragment sequences showed that the 33 virus isolates from Democratic Republic of the Congo (n = 426), and from 1984 through patients resolved into exactly the same 9 groups as did the 1994 in Kruger National Park, South Africa (n = 188). The cutoff PP VP35 gene fragments of the same isolates (1). Nucleotide value of 16.4 was fi xed as 3 × (mean + 3 SD) of values observed in sequence divergences of up to 21% observed among the the Kruger National Park samples. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007 1849 RESEARCH separate incidents in 1980 and 1987, infection with MARV was putatively linked with entry into Kitum Cave on the slopes of Mount Elgon in Kenya, where fruit and insectivo- rous bats are present (8,9). In 1994, a clan of chimpanzees in a forest reserve in Côte d’Ivoire had been observed feed- ing in a wild fi g tree with fruit bats for 2 weeks before an outbreak of fatal disease, caused by a new strain of Ebola vi- rus, occurred (10). The Reston strain of Ebola virus, which is apparently nonpathogenic for humans, was imported into the United States and Europe in infected monkeys from the Philippines; on each occasion, the animals came from a holding facility where they were potentially exposed to the excretions of large numbers of fruit bats (11). The circumstantial evidence in the Marburg hemor- rhagic fever outbreak in Durba strongly implicates Gor- oumbwa Mine as the source of human infection. At least 9 genetic variants of MARV circulated in humans during the outbreak. And because laboratory testing was limited to a few patients, additional variants could have been un- detected, as substantiated by our evidence of 6 more vari- ants in bats. The evolution and perpetuation of multiple genetic variants of virus in a fi xed location would require a suitably large reservoir host population with constant re- Figure 2. Phylogenetic analysis created by using a neighbor-joining cruitment through reproduction or migration of susceptible algorithm (MEGA version 3.1, [3]) that related sequences of 302-nt individuals, as generally occurs in small vertebrate and in- fragments of Marburg viral protein 35 gene detected in 12 bats in vertebrate populations such as the bat population of Gor- Durba Mine (boldface) to sequences determined for isolates from human patients in the Durba plus previous outbreaks of the disease. oumbwa Mine. Failure to isolate live virus may be because Six bat-derived sequences were identical to sequences from human it was present in very low concentrations, either early or isolates during the outbreak; 1 corresponded to a 1975 human late in the course of infection. This was the fi rst detection of isolate from Zimbabwe, and the remaining 5 represented novel fi lovirus nucleic acid and antibody in bats, a phenomenon sequences, making a total of 15 distinct MARV sequences found to which was subsequently demonstrated with Ebola virus be in circulation during the Durba epidemic. Bootstrap values were determined by 500 replicates. DRC, Democratic Republic of the and MARV nucleic acids and antibodies in fruit bats col- Congo; GER, Germany; KEN, Kenya; ZIM, Zimbabwe. lected in 2002 and 2005 in Gabon, where it again proved impossible to isolate live virus (12,13). The nature of fi lovirus infection in bats may vary with age and reproductive status. A seasonal pattern in the oc- virus in bat colonies, as would occur if the species involved currence of human disease was noted over the 2 years of existed as metapopulations, spatially discrete subgroups of the epidemic in Durba; transmission began in October–No- the same species, as opposed to panmictic populations in vember and peaked in January–February (1). In the caves of which there are no mating restrictions (5). Alternatively, Mount Elgon in Kenya, Egyptian fruit bats breed in March bats could be intermediate hosts of the virus. and September; at other sites in Kenya, the timing varies The history of fi lovirus outbreaks shows several in- markedly; no data are available for the Durba area (14). The stances from which it can be inferred that bats may have remaining species of bats found in Goroumbwa Mine breed served as the source of infection. Anecdotal evidence in- annually, but details for this location are unknown. Thus, dicates that during