Emerg Infect DisEmerging Infect. DisEIDEmerging Infectious Diseases1080-60401080-6059Centers for Disease Control and Prevention22607717335814311-094810.3201/eid1806.110948DispatchDispatchMolecular Epidemiology of Laguna Negra Virus, Mato Grosso State, BrazilLaguna Negra Virus, BrazilTravassos da RosaElizabeth S.MedeirosDaniele B.A.NunesMárcio R.T.SimithDarlene B.PereiraArmando de S.ElkhouryMauro R.SantosElizabeth DaviLavocatMaríliaMarquesAparecido A.ViaAlba V.G.KohlVânia A.TerçasAna C.P.D`AndreaPauloBonvícinoCibele R.Sampaio de LemosElba R.VasconcelosPedro F.C.Evandro Chagas Institute, Ministry of Health, Ananindeua, Brazil (E.S. Travassos da Rosa, D.B. Medeiros, M.R. Nunes, D.B. Simith, A.S. Pereira, P.F. Vasconcelos);National Foundation of Health, Brasília, Brazil (M.R. Elkhoury);Surveillance Health Secretariat, Ministry of Health, Brasília (E.D. dos Santos, M. Lavocat);Mato Grosso State Health Secretariat, Cuiabá, Brazil (A.A. Marques; A.V. Via);Universitary Center Cândido Rondon, Cuiabá (V.A. Kohl);Rural Producers Sindicate, Campo Novo do Parecis, Brazil (A.C. Terças);Oswaldo Cruz Foundation, Rio de Janeiro, Brazil (P. D’andrea, C.R. Bonvicino);National Câncer Institute, Rio de Janeiro (C.R. Bonvicino);Pará State University, Belém, Brazil (P.F. Vasconcelos)Address for correspondence: Pedro F. C. Vasconcelos, Departamento de Arbovirologia e Febres Hemorrágicas. Instituto Evandro Chagas, SVS/MoH. Rodovia BR 316, Km 07, S/Nº , Bairro – Levilândia, 66030-000, Ananindeua, Pará, Brazil; email: pedrovasconcelos@iec.pa.gov.br62012186982985
We associated Laguna Negra virus with hantavirus pulmonary syndrome in Mato Grosso State, Brazil, and a previously unidentified potential host, the Calomys callidus rodent. Genetic testing revealed homologous sequencing in specimens from 20 humans and 8 mice. Further epidemiologic studies may lead to control of HPS in Mato Grosso State.
Hantavirus pulmonary syndrome (HPS) is a manifestation of an emerging zoonosis caused by New World viruses of the family Bunyaviridae, genus Hantavirus. Hantavirus is transmitted to humans by inhalation of aerosols of excreta from infected rodents of the subfamily Sigmodontinae (Rodentia, Cricetidae) (1,2). HPS was initially reported during an epidemic of severe respiratory disease that occurred in the southwestern United States in 1993 (1). HPS was subsequently identified in Brazil and other Latin American countries, which facilitated the recognition of new hantavirus species such as Laguna Negra virus (LNV), Andes virus, Choclo virus, Juquitiba virus, Araraquara virus, Castelo dos Sonhos virus, Anajatuba virus, as well as several other viruses detected in wild rodents which are not associated with HPS (3–9). Like particles of other bunyaviruses, hantavirus particles are spherical or pleomorphic and measure 80–120 nm in diameter; their genome comprises 3 RNA segments, and the small RNA fragment is used to characterize the nucleoprotein (N) gene and the hantavirus species (2).
During 1993–2009, a total of 1,246 cases of HPS were reported in Brazil; the state of Mato Grosso reported the fourth highest case count, diagnosed mainly in the municipalities of Tangará da Serra and Campo Novo do Parecis. However, the circulating hantavirus species and its host remained unknown, and identification of these factors were the main objectives of this study.
The Study
Mato Grosso comprises 903,357.9 km2 and has an estimated population of 2,803,274 inhabitants living in 141 municipalities. Nineteen municipalities have reported cases of HPS, mainly near Brazil’s BR-364 highway, located between the north and southwestern sections of the state. The climate is equatorial subhumid, with an annual rainfall of 1,700 mm, and temperature range 24°–40°C; the landscape consists of savannah (Cerrado) and pre-Amazon rainforest. The economic activities are agricultural production and ecologic tourism.
