INTRODUCTION
Sin Nombre virus, a New World hantavirus, was the causative agent for an outbreak of severe respiratory illness with a case fatality initially exceeding 75% of the cases in the southwestern United States in 1993.1,2 Subsequent studies resulted in the discovery of approximately 30 hantaviruses throughout the Americas.2–4 Although retrospective surveys found evidence of hantaviral infection in South America as early as 1985,5,6 the first HPS-associated hantavirus for the region was discovered in 1993 in Brazil, and the first genetic characterization of a South American hantavirus was reported in 1996 following an outbreak of hantavirus pulmonary syndrome (HPS) in southwestern Argentina.6–8 Since then, hundreds of human infections have been reported in Argentina, Brazil, Chile, Paraguay, and Uruguay, with mortality ranging from 30% to 70%.6,9–11 The first hantavirus in Bolivia, Río Mamoré virus (RIOMV), was isolated from a small-eared pygmy rice rat (Oligoryzomys microtis) trapped in 1996.12,13 This virus has not been associated with human disease. In 1997, Laguna Negra virus (LNV), hosted by the small vesper mouse (Calomys laucha), was identified in a patient in Santiago, Chile who had traveled extensively in Bolivia prior to the onset of illness.14,15 In 2000, six HPS cases in southern Bolivia were linked to Bermejo virus, the first association of HPS with this hantavirus.16
In June and July 2002, two men living or working 100–250 km north of Santa Cruz, Bolivia (Figure 1) developed an illness compatible with HPS; one died. Laboratory testing (serology) in Bolivia showed that both patients were infected with a hantavirus. The Bolivian National Center for Tropical Diseases received reports of at least two additional suspected cases of HPS-like illness in the area. Herein, we report the results of an investigation to identify the virus and the rodent reservoir associated with HPS in Santa Cruz Department. We also describe the circumstances surrounding the transmission of hantaviruses to humans in this region, the prevalence of hantavirus infection in rodent populations in the disease endemic area, and the habitat associations of host species. A detailed description of the epidemiology of the human cases and results of a concurrent serosurvey conducted in the same region will be presented separately.
MATERIALS AND METHODS
Ecologic investigation.
From August 16–21, 2002, small mammals were collected from four localities in the Department of Santa Cruz, Bolivia. Three collection sites (Dina marca, Universal Transverse Mercator coordinates (UTM) 20K 0457277 8113921; Las Empalizadas, 20K 0468038 8107777; and La Patria, 20K 0463980 8106647) were near the town of Mineros, approximately 100 km northeast of Santa Cruz de la Sierra (Figure 1). Four habitat types (forest, cultivated sugarcane fields, harvested sugarcane fields, and peridomestic areas) were studied at these sites. The fourth collection locality was near the town of Concepción, (20L0603157 8269311), approximately 250 km northeast of Santa Cruz (Figure 1). Disturbed forest and peridomestic environments were sampled at the fourth location.
Las Empalizadas was the abandoned sugarcane camp where a patient (fatal case) worked for eight days before his illness. Dinamarca was the relocation site for workers from Las Empalizadas after HPS was diagnosed there. La Patria was the closest permanent human settlement to the previously mentioned sugarcane camps. Concepción, the site of the non-fatal case, consisted of peridomestic habitats (adobe houses) and dense forests punctuated by areas cleared for cattle pasture. Processing of rodents followed standardized procedures.17 Rodent carcasses were fixed in 10% formalin and preserved in 70% ethanol for permanent storage in the M. L. Bean Life Science Museum at Brigham Young University (BYU). Genbank accession numbers (AY439000–AY439002) were obtained for the cytochrome b (cyt b) sequences for representatives of the hantavirus antibody-positive rodents, and BYU voucher numbers were assigned for all individuals.
Rodent sequencing and identification.