shipment from Uganda, the monkeys although the reproductive status of bats differed in May and associated with the fi rst outbreak of Marburg hemorrhagic October, evidence is insuffi cient to establish a clear link fever in Europe in 1967 were kept in a holding facility on between breeding patterns of bats in Goroumbwa Mine and a Lake Victoria island that had large numbers of fruit bats. the occurrence of Marburg hemorrhagic fever. Neverthe- In the second fi lovirus outbreak in 1975, Marburg hemor- less, many examples in human and veterinary medicine rhagic fever developed in 2 tourists who had slept in rooms indicate that the outcome of virus infection, development with insectivorous bats at 2 locations in Zimbabwe (6). In of carrier status, and shedding of virus are infl uenced by the fi rst recognized outbreak of Ebola hemorrhagic fever age and reproductive status, including stage of gestation at in 1976, the fi rst 6 patients had worked in a cotton factory which infection occurs and the conferral to and duration of in Sudan in which insectivorous bats were present (7). In 2 1850 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007 Studies of Reservoir Hosts for Marburg Virus 5. Calisher CH, Childs JE, Field HE, Holmes KE, Schountz T. Bats: maternal immunity in progeny (15). Likewise, whether in- important reservoir hosts of emerging viruses. Clin Microbiol Rev. sectivorous bats, fruit bats, or both, are likely to serve as the 2006;19:531–45. primary source of infection and whether particular species 6. Conrad JL, Isaacson M, Smith EB, Wulff H, Crees M, Geldenhuys P, are involved with secondary transmission of infection to et al. Epidemiologic investigation of Marburg virus disease, South- ern Africa, 1975. Am J Trop Med Hyg. 1978;27:1210–5. other species is unclear. The evolutionary distinction may 7. Arata AA, Johnson B. Approaches towards studies on potential exist between cave-roosting bats as hosts of MARV and reservoirs of viral haemorrhagic fever in Southern Sudan (1977). forest bats as hosts of Ebola virus. Moreover, the ultimate In: Pattyn SR, editor. Ebola virus haemorrhagic fever. Amsterdam: source of infection could prove to be external, such as bat Elsevier/North Holland Biomedical Press; 1978. p. 134–9. 8. Smith DH, Johnson BK, Isaacson M, Swanepoel R, Johnson KM, parasites or seasonally active insects in the bats’ diet. Ex- Kiley M, et al. Marburg-virus disease in Kenya. Lancet. 1982;1: perimental infections in colonized bats could answer some 816–20. of these questions (16). 9. Johnson ED, Johnson BK, Silverstein D, Tukei P, Geisbert TW, Sanchez AN, et al. Characterization of a new Marburg virus iso- lated from a 1987 fatal case in Kenya. Arch Virol Suppl. 1996;11: Acknowledgments 101–14. We are indebted to the administration of the Kilo-Moto 10. Formenty P, Boesch C, Wyers M, Steiner C, Donati F, Dind F, et al. (OKIMO) mining company in Durba for their assistance and to T. Ebola outbreak in wild chimpanzees living in a rainforest of Côte Kearney, Pretoria, South Africa, for help in identifying bats. d’Ivoire. J Infect Dis. 1999;179(Suppl 1):S120–6. 11. Miranda ME, Ksiazek TG, Retuya TJ, Khan AS, Sanchez A, Ful- The project was supported by the Department of Communi- horst CF, et al. Epidemiology of Ebola (subtype Reston) virus in the cable Disease Surveillance and Response, World Health Organi- Philippines, 1996. J Infect Dis. 1999;179(Suppl 1):S115–9. 12. Leroy EM, Kumulungui B, Pourrut X, Rouquet P, Hassanin A, Yaba zation, Geneva. P, et al. Fruit bats as reservoirs of Ebola virus. Nature. 2005;438: 575–6. Dr Swanepoel is a consultant in the Special Pathogens Unit, 13. Towner JS, Pourrut X, Albarino CG, Nkogue CN, Bird BH, Grard National Institute for Communicable Diseases, South Africa, with G, et al. Marburg virus infection detected in a common African bat. a special interest in fi lovirus ecology. PLoS ONE. 2007;2:e764. 14. Kingdon J. East African mammals. Vol. 2A. Insectivores and bats. Chicago: University of Chicago Press; 1984. References 15. Hyatt AD, Daszak P, Cunningham AA, Field H, Gould AR. Henipa- viruses: gaps in the knowledge of emergence. EcoHealth. 