HPS was diagnosed in 24 persons who were IgM positive for LNV during 2001–2006 in the municipalities of Barra do Bugres (n = 1), Campo Novo do Parecis (n = 13), Diamantino (n = 3), Nova Olímpia (n = 1), Santo Afonso (n = 1), São José do Rio Claro (n = 1), and Tangará da Serra (n = 4) (Figure 1). Detailed information of patient samples submitted for nucleotide sequencing is provided in the Table.
State of Mato Grosso, Brazil, indicating municipalities where hanta pulmonary syndrome cases occurred.
During a 2001 ecological–epidemiologic study conducted in the municipalities of Tangará da Serra and Campo Novo do Parecis, researchers obtained blood and viscera samples from wild rodents (10). The researchers followed Brazilian Institute for the Environment and Renewable Natural Resources guidelines for the capture and handling of rodents and using biosafety level 3 protocols. The samples were tested for hantavirus; animals with positive test results were identified taxonomically by morphometry and molecular analysis of mitochondrial DNA (cytochrome b gene) (10,11).
For hantavirus detection, we conducted reverse transcription PCR to synthesize complementary DNA with generic hantavirus primers as described (1,2). We obtained N gene partial nucleotide sequences by using the Sanger method with the same primers (3,4,6,9). At least 3 amplicons per sample were sequenced in both directions to improve coverage and confidence for results. The obtained sequences were aligned with other hantavirus sequences available at the GenBank database (www.ncbi.nlm.nih.gov) with ClustalW software in BioEdit version 7.1.3 (www.mbio.ncsu.edu/BioEdit/bioedit.html). We implemented the maximum-likelihood and Bayesian methods by using PHYML (www.atgc-montpellier.fr/phyml/versions.php) and Mr. Bayes version 3.2 (http://mrbayes.scs.fsu.edu) software, respectively, for phylogenetic reconstructions. We used Modeltest version 3.7 (http://gel.ahabs.wisc.edu/mauve) to determine the best nucleotide substitution model. We analyzed 2 million replicates, with the sample fixed at every 1,000 trees generated, and used TRACER (www.evolve.zoo.ox.ac.uk) to determine whether the Bayesian analysis reached appropriate convergence (3,6,9,12).
We obtained amplicons from 20 of the 24 samples from persons with HPS and partial sequence of the N gene (≈434 bp) from 16 of the 24 samples from patients who were symptomatic at the time of sampling. During the ecologic study, 126 rodents were captured: 68 (53.9%) commensal synanthropic species, 49 (38.8%) wild rodents [Calomys callidus (n = 46), Proechimys sp. (n = 1), and Necromys lasiurus (n = 2)], and 9 (7.1%) unidentified species. IgG was detected in 8 (17.4%) C. callidus rodents (2 captured in Campo Novo do Parecis, 6 in Tangará da Serra). Amplicons were produced in lung/heart samples from 7 of the 8 IgG-positive rodents; 3 of those were selected for nucleotide sequencing of the N gene (Table).
All strains recovered from human and C. callidus rodent specimens were related and formed a monophylogenetic cluster with the LNV (GenBank accession no. AF005727), with a mean genetic divergence of 4.8%. These strains were included in subclade II, which comprises Anajatuba, Rio Mamore, Rio Mearim, and Alto Paraguay viruses (Figure 2). The genetic distance between strains recovered from rodents and humans was 5.5%, whereas the genetic distance between the human strains was 6.8%. Analysis of homology showed no difference between the partial amino acid sequences of human and rodent strains and LNV (100% homology). The homology of nucleotide sequences between the LNV strains was 89.9%–93.4% (Table A1). Most changes were silent mutations in the nucleotide sequences, indicated by the genetic divergence between LNV strains (Δdiv = 0.2%–9.8%).
Phylogenetic comparison of the partial nucleotide sequences of nucleoprotein (N) gene of the small (S) RNA segment of different hantavirus strains from the Old World and New World by using the maximum-likelihood method and Bayesian analysis, with detail of the phylogenetic relationship between LNV strains isolated from humans and rodents in the state of Mato Grosso, Brazil. Bootstrap and Bayesian values (within parentheses) are shown for each respective knot. The arrows indicate the exact position of the values. Numbers within brackets correspond to the divergence between groups. Scale bar indicates 30% divergence of nucleotide sequences. APV, Alto Paraguay; ANAJV, Anajatuba, ANDV, Andes; BMJV, Andes Bermejo, NEMV, Andes Neembuco, LECV, Andes Lechiguanas; ORNV, Andes Oran; ARAV, Araraquara; BAYV, Bayou; BCCV, Black Creek Canal; CANOV, Cano Delgadito; CASV, Castelo dos Sonhos; CHOV, Choclo; EMCV, El Moro Canyon; HTNV, Hantaan; JUQV; Juquitiba-Araucaria; LNV, Laguna Negra; LSCV, Limestone Canyon; MULV, Muleshoe; NYV, New York; PRNV, Pergamino; PUUV, Puumala; RIOMV, Rio Mamoré; RIMEV, Rio Mearim; RIOSV, Rio Segundo; SEOV, Seoul; SNV, Sin Nombre; TULV, Tula.