Total genomic DNA was extracted from liver tissue preserved in 95% ethanol using the DNeasy Tissue Kit (Qiagen, Valencia, CA). The primers in Table 1 were used to amplify and/or obtain the nucleotide sequence of the mitochondrial cyt b gene.18–22
The cyt b gene was amplified using a polymerase chain reaction (PCR) in a master mixture containing 1.0 μL of template DNA (approximate concentration estimated on a 2% agarose gel), 4 μL of dNTPs (1.25 mM), 2 μL of 10× Taq buffer, 0.5 μL of each primer (100 μM), 3 μL of MgCl2 (25 mM), 14 μL of distilled water, and 0.25 μL of Taq polymerase (5 units/μL; Promega Corp., Madison, WI). The following thermal profiles were used for the majority of the PCRs: two minutes at 94°C, 39 cycles of one minute at 94°C, one minute at 45°C, and one minute at 72°C, plus five minutes at 72°C. The annealing temperature was increased from 45°C to 49°C for the three primers designed specifically for this study (Table 1). Sequences were determined using an ABI 570 Genetic Analyzer (Applied Biosystems, Inc., Foster City, CA) and edited and aligned using Sequencher version 4.1.1 (Gene Codes Corp., Ann Arbor, MI).
To confirm the field identifications of the hantavirus antibody-positive rodents, we obtained reference sequences from GenBank for representative genera of sigmodontine rodents and compared sequence data from the cyt b gene of the three voucher specimens analyzed in this study. Missing data were coded as ?. Phylogenetic analyses, including maximum parsimony (MP) and neighbor-joining (NJ), were performed using PAUP*.23 For the maximum parsimony analysis all characters were weighted equally, and we performed a heuristic search with 100 replicates of random taxon addition with tree bisection-reconnection branch swapping. Confidence in nodes was assessed using the bootstrap procedure with 1,000 resampling replicates. Bootstrap values ≥ 70% were considered well-supported.22,24,25
Extraction of viral RNA, reverse transcriptase-PCR (RT-PCR), and sequencing.
Tissues were frozen on dry ice in the field and stored at −70°C until processed. To extract RNA, 50–100 mg of rodent lung or spleen tissue was homogenized with 1 mL of TRIzol (Invitrogen Life Technologies, Frederick, MD). Total RNA was prepared as previously described,26 and the RT-PCR was conducted using an Access RT-PCR kit (Promega Corp.) following the manufacturer’s instructions. Briefly, the RT products were amplified using primers (Table 1) specific for the S segment of the hantavirus genome, and hemi-nested reactions were generated using 1 μL of first-round product.14,26 Purified PCR amplicons were sequenced directly with the Big Dye Sequencing kit (Applied Biosystems, Inc.) and analyzed on an ABI 377 Genetic Analyzer (Applied Biosystems, Inc.). Nucleotide sequences were aligned and analyzed as described in the rodent cyt b analyses.
RESULTS
Ecologic investigation and rodent identification.
Rodent trapping results for each site are listed in Table 2. Relative densities in each habitat and trap site are provided as trap success (numbers of captures per 100 trap nights, where trap nights is the number of traps set multiplied by the number of nights set). It is likely that some of the currently recognized hantavirus rodent host species are composites of cryptic species that are difficult to identify morphologically.18 Therefore, field identifications of the hantavirus antibody-positive rodents were confirmed through phylogenetic analysis of nucleotide sequence data. The entire cyt b gene (1,143 basepairs) was sequenced for BYU 19014 (identified in the field as O. microtis) from the Dinamarca site. This sequence was compared with GenBank cyt b sequences for 18 samples of Oligoryzomys representing eight species. Both NJ and MP analyses associated BYU 19014 with O. microtis (GenBank assession no. U58381). The Bootstrap value for this node was 100%. Cytochrome b sequences were obtained for two samples identified in the field as Calomys callosus (BYU 19015 and 19016, 1,130 and 1,143 basepairs, respectively). These sequences were compared with those of 61 samples of Calomys available from GenBank representing nine species. Both NJ and MP analyses associated BYU 19015 and 19016 with samples identified as C. callosus with 95% nodal support.
Viral RT-PCR and DNA sequencing.