2004;1: 1. Bausch DG, Nichol ST, Muyembe-Tamfum JJ, Borchert M, Rollin 25–38. PE, Sleurs H, et al. Marburg hemorrhagic fever associated with mul- 16. Swanepoel R, Leman LA, Burt FJ, Zachariades NA, Braack LEO, tiple genetic lineages of virus. N Engl J Med. 2006;355:909–19. Ksiazek TG, et al. Experimental inoculation of plants and animals 2. Venter M, Smit S, Leman P, Swanepoel R. Phylogenetic evidence with Ebola virus. Emerg Infect Dis. 1996;2:321–5. of widespread distribution of genotype 3 JC virus in Africa and identifi cation of a type 7 isolate in an AIDS patient. J Gen Virol. Address for correspondence: Robert Swanepoel, National Institute for 2004;85:2215–9. Communicable Diseases, Private Bag X4, Sandringham 2131, South 3. Kumar S, Tamura K, Nei M. MEGA3: integrated software for mo- lecular evolutionary genetics analysis and sequence alignment. Brief Africa; email: [email protected] Bioinform. 2004;5:150–63. The opinions expressed by authors contributing to this journal do 4. Towner JS, Khristova ML, Sealy TK, Vincent MJ, Erickson BR, Bawiec DA, et al. Marburgvirus genomics and association with not necessarily refl ect the opinions of the Centers for Disease Con- trol and Prevention or the institutions with which the authors are a large hemorrhagic fever outbreak in Angola. J Virol. 2006;80: 6497–516. affi liated. The Public Health Image Library (PHIL) The Public Health Image Library (PHIL), Centers for Disease Control and Prevention, contains thousands of public health-related images, including high-resolution (print quality) photographs, illustrations, and videos. PHIL collections illustrate current events and articles, supply visual content for health promotion brochures, document the effects of disease, and enhance instructional media. PHIL Images, accessible to PC and Macintosh users, are in the public domain and available without charge. Visit PHIL at http://phil.cdc.gov/phil. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007 1851 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Emerging Infectious Diseases Pubmed Central

Studies of Reservoir Hosts for Marburg Virus

Download PDF
5 pages

Loading next page...
 
/lp/pubmed-central/studies-of-reservoir-hosts-for-marburg-virus-JlREIF4wNt

References (34)

Publisher
Pubmed Central
ISSN
1080-6040
eISSN
1080-6059
DOI
10.3201/eid1312.071115
Publisher site
See Article on Publisher Site

Abstract

Studies of Reservoir Hosts for Marburg Virus Robert Swanepoel,* Sheilagh B. Smit,* Pierre E. Rollin,† Pierre Formenty,‡ Patricia A. Leman,* Alan Kemp,* Felicity J. Burt,§ Antoinette A. Grobbelaar,* Janice Croft,* Daniel G. Bausch,¶ Hervé Zeller,# Herwig Leirs,** †† L.E.O. Braack,‡‡ Modeste L. Libande,§§ Sherif Zaki,† Stuart T. Nichol,† Thomas G. Ksiazek,† and Janusz T. Paweska,* on behalf of the International Scientifi c and Technical Committee for Marburg Hemorrhagic Fever Control in the Democratic Republic of the Congo To determine reservoir hosts for Marburg virus (MARV), 1998 through September 2000. The outbreak involved 154 we examined the fauna of a mine in northeastern Demo- patients (48 confi rmed and 106 suspected cases); the case- cratic Republic of the Congo. The mine was associated with fatality ratio was 83% (1). Primary cases occurred in young a protracted outbreak of Marburg hemorrhagic fever during male miners and spread as secondary cases to family mem- 1998–2000. We found MARV nucleic acid in 12 bats, com- bers and, less frequently, to healthcare workers and others prising 3.0%–3.6% of 2 species of insectivorous bat and 1 in the community. Most cases occurred in Durba, but a few species of fruit bat. We found antibody to the virus in the se- secondary cases occurred elsewhere, including nosocomial rum of 9.7% of 1 of the insectivorous species and in 20.5% infections in nearby Watsa village, where severely ill pa- of the fruit bat species, but attempts to isolate virus were tients sought care. The occurrence of sporadic cases and unsuccessful. short chains of human-to-human transmission suggested that infection had been repeatedly introduced into the hu- arburg virus (MARV) and Ebola virus, members of man population; this suggestion was substantiated by the Mthe family Filoviridae, cause outbreaks of severe detection of at least 9 genetically distinct viruses circulat- hemorrhagic fever in Africa. Although humans have on ing during the outbreak. Identical sequences of MARV occasion