Studies of reservoir hosts for Marburg virus

Emerging Infectious Diseases, Dec, 2007 by 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, Herve Zeller, Herwig Leirs, L.E.O. Braack, Modeste L. Libande, Sherif Zaki, Stuart T. Nichol, Thomas G. Ksiazek, Janusz T. Paweska

To determine reservoir hosts for Marburg virus (MARV), we examined the fauna of a mine in northeastern Democratic Republic of the Congo. The mine was associated with a protracted outbreak of Marburg hemorrhagic fever during 1998-2000. We found MARV nucleic acid in 12 bats, comprising 3.0%-3.6% of 2 species of insectivorous bat and 1 species of fruit bat. We found antibody to the virus in the serum of 9.7% of 1 of the insectivorous species and in 20.5% of the fruit bat species, but attempts to isolate virus were unsuccessful.

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Marburg virus (MARV) and Ebola virus, members of the family Filoviridae, cause outbreaks of severe hemorrhagic fever in Africa. Although humans have on occasion acquired infection from contact with tissues of diseased nonhuman primates and other mammals, the reservoir hosts of the viruses in nature remain unknown.

An outbreak of Marburg hemorrhagic fever ran a protracted course in the gold-mining village of Durba, northeastern Democratic Republic of the Congo, from October 1998 through September 2000. The outbreak involved 154 patients (48 confirmed and 106 suspected cases); the case-fatality ratio was 83% (1). Primary cases occurred in young male miners and spread as secondary cases to family members and, less frequently, to healthcare workers and others in the community. Most cases occurred in Durba, but a few secondary cases occurred elsewhere, including nosocomial infections in nearby Watsa village, where severely ill patients sought care. The occurrence of sporadic cases and short chains of human-to-human transmission suggested that infection had been repeatedly introduced into the human population; this suggestion was substantiated by the detection of at least 9 genetically distinct viruses circulating during the outbreak. Identical sequences of MARV were found in patients within but not across clusters of epidemiologically linked cases, although viruses with the same sequences reappeared at irregular intervals during the outbreak. Most (94%) affected miners worked underground in Goroumbwa Mine, rather than in the 7 opencast mines in the village. Cessation of the outbreak coincided with the flooding of Goroumbwa Mine. Interviews with long-term residents and healthcare workers and review of hospital records showed that a syndrome hemorragique de Durba [hemorrhagic syndrome of Durba] had been associated with the mine since at least 1987, and a survivor of a 1994 outbreak was found to have antibodies against MARV. The fauna of Goroumbwa Mine included bats, rodents, shrews, frogs, snakes, cockroaches, crickets, spiders, wasps, and moth flies (1). We present the results of virus reservoir host studies conducted during the outbreak.

Methods

In parallel with human epidemiologic studies, visits were made to Durba in May and October 1999 to collect specimens for virus ecostudies. The ecostudies were approved by the International Scientific and Technical Committee for Marburg Hemorrhagic Fever Control, which was coordinated by the World Health Organization on behalf of the government of the Democratic Republic of the Congo. In view of the epidemiologic findings during the outbreak, emphasis was placed on the fauna of Goroumbwa Mine. Bats were caught with mist nets at mine entrances; rodents and shrews were caught live with Sherman traps within and close to the mine; and arthropods (cockroaches, crickets, spiders, wasps, and moth flies, plus streblid, nycteribiid, and mite parasites of bats) were collected by hand or with sweepnets. Vertebrates were euthanized and dissected on site. Blood samples were collected; and samples of liver, lung, spleen, kidney, testes, brain, salivary glands, and fetuses of pregnant females were preserved along with the arthropods in liquid nitrogen dry-shipping containers for transport to the National Institute for Communicable Diseases in South Africa. Extra liver samples were collected for phylogenetic studies on bats and rodents, and formalin-fixed tissue samples were kept for possible histopathologic and immunohistochemical examination. Carcasses were fixed in formalin for [alpha]-taxonomy purposes.

Vertebrate tissue and arthropod suspensions were processed and tested for filovirus nucleic acids by reverse transcription--PCR (RT-PCR) and nested PCR by using filovirus-specific large (L) protein gene primers and nested MARV-specific viral protein 35 (VP35) primers as described for samples from human patients during the outbreak (1). Nucleotide sequencing of amplicons and sequence data analysis were also performed as described previously (2), except that MEGA version 3.1 software was used (3). Initial RT-PCR and nested PCR were performed with pooled tissue samples of individual vertebrates; when possible, for specimens that produced positive results, all tissues were retested separately. In attempts to isolate virus as detected by indirect immunofluorescence, suspensions ([approximately equal to] 10%) of vertebrate tissues pooled for individual animals and arthropods pooled by species were subjected to 3 serial passages in Vero 76 cell cultures. Serum samples from bats and rodents were tested for antibody to MARV by ELISA by using a modification of the technique described previously for human serum (1). ELISA antigen consisted of lysate of Vero cell cultures infected with the Musoke strain of MARV. Bat antibody was detected with antibat immunoglobulin--horseradish peroxidase conjugate (Bethyl, Montgomery, AL, USA) and rodent antibody with antimouse immunoglobulin conjugate (Zymed Laboratories, San Francisco, CA, USA). Net ELISA optical density values were expressed as percent positivity (PP) of a human serum sample confirmed positive for MARV and used as an internal control. Cutoff values for recording positive results were deliberately selected to be stringent at 3 x (mean + 3SD) PP values determined for stored bat (n = 188) and rodent (n = 360) serum samples that had been collected for unrelated purposes in Kruger National Park, South Africa, from 1984 through 1994, and tested at a dilution of 1:100. The Kruger bat samples were collected from 3 species of fruit bats (Megachiroptera) and 12 species of insectivorous bats (Microchiroptera), including samples from 56 Chaerephon pumila, 32 Rousettus aegyptiacus, 27 Mops condylurus, 16 Hipposideros caffer, plus 57 samples from 11 other species.