The nucleotide sequence generated from the antibody-positive O. microtis captured in Mineros was 90% similar to those of RIOMV (GenBank accession no. U52137-39). Sequencing of PCR products from two C. callosus captured in Concepción identified a virus that was 87–88% similar to published sequences for LNV. In addition, the topology produced by the NJ analysis shows that the viral RNA amplicons from C. callosus and O. microtis each form a monophyletic cluster with sequences obtained from LNV and RIOMV when compared with sequences from all other hantaviruses known from this region (Figure 2).
DISCUSSION
Viral RNA sequencing demonstrated that one case patient was infected with an LNV-like virus, which implicates a member of the genus Calomys as the reservoir. In addition, the comparison of the viral sequences from the case patient and the C. callosus captured in the same area (> 99% identity) links the rodent and human viruses. This is congruent with the recent collection of hantavirus antibody-positive C. callosus in northern Argentina.27 Calomys laucha is recognized as the primary rodent reservoir for LNV.14 It is not common for a New World hantavirus to be hosted by multiple species of rodents. Spillover is unlikely since the Concepción study site is outside of the recognized range of C. laucha, and no individuals of this species were collected at this location. It is possible that LNV infected the common ancestor to both C. laucha and C. callosus and has remained associated with both species with minimal genetic divergence.
The identification of O. microtis as the host of the only hantavirus detected in the Mineros region at the location of the human case is consistent with the previous recognition of this rodent as the host of RIOMV.12 Unfortunately, no sample was available from the fatal human case for comparison with the rodent virus sequence. Given the phylogenetic similarity of the rodent virus amplicon to that of RIOMV, the geographic proximity of the captured RIOMV PCR-positive rodent to the site where the patient became ill, and the lack of detection of any other hantaviruses in the vicinity of the human case, it appears possible that this variant of RIOMV may be the pathogen responsible for the HPS case in this region. Río Mamoré virus has not been associated with human disease; however, another South American hantavirus (Bermejo virus) was shown to be associated with human disease several years after the host and virus were described.16 Alternatively, trap success, and therefore the number of rodents tested, were much lower in Mineros than Concepción. It is possible that another hantavirus circulating in the area is responsible for human disease, but was undetected by our sampling. Continued human and rodent surveillance are needed to positively identify the virus and host responsible for HPS near Mineros.
The potential for intermittent HPS cases in these regions likely will continue due to the presence of housing that is highly permeable to rodents, and the constant availability of food, water, and shelter for wild rodents in the vicinity of human habitations. These conditions could contribute to disease outbreaks should environmental circumstances lead to a sudden increase in reservoir population density, increased transmission of the virus within the rodent population, or higher levels of virus infection and shedding by the host population.
These data were collected during a case investigation to identify high-risk areas for transmission of hantaviruses to humans. Thus, all sites sampled were somewhat disturbed. Changes in environmental conditions can contribute to an increase in populations of some hantavirus reservoir species and, in turn, an increased risk of human disease.28,29 In this study, the HPS cases occurred in areas of extensive anthropogenic perturbation. Dinamarca was at the boundary of natural forest being cleared to cultivate sugarcane. The study site near Concepción was adjacent to slash and burn deforestation. These disturbances decrease species diversity and simplify local ecosystems, including the rodent assemblages.30,31 Calomys and Oligoryzomys are murid genera that contain species that adapt well to disturbance. In this sense, the situation in Santa Cruz Department is similar to that associated with a recent HPS outbreak in Panama and suggests that environmental perturbations create conditions that favor hantavirus reservoir species and result in increased risk for HPS.32 In Santa Cruz Department, long-term sampling of these rodent populations could be used to test the hypothesized relationship between decreasing biodiversity and increasing numbers of hantavirus-infected rodents. These studies should include the sampling of disturbed and undisturbed habitats with comparisons of the species composition and prevalence of hantavirus infection in host populations.
