Figure 1

Figure 1. A) Regions of Anajatuba (red) (Maranhão River Microregion) and Santa Rita (blue) (Rosario Microregion), Maranhão, Brazil, where hantavirus pulmonary syndrome (HPS) cases were found. PA, Para; TO, Tocantins; PI, Piaui; 1,...

Anajatuba (3°16′S, 44°37′W; population 23,907) and Santa Rita (3°9′S, 44°20′W; population 31,033) (www.ibge.gov.br), are located in the western floodplain of the Maranhão River in Maranhão State, Brazil (Figure 1, panel A). The region has chains of lakes with extensive swamps and flooded fields, forest areas, and rice fields extending from the outskirts of the urban area. The climate is tropical and humid (average temperature range 26°C–28°C), and the rainy season is during January–July (5,6).

Data for 5 cases of HPS in men (age range 25–30 years, 3 from Anajatuba and 2 from Santa Rita) are shown in Table 1. In a cross-sectional serologic survey in residents of Anajatuba, 293 serum samples (8.1% of the population studied and 1.2% of the total population of the municipality) were obtained; 153 (52%) residents were women. Fifty-four samples were obtained from urban residents, and 239 samples were obtained from rural residents. All samples were tested by using an ELISA to detect IgM and IgG as described (7).

The main findings of the serologic study are shown in Table 2. A male:female ratio of 2:1 was observed in urban and rural areas. Factors investigated for increasing risk for exposure to hantaviruses included living near rice paddies; engaging in farming or fishing; having wild rodents around the household; having contact with wild rodents in the workplace, school, or domestic surroundings; and storing rice in the household.

In May 2003 and May 2005, two rodent captures approved by the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis/Instituto Chico Mendes de Conservação da Biodiversidade were conducted in São Roque, Anajatuba (Figure 1, panel B). Trapping was conducted <50 m from residences of 3 deceased HPS case-patients in accordance with accepted rodent capture and handling procedures and standard biosafety protocols for anesthetizing and killing rodents, and biometric analysis was conducted (8). Fragments of liver, lung, spleen, heart, and kidney were obtained. Taxonomic identification was performed according to procedures of Bonvincino and Moreira (9).

Biologic samples (blood and viscera fragments) were obtained from 216 captured rodents: 96 (44%) captured in 2003 and 120 (56%) captured in 2005. The most common species captured in 2003 were Necromys lasiurus rodents (n = 62, 64%) and Akodon sp. rodents (n = 27, 28%). The most common species captured in 2005 were N. lasiurus rodents (n = 105, 87%) and O. fornesi rodents (n = 2, 2%); the remaining rodents were from other genera.

Blood samples collected from wild rodents were also tested by using an IgG ELISA (10). IgG against hantavirus was detected in 2 (100%) of 2 O. fornesi rodents captured in 2005 and 6 (4%) of 167 N. lasiurus rodents (3 of 62 captured in 2003 and 3 of 105 captured in 2005) (Table 1).

Virus RNA was extracted from IgM-positive human serum or blood samples and lung fragments from IgG-positive rodents by using the QIAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. Nested reverse transcription–PCR and hemi-nested reverse transcription–PCR were used for amplification of partial nucleocapsid gene sequences from human and rodent samples, respectively, by using primers described (11). Purified amplicons were obtained by using the GFX PCR DNA and Gel Band Purification Kit (Healthcare, Little Chalfont, UK) and sequenced. Amplicons (434 bp) generated from HPS cases in humans (2 from Anajatuba and 1 from Santa Rita) and from 3 of 8 lung samples from hantavirus IgG-positive rodents were sequenced (Table 1).

Phylogenetic trees were constructed by using neighbor-joining, maximum-parsimony, maximum-likelihood, and Bayesian methods implemented in PAUP 4.0b.10 (12), PHYML (13), and BEAST (14). Modeltest version 3.6 (15) was used to determine the best nucleotide substitution model based on Akaike information criteria. Analyses were conducted by using confidence values estimated from mean nucleotide divergence obtained for different Old World and New World hantavirus sequences by using MEGA version 3.0 software (www.megasoftware.net) Estimated values were <45%, <25%, 22%, and 15% and were used for grouping viruses in clusters, clades, subclades, and species, respectively.

Figure 2

Figure 2. Phylogenetic analysis of partial small RNA segments of hantaviruses, Maranhão, Brazil, by using maximum-likelihood and Bayesian methods. Bayesian and bootstrap values (in parentheses) are shown over each main tree node. Values...

All phylogenetic methods showed similar topologies, and the ML maximum-likelihood construction was selected for representing the final tree. Bootstrap and Bayesian posterior probability values are shown in Figure 2.

Two major clusters were observed (New World and Old World hantavirus groups) and had a genetic distance of 28.2% (inclusion value 25%). The New World group was divided into clades I and II. Clade I was divided into 3 subclades (genetic divergence 23.5%), Ia, Ib, and Ic. Clade II was divided into 2 subclades (genetic divergence 23.7%), IIa and IIb. The strains used in this study were closely related to Anajatuba virus and were included in the IIa subclade (genetic divergence 2%) (Technical Appendix ).

Nucleotide and amino acid homology between Anajatuba virus (5) and the strains isolated in this study in Maranhão were 98.3% and 100%, respectively. These strains were included in a group related to rodents belonging to the genus Oligoryzomys, although sample Be AN 669104 was obtained from an N. lasiurus rodent, which suggests spillover transmission between rodent species.

We showed that Anajatuba virus is responsible for human HPS cases and that O. fornesi rodents are its likely reservoir. Anajatuba virus infections of N. lasiurus were spillover infections. Human hantavirus infections are common among persons in the Baixada Maranhense region, but cases of HPS are rare. However, educational and health surveillance programs are needed to prevent hantavirus transmission.