acquired infection from contact with tissues of were found in patients within but not across clusters of diseased nonhuman primates and other mammals, the res- epidemiologically linked cases, although viruses with the ervoir hosts of the viruses in nature remain unknown. same sequences reappeared at irregular intervals during the An outbreak of Marburg hemorrhagic fever ran a pro- outbreak. Most (94%) affected miners worked underground tracted course in the gold-mining village of Durba, north- in Goroumbwa Mine, rather than in the 7 opencast mines eastern Democratic Republic of the Congo, from October in the village. Cessation of the outbreak coincided with the fl ooding of Goroumbwa Mine. Interviews with long-term *National Institute for Communicable Diseases, Sandringham, Re- residents and healthcare workers and review of hospital public of South Africa; †Centers for Disease Control and Preven- records showed that a syndrome hémorragique de Durba tion, Atlanta, Georgia, USA; ‡World Health Organization, Geneva, [hemorrhagic syndrome of Durba] had been associated Switzerland; §University of the Free State, Bloemfontein, South Af- with the mine since at least 1987, and a survivor of a 1994 rica; ¶Tulane School of Public Health and Tropical Medicine, New outbreak was found to have antibodies against MARV. The Orleans, Louisiana, USA; #Institut Pasteur, Lyon, France; **Uni- fauna of Goroumbwa Mine included bats, rodents, shrews, versity of Antwerp, Antwerp, Belgium; ††University of Aarhus, Kon- frogs, snakes, cockroaches, crickets, spiders, wasps, and gens Lyngby, Denmark; ‡‡Conservation International, Cape Town, moth fl ies (1). We present the results of virus reservoir host South Africa; and §§Department of Health, Watsa, Democratic Re- studies conducted during the outbreak. public of the Congo Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007 1847 RESEARCH serum sample confi rmed positive for MARV and used as an Methods internal control. Cutoff values for recording positive results In parallel with human epidemiologic studies, visits were deliberately selected to be stringent at 3 × (mean + were made to Durba in May and October 1999 to collect 3SD) PP values determined for stored bat (n = 188) and specimens for virus ecostudies. The ecostudies were ap- rodent (n = 360) serum samples that had been collected for proved by the International Scientifi c and Technical Com- unrelated purposes in Kruger National Park, South Africa, mittee for Marburg Hemorrhagic Fever Control, which was from 1984 through 1994, and tested at a dilution of 1:100. coordinated by the World Health Organization on behalf of The Kruger bat samples were collected from 3 species of the government of the Democratic Republic of the Congo. fruit bats (Megachiroptera) and 12 species of insectivorous In view of the epidemiologic fi ndings during the outbreak, bats (Microchiroptera), including samples from 56 Chae- emphasis was placed on the fauna of Goroumbwa Mine. rephon pumila, 32 Rousettus aegyptiacus, 27 Mops con- Bats were caught with mist nets at mine entrances; rodents dylurus, 16 Hipposideros caffer, plus 57 samples from 11 and shrews were caught live with Sherman traps within and other species. close to the mine; and arthropods (cockroaches, crickets, spiders, wasps, and moth fl ies, plus streblid, nycteribiid, Results and Discussion and mite parasites of bats) were collected by hand or with The numbers of specimens collected, plus the results sweepnets. Vertebrates were euthanized and dissected on of RT-PCR, nested PCR, attempts to isolate virus in cell site. Blood samples were collected; and samples of liver, culture, and ELISA antibody determinations, are summa- lung, spleen, kidney, testes, brain, salivary glands, and fe- rized in the Table. With the exception of a Nycteris hispida tuses of pregnant females were preserved along with the bat, which was caught near a house in Durba, all specimens arthropods in liquid nitrogen dry-shipping containers for were collected within Goroumbwa Mine or its immediate transport to the National Institute for Communicable Dis- surroundings. An estimated minimum of 10,000 Egyptian eases in South Africa. Extra liver samples were collected fruit bats (R. aegyptiacus) roosted in the mine, clustered for phylogenetic