Primer sequences used to amplify viral (S-segment) and rodent mitochondrial (cytochrome b) fragments
| Virus primers | |
| Sense strand SS143C-N | 5′-TGG A(C/G)C CIG ATG AIG TTA ACA C-3′ |
| Anti-sense SS743R-N | 5′-TCI ATC CA(G/A) TC(C/T) TTI ACA AA-3′ |
| Sense strand SS283C-N | 5′-CCA ACA GGG ITT GA(A/G) CC(T/A) GAT GA-3′ |
| Anti-sense PPT716R | 5′-AAI CCI ATI ACI CCC AT-3′ |
| Rodent cytochrome b primers | |
| L1472419 | 5′-CGA AGC TTG ATA TGA AAA ACC ATC GTT G-3′ |
| L1464821 | 5′-TGA ATY TGA GGR GGC TTC TCA GTA-3′ |
| 700L18 | 5′-CCC CAG CAC ATA TTA AAC CAG AAT G-3′ |
| F120 | 5′-TGA GGA CAR ATA TCH TTY TGR GG-3′ |
| H1591519 | 5′-AAC TGC AGT CAT CTC CGG TTT ACA AGA C-3′ |
| H1514919 | 5′-AAA CTG CAG CCC CTC AGA ATG ATA TTT GTC CTC A-3′ |
| CBH322 | 5′-GGC AAA TAG GAA RTA TCA TTC-3′ |
| L738-Calomys | 5′-CCC AGA TGT GCT CGG AGA CCC-3′ |
| H738-Calomys | 5′-GGG TCT CCG AGC ACA TCT GGG-3′ |
| H1105-Calomys | 5′-GGA TAT CAT TCT CAA TTA TGC TGG C-3′ |
Sites and habitats of rodent collections in Santa Cruz Province, Bolivia, August 2002*
| Mineros; Dinamarca August 16–17, 2002 | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| General habitat | T | A sp | B sp | Cc | Or sp | Olm | O(i) | Hs | TC | TS |
| Peridomestic | 99 | 0 | 0 | 0 | 0 | 2 13 | 1 | 0 | 3 | 3.0% |
| Disturbed forest | 198 | 8 | 8 | 1 | 2 | (1/7.7%) | 4 | 1 | 37 | 18.7% |
| Cultivated sugarcane | 40 | 0 | 0 | 1 | 0 | 1 | 0 | 0 | 2 | 5.0% |
| Total | 337 | 8 | 8 | 2 | 2 | 16 | 5 | 1 | 42 | 12.46% |
| Mineros; Las Empalizadas August 2002 | |||||
|---|---|---|---|---|---|
| General habitat | T | A sp | Cc | TC | TS |
| Cleared sugarcane | 97 | 1 | 16 | 17 | 17.5% |
| Cultivated sugarcane | 80 | 5 | 4 | 9 | 11.3% |
| Total | 117 | 6 | 20 | 26 | 14.69% |
| Mineros; La Patria August 17–18, 2002 | ||||||||
|---|---|---|---|---|---|---|---|---|
| General habitat | T | M(m) | A sp | Cc | Or sp | Olm | TC | TS |
| Peridomestic | 226 | 0 | 1 | 1 | 0 | 0 | 2 | 0.9% |
| Disturbed forest | 200 | 1 | 0 | 13 | 1 | 2 | 17 | 8.5% |
| Total | 426 | 1 | 1 | 14 | 1 | 2 | 19 | 4.46% |
| Concepción; August 21–22, 2002 | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| General habitat | TS | A sp | Cc | Cf | Or sp | Olm | O(i) | M(m) | TC | TS |
| * T = number of traps set; A sp = Akodon species; B sp = Bolomys species; Cc = Calomys callosus; Or sp = Oryzomys species; Olm = Oligoryzomys microtis; O(i) = Oxymycterus inca; Hs = Holochilus sciureus; TC = total captures; TS = percent trap success; M(m) = Marmosa murina (field indentification); Cf = Calomys fecundus. Numbers in parentheses are the number of hantavirus-positive individuals/hantavirus antibody prevalence for that species in that habitat. | ||||||||||
| Peridomestic | 110 | 0 | 6 37 | 0 | 5 | 6 | 0 | 0 | 17 | 15.5% |
| Disturbed forest | 480 | 38 | (3/8.1%) | 1 | 28 | 27 | 5 | 1 | 137 | 28.5% |
| Total | 590 | 38 | 43 | 1 | 33 | 33 | 5 | 1 | 154 | 26.10% |
Map of Bolivia depicting areas of epidemiologic and ecologic/rodent reservoir investigations. The two areas investigated were Mineros and Concepción, as indicated by the white crosses.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 1; 10.4269/ajtmh.2005.72.42
Map of Bolivia depicting areas of epidemiologic and ecologic/rodent reservoir investigations. The two areas investigated were Mineros and Concepción, as indicated by the white crosses.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 1; 10.4269/ajtmh.2005.72.42
Map of Bolivia depicting areas of epidemiologic and ecologic/rodent reservoir investigations. The two areas investigated were Mineros and Concepción, as indicated by the white crosses.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 1; 10.4269/ajtmh.2005.72.42