LNV was initially confirmed in 1997 by serologic testing of a patient with HPS who died. The patient lived in Santiago, Chile, but was probably infected in Santa Cruz, Bolivia (13). In 1999, molecular analysis of the small N gene and medium Gn and Gc gene segments of the hantavirus genome in samples from HPS patients from Bolivia, western Paraguay, and Chile facilitated the genetic characterization of LNV and its association with the small vesper mouse Calomys laucha, which is considered the primary host of LNV. Subsequent studies in Argentina have also demonstrated the circulation of LNV in patients with HPS and in the large vesper mouse Calomys callosus (4,9,13–15).
Conclusions
Our phylogenetic analysis of partial sequences of the N gene showed LNV as the cause of HPS, and the possible association of the organism with C. callidus rodents in western Brazil. These findings highlight the intense circulation of LNV in Matto Grosso municipalities located near the BR-364 highway. The vegetation and the equatorial climate of the area provide an excellent microenvironment for the maintenance of C. callidus rodents, as do areas in Bolivia Paraguay, and northern Argentina, where HPS caused by LNV has been reported (4,9,13–15).
The high nucleotide and amino acid homology between strains recovered from humans and the C. callidus rodent in Matto Grosso and the LNV prototype detected in Paraguay and Argentina suggest that LNV was transmitted by the rodent host C. callidus and led to the HPS cases that occurred in the vicinity of the highway BR-364 in southwestern Matto Grosso. No correlation was observed between the human LNV strains and year, geographic distribution, or between the severity of disease and the genetic diversity of LNV found in Brazil. The genetic data obtained in this study provide a better understanding of the molecular characterization of LNV and its association with HPS in southwestern Matto Grosso. Finally, on the basis of the phylogenetic analysis, the rodent species C. callidus is suggested as a potential reservoir for LNV. Further analyses of complete genome data are needed to confirm this result and to assess whether the C. callidus rodent is the sole carrier of of LNV in Matto Grosso.
Suggested citation for this article: Travassos da Rosa ES, Medeiros DBA, Nunes MRT, Simith DB, Pereira A, Elkhoury MR, et al. Molecular epidemiology of Laguna Negra virus, Mato Grosso State, Brazil. Emerg Infect Dis [serial on the Internet]. 2012 Jun [date cited]. http://dx.doi.org/10.3201/eid1806.110948
Acknowledgments
We are grateful to João Paulo da Silva, Reginaldo Alves da Silva, Irene Maria Mendes, Jason Batista, and Noeli Negrão; and Mato Grosso State Health Department for logistical support. Thanks are also due to the LabGeo at Instituto Evandro Chagas for helping with Figure 1.
This study was partially supported by Conselho Nacional para o Desenvolvimento Científico e Tecnológico (grants 301641/2010-2 8 and 302987/2008-8), Conselho Nacional para o Desenvolvimento Científico e Tecnológico/Fundação de Amparo à Pesquisa do Estado do Pará/Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior grant 573739/2008-0, Instituto Nacional de Ciência e Tecnologia para Febres Hemorrágicas Virais, Secretaria de Ciência Tecnologia e Meio Ambiente/Fundo Estadual de Ciência e Tecnologia/EDITAL PPRH 2003 CT, and by Instituto Evandro Chagas, Fundação Oswaldo Cruz, and Secretaria de Vigilância em Saúde.