studies on bats and rodents, and formalin- within the upper galleries. Although the numbers of insec- fi xed tissue samples were kept for possible histopathologic tivorous bats were diffi cult to estimate because these bats and immunohistochemical examination. Carcasses were roosted mainly in the deeper recesses of the mine, the catch fi xed in formalin for α-taxonomy purposes. rates indicated substantial numbers of the eloquent horse- Vertebrate tissue and arthropod suspensions were shoe bat (Rhinolophus eloquens) and the greater long-fi n- processed and tested for fi lovirus nucleic acids by reverse gered bat (Miniopterus infl atus). Few microchiropterans transcription–PCR (RT-PCR) and nested PCR by us- were caught in May, but catch rates improved in October ing fi lovirus-specifi c large (L) protein gene primers and after adjustment of trapping hours and the gauge of mist nested MARV-specifi c viral protein 35 (VP35) primers nets used. Pregnancy was recorded in 12 (24%) of 50 R. as described for samples from human patients during the aegyptiacus females in May and in 2 (4.2%) of 47 females outbreak (1). Nucleotide sequencing of amplicons and se- in October; descended testes were found in 2 (6%) of 33 quence data analysis were also performed as described pre- males in May and 19 (25%) of 76 in October. The only in- viously (2), except that MEGA version 3.1 software was dication of breeding activity observed in microchiropterans used (3). Initial RT-PCR and nested PCR were performed was that 1/7 Rh. eloquens females was pregnant in May. with pooled tissue samples of individual vertebrates; when The L primer RT-PCR, which was applied to all speci- possible, for specimens that produced positive results, all mens, produced no positive result. In contrast, the nested tissues were retested separately. In attempts to isolate virus MARV VP35 PCR, which was applied only to specimens as detected by indirect immunofl uorescence, suspensions collected in October 1999, produced positive results on (≈10%) of vertebrate tissues pooled for individual animals specimens from 12 bats: 1 (3.0%) of 33 M. infl atus, 7 and arthropods pooled by species were subjected to 3 se- (3.6%) of 197 Rh. eloquens, and 4 (3.1%) of 127 R. ae- rial passages in Vero 76 cell cultures. Serum samples from gyptiacus. Nested VP35 PCR on individual tissues of the bats and rodents were tested for antibody to MARV by positive bats produced positive results for liver, spleen, ELISA by using a modifi cation of the technique described kidney, lung, salivary gland (3/5 bats), and heart (2/5 bats). previously for human serum (1). ELISA antigen consisted Attempts to isolate virus in cell cultures from pooled or- of lysate of Vero cell cultures infected with the Musoke gans were uniformly negative. Applying an ELISA cutoff strain of MARV. Bat antibody was detected with antibat value of 16.4 PP, determined as 3 × (mean + 3 SD) of val- immunoglobulin–horseradish peroxidase conjugate (Beth- ues recorded for 188 bat serum samples from Kruger Na- yl, Montgomery, AL, USA) and rodent antibody with an- tional Park, antibody activity to MARV was detected by timouse immunoglobulin conjugate (Zymed Laboratories, ELISA in 20 (9.7%) of 206 Rh. eloquens and in 32 (20.5%) San Francisco, CA, USA). Net ELISA optical density val- of 156 R. aegyptiacus serum specimens from Durba (Table; ues were expressed as percent positivity (PP) of a human 1848 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007 Studies of Reservoir Hosts for Marburg Virus Table. Results from Marburg virus testing of specimens collected in Durba, northeastern Democratic Republic of the Congo, May and October 1999 Marburg ELISA Marburg nested VP35 Total no. antibody, no. Filovirus L RT-PCR RT-PCR, no. Species sampled positive/no. tested (%) and virus isolation positive/no. tested (%) Chiroptera: Microchiroptera Hipposideros caffer 13 0/10 0/13 0/7 H. commersoni 17 0/16 0/17 0/13 Miniopterus inflatus 38 0/34 0/38 1/33 (3.0) Nycteris hispida 1 0/1 0/1 0/1 Rhinolophus eloquens 222 20/206 (9.7) 0/222 7/197 (3.6) Rh. landeri 10/1 Chiroptera: Megachiroptera Lissonycteris angolensis 3 0/3 0/3 0/3 Rousettus aegyptiacus 230 32/156 (20.5) 0/230 4/127 (3.1) Rodentia Lemniscomys striatus 10 0/10 0/10 Lophuromys sikapusi 20/2 0/2 