Neighbor-joining tree using 288 basepairs of the N gene from the S segment, depicting the phylogenetic relationship of hantaviruses isolated from central Bolivia compared with Genbank nucleotide sequences from previously identified South American hantavirus species. Sin Nombre virus was used as the outgroup taxon. The values with decimals represent the average absolute genetic divergence of the taxa united by the indicated node. The whole number values are bootstrap values (1,000 replicates) taken from a maximum parsimony tree. Nodes without bootstrap values were collapsed in the parsimony analysis.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 1; 10.4269/ajtmh.2005.72.42
Neighbor-joining tree using 288 basepairs of the N gene from the S segment, depicting the phylogenetic relationship of hantaviruses isolated from central Bolivia compared with Genbank nucleotide sequences from previously identified South American hantavirus species. Sin Nombre virus was used as the outgroup taxon. The values with decimals represent the average absolute genetic divergence of the taxa united by the indicated node. The whole number values are bootstrap values (1,000 replicates) taken from a maximum parsimony tree. Nodes without bootstrap values were collapsed in the parsimony analysis.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 1; 10.4269/ajtmh.2005.72.42
Neighbor-joining tree using 288 basepairs of the N gene from the S segment, depicting the phylogenetic relationship of hantaviruses isolated from central Bolivia compared with Genbank nucleotide sequences from previously identified South American hantavirus species. Sin Nombre virus was used as the outgroup taxon. The values with decimals represent the average absolute genetic divergence of the taxa united by the indicated node. The whole number values are bootstrap values (1,000 replicates) taken from a maximum parsimony tree. Nodes without bootstrap values were collapsed in the parsimony analysis.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 1; 10.4269/ajtmh.2005.72.42
Authors’ addresses: Darin S. Carroll, James N. Mills, Joel M. Montgomery, Stuart T. Nichol, and Thomas G. Ksiazek, Special Pathogens Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Mailstop A-26, 1600 Clifton Road NE, Atlanta, GA 30333, E-mail: dcarroll@cdc.gov. Daniel G. Bausch, Tulane School of Public Health and Tropical Medicine, SL-17, J. B. Johnston Building, Room 511, 1430 Tulane Avenue, New Orleans, LA 70112. Patrick J. Blair, James P. Burans, Vidal Felices, James G. Olson, and Milagros Salazar, U.S. Navy, Medical Research Detachment Lima, Peru, Unit Number 3800, American Embassy, APO AA 34031–3800. Alberto Gianella and Naomi Iihosi, Centro Nacional de Enfermedades Tropicales, Bolivian National Center for Tropical Diseases, 2do. Anillo esq. Av. Centenario, Casilla 2974, Santa Cruz de la Sierra, Bolivia. Duke S. Rogers, 401 WIDB, Department of Integrative Biology and M. L. Bean Life Science Museum, Brigham Young University, Provo, UT 84602–5181.
Acknowledgments: We are grateful for technical help provided by Rosa Rios and Tania Garron (Bolivian National Center for Tropical Diseases) and Roger Castillos (Naval Medical Research Center Detachment, Lima, Peru), as well as administrative support from Gloria Talledo and Carolina Guevara (Naval Medical Research Center Detachment, Lima, Peru).
Financial support: This work was supported in part by The Department of Defense Global Emerging Infectious Surveillance Program and Brigham Young University.
Disclaimer: The views expressed in this article are those of the authors and do not reflect the official policy or position of the Department of Defense, the Department of the Navy, or the U.S. Government.
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