Homology between hantavirus strains originating from Mato Grosso, Brazil, and other hantaviruses from South America*
Strains
H 651686
H 650736
H 695325
H 713175
H 712518
H 671696
H 653486
H 682807
H 657848
H 660462
H 706738
H 695689
H 696558
H 711891
H 678213
H 710031
AN 649993
AN 650204
AN 650228
LNV
APV
RIMEV
RIOM
ANAJV
CASV
JUQV
ARAV
H 651686
98.1
96.8
95.1
95
94.1
95.6
95
95.7
93.5
94.6
92.9
91.6
91.6
92.1
90.1
92.4
93.4
92.8
96.8
71.1
72.7
76.9
72.7
75
72.7
77.2
H 650736
100
96.9
95.3
95.6
94.7
95.9
95.6
94.9
92.8
93.8
92.4
91.3
91.3
91.8
89.7
92.1
92.8
92.1
96.5
70.5
72.5
76.1
73.2
75.2
72.9
77.2
H 695325
100
100
97.1
95.2
94.6
96
95.7
95.9
93.8
94.9
92.7
91.6
91.6
92.1
90
94.3
95.2
94.4
94.1
71.3
71.8
76.3
72.5
74.8
73
76.7
H 713175
100
100
100
96.6
95.7
97.2
96.9
97.1
95.9
97.8
94.7
94.6
94.6
94
92.1
92.2
93.1
92.4
92.8
70.7
73.3
75.6
75
74.5
72.6
78.4
H 712518
100
100
100
100
98.5
99.1
98.5
98.7
96.8
97.8
96.3
95.3
95.3
95.7
93.7
91.8
91.8
91.8
93.8
70.8
73
76.3
77.4
75.5
73
78.2
H 671696
100
100
100
100
100
98.5
97.9
97.8
95.7
96.8
95.3
94.4
94.4
94.9
92.8
90.9
90.9
90.9
93.1
71.3
73.3
76.4
77.1
75.2
72.1
77.2
H 653486
100
100
100
100
100
100
99.4
99
96.9
97.9
96.3
95.3
95.3
95.7
93.7
91.8
91.8
91.9
93.5
71
73.5
76.4
76.9
75.5
73.3
77.2
H 682807
100
100
100
100
100
100
100
98.4
96.3
97.4
95.7
94.7
94.7
95.2
93.1
91.2
91.2
91.3
93.4
70.8
73.3
76
76.8
75.7
73
77.5
H 657848
100
100
100
100
100
100
100
100
97.8
98.8
96.6
95.6
95.6
96
94
91.9
92.2
92.1
92.8
70.7
73
75.9
77.1
75.2
72.6
77.7
H 660462
100
100
100
100
100
100
100
100
100
98.1
95.4
94.9
94.9
95.2
96.2
90.7
91
90.9
90.9
70.4
72.2
75.9
79.2
76
73.2
78.2
H 706738
100
100
100
100
100
100
100
100
100
100
96.6
96.8
96.8
96.2
94.3
91.9
92.2
92.1
91.9
70.5
72.7
76
77.2
75
72.6
77.5
H 695689
100
100
100
100
100
100
100
100
100
100
100
96.2
96.2
96.8
95.3
93.7
95.3
94.6
90.9
71.3
72.5
76.4
78
75
73.6
77.5
H 696558
100
100
100
100
100
100
100
100
100
100
100
100
100
99
96.8
94.4
92.5
92.5
90.2
71.1
73.5
76.7
78.3
76.2
73.2
77.7
H 711891
100
100
100
100
100
100
100
100
100
100
100
100
100
99
96.8
94.4
92.5
92.5
90.2
71.1
73.5
76.7
78.3
76.2
73.2
77.7
H 678213
100
100
100
100
100
100
100
100
100
100
100
100
100
100
96.6
94.6
93.1
93.1
90.6
71.1
73
76.6
78.1
75.5
73.3
77.5
H 710031
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
94.6
92.6
92.6
88.7
71.3
72.9
77
81
76.5
74.2
79.4
AN 649993
99.6
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
97.5
98.1
90.9
71.9
71.3
77.3
74.8
75.5
74.2
78.2
AN 650204
99.6
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
98.4
91.6
72.3
71.5
77.5
75
74.3
74.4
77.2
AN 650228
99.6
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
90.7
72.5
71.3
78.6
75
74
74.6
77
LNV
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
72.3
72.7
77.9
74.4
75.5
75.3
77.7
APV
96.7
96.7
96.7
96.7
96.7
96.7
96.7
96.7
96.7
96.7
96.7
96.7
96.7
96.7
96.7
96.7
96.3
96.3
96.3
96.7
76.6
76.6
79
76
72.6
76.2