Mastomys natalensis 4 0/4 0/4 0/1 Mus (Nannomys) minutoides 11 0/11 0/11 0/2 Praomys delectorum 14 0/14 0/14 0/4 Taterillus emini 1 0/1 0/1 0/1 Rattus norvegicus 5 0/5 0/5 0/1 Insectivora: Sorcidae (Crocidura spp.) 3 0/3 0/3 Amphibia: Anura (unidentified frog) 1 0/1 0/1 Arthropoda: Crustacea (unidentified crab) 4 0/4 0/4 Arthropoda: Hexapoda, Arachnida* ≈2,000 0/22† *Cockroaches, crickets, spiders, wasps, moth flies, streblids, nycteribiids, mites. †Pooled specimens. Figure 1). Prevalence of nucleic acid or antibody did not VP35 gene fragments detected in the Durba patients and differ signifi cantly between male and female bats or adults bats are representative of the diversity of the complete and juveniles (determined on the basis of body mass) or MARV genome and encompass the entire genetic spectrum between bats collected in May and October. The only RT- of isolates obtained over the past 40 years (1,4). This fact PCR–positive bat that had antibody was a Rh. eloquens indicates that the virus evolves slowly and that any possible male collected in October. All other investigations pro- relationship with bats in the Goroumbwa Mine must have duced negative results. extended over a long period. The diversity of MARV se- Phylogenetic analysis of the sequences determined for quences detected suggests compartmentalized circulation of the twelve 302-nt MARV VP35 gene fragments amplifi ed from bat specimens (GenBank accession nos. EU11794– EU118805) showed that 6 corresponded to sequences pre- viously determined for virus isolates from humans during the epidemic (1), 1 corresponded to a 1975 human isolate from Zimbabwe, and the remaining 5 represented novel se- quences; these last 6 variants from bats, combined with the 9 variants from humans, make a total of 15 distinct MARV sequences found to have been in circulation during the Durba epidemic (Figure 2). Although the differences ob- served between MARV sequences during the 1999 Durba outbreak were minor, the sequences were consistent in se- quential isolates from individual patients and within groups of epidemiologically linked patients (e.g., intrafamilial transmission). In addition, phylogenetic analysis on L gene Figure 1. Marburg virus ELISA percent positivity (PP) values recorded on bat serum samples collected in 1999 in Durba, fragment sequences showed that the 33 virus isolates from Democratic Republic of the Congo (n = 426), and from 1984 through patients resolved into exactly the same 9 groups as did the 1994 in Kruger National Park, South Africa (n = 188). The cutoff PP VP35 gene fragments of the same isolates (1). Nucleotide value of 16.4 was fi xed as 3 × (mean + 3 SD) of values observed in sequence divergences of up to 21% observed among the the Kruger National Park samples. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007 1849 RESEARCH separate incidents in 1980 and 1987, infection with MARV was putatively linked with entry into Kitum Cave on the slopes of Mount Elgon in Kenya, where fruit and insectivo- rous bats are present (8,9). In 1994, a clan of chimpanzees in a forest reserve in Côte d’Ivoire had been observed feed- ing in a wild fi g tree with fruit bats for 2 weeks before an outbreak of fatal disease, caused by a new strain of Ebola vi- rus, occurred (10). The Reston strain of Ebola virus, which is apparently nonpathogenic for humans, was imported into the United States and Europe in infected monkeys from the Philippines; on each occasion, the animals came from a holding facility where they were potentially exposed to the excretions of large numbers of fruit bats (11). The circumstantial evidence in the Marburg hemor- rhagic fever outbreak in Durba strongly implicates Gor- oumbwa Mine as the source of human infection. At least 9 genetic variants of MARV circulated in humans during the outbreak. And because laboratory testing was limited to a few patients, additional variants could have been un- detected, as substantiated by our evidence of 6 more vari- ants in bats. The evolution and perpetuation of multiple genetic variants of virus in a fi xed location would require a suitably large reservoir host population with constant re- Figure 2. Phylogenetic analysis created by using a neighbor-joining cruitment through reproduction or migration of susceptible