RIMEV
94.8
94.8
94.8
94.8
94.8
94.8
94.8
94.8
94.8
94.8
94.8
94.8
94.8
94.8
94.8
94.8
94.2
94.2
94.2
94.8
96.2
76.8
78.9
72.8
70
72.8
RIOMV
97.7
97.7
97.7
97.7
97.7
97.7
97.7
97.7
97.7
97.7
97.7
97.7
97.7
97.7
97.7
97.7
97.1
97.1
97.1
97.7
99.1
97.3
81
74.3
73.2
79.9
ANAJV
96.2
96.2
96.2
96.2
96.2
96.2
96.2
96.2
96.2
96.2
96.2
96.2
96.2
96.2
96.2
96.2
96
96
96
96.2
96.7
95
93.8
76.2
75.7
77.2
CASV
91.5
91.5
91.5
91.5
91.5
91.5
91.5
91.5
91.5
91.5
91.5
91.5
91.5
91.5
91.5
91.5
91
91
91
91.5
89.7
56.6
56
57.5
79.4
77
JUQV
92
92
92
92
92
92
92
92
92
92
92
92
92
92
92
92
91.5
91.5
91.5
92
90.6
86.4
86.7
89.1
96.3
79.2
ARAV
90.6
90.6
90.6
90.6
90.6
90.6
90.6
90.6
90.6
90.6
90.6
90.6
90.6
90.6
90.6
90.6
90
90
90
90.6
90.6
56.1
56
57.5
95.6
93.9
*Numbers above the diagonal indicate nucleotide sequence identity and numbers below the diagonal indicate amino acid sequence identity. Gray shading corresponds to the genetic relationship (nucleotides and amino acids) of hantavirus strains isolated from humans (H) and rodents (AN) with the Laguna Negra virus. APV, Alto Paraguay virus; ARAV, Araraquara virus; ANAJV, Anajatuba virus; CASV, Castelo dos Sonhos virus; JUQV, Juquitiba-Araucaria virus; LNV, Laguna Negra virus; RIOMV, Rio Mamore virus; RIMEV, Rio Mearim virus.
Characteristics of human patients and <italic>Calomys callidus</italic> rodents with positive serologic results for hantavirus and partial nucleotide sequence of gene N, Mato Grosso, Brazil*
ID no.
Age, y/sex
Sample
Date of sample collection
Outcome
GenBank accession no.
Municipality
Humans
H 678213
38/M
Serum
2005 Mar 10
Cure
JQ775513
Barra do Bugres (15°4′21′′S; 57°10′52′′W)
H 650736
33/M
Serum
2001 Dec 19
Death
JQ775504
Campo Novo do Parecis (13°40′31′′S; 57°53′31′′W)
H 660462
23/F
Serum
2002 Jul 8
Cure
JQ775506
H 671696
20/F
Serum
2003 Aug 23
Death
JQ775505
H 682807
20/M
Serum
2004 Aug 22
Death
JQ775507
H 695689
27/F
Serum
2005 Aug 22
Cure
JQ775508
H 696558
22/M
Blood
2005
Cure
JQ775512
H 711891
42/M
Serum
2006 Aug 17
Death
JQ775503
H 657848
42/M
Serum
2002 May 14
Cure
JQ775517
Diamantino (14°24′31′′S; 56°26′46′′W)
H 706738
13/F
Serum
2006 May 22
Cure
JQ775516
H 712518
33/M
Serum
2006 Aug 29
Cure
JQ775518
H 653486
13/F
Serum
2002 Feb 22
Cure
JQ775514
Nova Olímpia (14°47′50′′S; 57°17′17′′W)
H 695325
24/M
Serum
2005 Nov 11
Cure
JQ775515
Santo Afonso (14°29′51′′S; 7°0′7′′W)
H 713175
17/M
Serum
2006 Sep 26
Cure
JQ775509
São José do Rio Claro (13°26′48′′S; 56°43′17′′W)
H 651686
31/M
Serum
2002 Jan 22
Cure
JQ775511
Tangará da Serra (14°37′10′′S; 57°29′9′′W)
H 710031
7/F
Serum
2006 Jul 18
Death
JQ775510
Rodents
AN 650204
NA
Lung
NA
NA
JQ775500
Campo Novo do Parecis
AN 650228
NA
Lung
NA
NA
JQ775502
AN 649993
NA
Heart
NA
NA
JQ775501
Tangará da Serra
*Source: Secretaria de Vigilância Epidemiológica do Estado do Mato Grosso and Instituto Evandro Chagas, Secretaria de Vigilância em Saúde), and Ministério da Saúde. NA, data not available.