algorithm (MEGA version 3.1, [3]) that related sequences of 302-nt individuals, as generally occurs in small vertebrate and in- fragments of Marburg viral protein 35 gene detected in 12 bats in vertebrate populations such as the bat population of Gor- Durba Mine (boldface) to sequences determined for isolates from human patients in the Durba plus previous outbreaks of the disease. oumbwa Mine. Failure to isolate live virus may be because Six bat-derived sequences were identical to sequences from human it was present in very low concentrations, either early or isolates during the outbreak; 1 corresponded to a 1975 human late in the course of infection. This was the fi rst detection of isolate from Zimbabwe, and the remaining 5 represented novel fi lovirus nucleic acid and antibody in bats, a phenomenon sequences, making a total of 15 distinct MARV sequences found to which was subsequently demonstrated with Ebola virus be in circulation during the Durba epidemic. Bootstrap values were determined by 500 replicates. DRC, Democratic Republic of the and MARV nucleic acids and antibodies in fruit bats col- Congo; GER, Germany; KEN, Kenya; ZIM, Zimbabwe. lected in 2002 and 2005 in Gabon, where it again proved impossible to isolate live virus (12,13). The nature of fi lovirus infection in bats may vary with age and reproductive status. A seasonal pattern in the oc- virus in bat colonies, as would occur if the species involved currence of human disease was noted over the 2 years of existed as metapopulations, spatially discrete subgroups of the epidemic in Durba; transmission began in October–No- the same species, as opposed to panmictic populations in vember and peaked in January–February (1). In the caves of which there are no mating restrictions (5). Alternatively, Mount Elgon in Kenya, Egyptian fruit bats breed in March bats could be intermediate hosts of the virus. and September; at other sites in Kenya, the timing varies The history of fi lovirus outbreaks shows several in- markedly; no data are available for the Durba area (14). The stances from which it can be inferred that bats may have remaining species of bats found in Goroumbwa Mine breed served as the source of infection. Anecdotal evidence in- annually, but details for this location are unknown. Thus, dicates that during shipment from Uganda, the monkeys although the reproductive status of bats differed in May and associated with the fi rst outbreak of Marburg hemorrhagic October, evidence is insuffi cient to establish a clear link fever in Europe in 1967 were kept in a holding facility on between breeding patterns of bats in Goroumbwa Mine and a Lake Victoria island that had large numbers of fruit bats. the occurrence of Marburg hemorrhagic fever. Neverthe- In the second fi lovirus outbreak in 1975, Marburg hemor- less, many examples in human and veterinary medicine rhagic fever developed in 2 tourists who had slept in rooms indicate that the outcome of virus infection, development with insectivorous bats at 2 locations in Zimbabwe (6). In of carrier status, and shedding of virus are infl uenced by the fi rst recognized outbreak of Ebola hemorrhagic fever age and reproductive status, including stage of gestation at in 1976, the fi rst 6 patients had worked in a cotton factory which infection occurs and the conferral to and duration of in Sudan in which insectivorous bats were present (7). In 2 1850 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007 Studies of Reservoir Hosts for Marburg Virus 5. Calisher CH, Childs JE, Field HE, Holmes KE, Schountz T. Bats: maternal immunity in progeny (15). Likewise, whether in- important reservoir hosts of emerging viruses. Clin Microbiol Rev. sectivorous bats, fruit bats, or both, are likely to serve as the 2006;19:531–45. primary source of infection and whether particular species 6. Conrad JL, Isaacson M, Smith EB, Wulff H, Crees M, Geldenhuys P, are involved with secondary transmission of infection to et al. Epidemiologic investigation of Marburg virus disease, South- ern Africa, 1975. Am J Trop Med Hyg. 1978;27:1210–5. other species is unclear. The evolutionary distinction may 7. Arata AA, Johnson B. Approaches towards studies on potential exist between cave-roosting bats as hosts of MARV and reservoirs of viral haemorrhagic fever in Southern Sudan (1977). forest bats as hosts of Ebola virus. Moreover, the ultimate In: Pattyn SR, editor. Ebola virus haemorrhagic fever. Amsterdam: source of infection could prove to be external, such as bat Elsevier/North Holland Biomedical Press; 1978. p. 134–9. 8. Smith DH, Johnson BK, Isaacson M, Swanepoel R, Johnson KM, parasites or seasonally active insects in the bats’ diet. Ex- Kiley M, et al. Marburg-virus disease in Kenya. Lancet. 1982;1: perimental infections in colonized bats could answer some 816–20. of these questions (16). 9. Johnson ED, Johnson BK, Silverstein D, Tukei P, Geisbert TW, Sanchez AN, et al. Characterization of a new Marburg virus iso- lated from a 1987 fatal case in Kenya. Arch Virol Suppl. 1996;11: Acknowledgments 101–14. We are indebted to the administration of the Kilo-Moto 10. Formenty P, Boesch C, Wyers M, Steiner C, Donati F, Dind F, et al. (OKIMO) mining company in Durba for their assistance and to T. Ebola outbreak in wild chimpanzees living in a rainforest of Côte Kearney, Pretoria, South Africa, for help in identifying bats. d’Ivoire. J Infect Dis. 1999;179(Suppl 1):S120–6. 11. Miranda ME, Ksiazek TG, Retuya TJ, Khan AS, Sanchez A, Ful- The project was supported by the Department of Communi- horst CF, et al. Epidemiology of Ebola (subtype Reston) virus in the cable Disease Surveillance and Response, World Health Organi- Philippines, 1996. J Infect Dis. 1999;179(Suppl 1):S115–9. 12. Leroy EM, Kumulungui B, Pourrut X, Rouquet P, Hassanin A, Yaba zation, Geneva. P, et al. Fruit bats as reservoirs of Ebola virus. Nature. 2005;438: 575–6. Dr Swanepoel is a consultant in the Special Pathogens Unit, 13. Towner JS, Pourrut X, Albarino CG, Nkogue CN, Bird BH, Grard National Institute for Communicable Diseases, South Africa, with G, et al. Marburg virus infection detected in a common African bat. a special interest in fi lovirus ecology. PLoS ONE. 2007;2:e764. 14. Kingdon J. East African mammals. Vol. 2A. Insectivores and bats. Chicago: University of Chicago Press; 1984. References 15. Hyatt AD, Daszak P, Cunningham AA, Field H, Gould AR. Henipa- viruses: gaps in the knowledge of emergence. EcoHealth. 2004;1: 1. Bausch DG, Nichol ST, Muyembe-Tamfum JJ, Borchert M, Rollin 25–38. PE, Sleurs H, et al. Marburg hemorrhagic fever associated with mul- 16. Swanepoel R, Leman LA, Burt FJ, Zachariades NA, Braack LEO, tiple genetic lineages of virus. N Engl J Med. 2006;355:909–19. Ksiazek TG, et al. Experimental inoculation of plants and animals 2. Venter M, Smit S, Leman P, Swanepoel R. Phylogenetic evidence with Ebola virus. Emerg Infect Dis. 1996;2:321–5. of widespread distribution of genotype 3 JC virus in Africa and identifi cation of a type 7 isolate in an AIDS patient. J Gen Virol. Address for correspondence: Robert Swanepoel, National Institute for 2004;85:2215–9. Communicable Diseases, Private Bag X4, Sandringham 2131, South 3. Kumar S, Tamura K, Nei M. MEGA3: integrated software for mo- lecular evolutionary genetics analysis and sequence alignment. Brief Africa; email: [email protected] Bioinform. 2004;5:150–63. The opinions expressed by authors contributing to this journal do 4. Towner JS, Khristova ML, Sealy TK, Vincent MJ, Erickson BR, Bawiec DA, et al. Marburgvirus genomics and association with not necessarily refl ect the opinions of the Centers for Disease Con- trol and Prevention or the institutions with which the authors are a large hemorrhagic fever outbreak in Angola. J Virol. 2006;80: 6497–516. affi liated. The Public Health Image Library (PHIL) The Public Health Image Library (PHIL), Centers for Disease Control and Prevention, contains thousands of public health-related images, including high-resolution (print quality) photographs, illustrations, and videos. PHIL collections illustrate current events and articles, supply visual content for health promotion brochures, document the effects of disease, and enhance instructional media. PHIL Images, accessible to PC and Macintosh users, are in the public domain and available without charge. Visit PHIL at http://phil.cdc.gov/phil. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 12, December 2007 1851

Journal

Emerging Infectious Diseases – Pubmed Central

Published: Dec 1, 2007

There are no references for this article.