Dr Travassos da Rosa is a senior researcher at the Evandro Chagas Institute, Ananindeua, Brazil. Her research interests are diagnosis, epidemiology, and molecular epidemiology of arboviruses, hantaviruses, and rabies virus.
ReferencesNicholST, SpiropoulouCE, MorzunovS, RollinPE, KsiazekTG, FeldmannH, Genetic identification of a hantavirus associated with an outbreak of acute respiratory illness.Science. 1993;262:914–710.1126/science.82356158235615SchmaljohnCS, NicholST Bunyaviridae. In: Knipe DM, Griffin DE, Lamb RA, Strauss SE, Howley PM, Martin MA, et al., editors. Fields virology, 5th ed. Philadelphia: Lippincott Williams & Wilkins; 2007 p. 1741–89.LevisS, MurzonovS, RoweJ, EnriaD, PiniN, CalderonG, Genetic diversity and epidemiology of hantaviruses in Argentina.J Infect Dis. 1998;177:529–3810.1086/5142219498428PadulaPJ, ColavecchiaSB, MartínezVP, ValleMO, EdelsteinA, MiguelSDL, Genetic diversity, distribution, and serological features of hantavirus infection in five countries in South America.J Clin Microbiol. 2000;38:3029–3510921972VincentMJ, QuirozE, GraciaF, SanchezAJ, KsiazekTG, KitsutaniPT, Hantavirus pulmonary syndrome in Panama: identification of novel hantaviruses and their likely reservoirs.Virology. 2000;277:14–910.1006/viro.2000.056311062031Travassos da RosaES, LemosERS, MedeirosDBA, SmithDB, PereiraAS, ElkhouryMR, Hantaviruses and hantavirus pulmonary syndrome, Maranhão, Brazil.Emerg Infect Dis. 2010;16:1952–521122229Travassos da RosaES, MillsJM, PadulaPJ, ElkhouryMR, KsiazekTG, MendesWS, Newly recognized hantaviruses associated with hantavirus pulmonary syndrome in northern Brazil: partial genetic characterization of viruses and serologic implication of likely reservoirs.Vector-B Zoon Dis.2005;5:11–910.1089/vbz.2005.5.11JohnsonAM, SouzaLTM, FerreiraIB, PereiraLE, KsiazekTG, RollinPE, Genetic investigation of novel hantaviruses causing fatal HPS in Brazil.J Med Virol. 1999;59:527–3510.1002/(SICI)1096-9071(199912)59:4<527::AID-JMV17>3.0.CO;2-Y10534737JohnsonAM, BowenMD, KsiazekTG, WilliamsRJ, BryanRT, MillsJN, Laguna Negra virus associate with HPS in western Paraguay and Bolivia.Virology. 1997;238:115–2710.1006/viro.1997.88409375015MillsJN, ChildsJEEcologic studies of rodent reservoirs: their relevance for human health.Emerg Infect Dis. 1998;4:529–3710.3201/eid0404.9804039866729BonvicinoCR, MoreiraMAMMolecular phylogeny of the genus Oryzomys (Rodentia: Sigmodontinae) based on cytochrome b DNA sequences.Mol Phylogenet Evol. 2001;18:282–9210.1006/mpev.2000.087811161762HuelsenbeckJP, RonquistFMrBAYES: Bayesian inference of phylogeny and its impact on evolutionary biology.Science. 2001;294:2310–410.1126/science.106588911743192EspinozaR, VialP, NoriegaLM, JohnsonA, NicholST, RollinPE, Hantavirus pulmonary syndrome in a Chilean patient with recent travel in Bolivia.Emerg Infect Dis. 1998;4:93–510.3201/eid0401.9801129452401LevisS, GarciaJ, PiniN, CalderónG, RamírezJ, BravoD, Hantavirus pulmonary syndrome in northwestern Argentina: circulation of Laguna Negra virus associated with Calomys callosus.Am J Trop Med Hyg. 2004;71:658–6315569801CarrollDS, MillsJN, MontgomeryJM, BauschDG, BlairPJ, BuransJP, Hantavirus pulmonary syndrome in central Bolivia: relationships between reservoir hosts, habitats, and viral genotypes.Am J Trop Med Hyg. 2005;72:42–615728866