Hantaviruses and Hantavirus Pulmonary Syndrome, Maranhão, Brazil

To confirm circulation of Anajatuba virus in Maranhão, Brazil, we conducted a serologic survey (immunoglobulin G ELISA) and phylogenetic studies (nucleocapsid gene sequences) of hantaviruses from wild rodents and persons with hantavirus pulmonary syndrome. This virus is transmitted by Oligoryzomys fornesi rodents and is responsible for hantavirus pulmonary syndrome in this region.


H antaviruses (family Bunyaviridae, genus Hantavirus)
cause a viral zoonosis transmitted by rodents belonging to the families Muridae and Cricetidae. Each hantavirus is predominantly associated with a specifi c rodent species in a specifi c geographic region. However, infection of other rodent species can occur as a spillover phenomenon (1).
Hantavirus disease has 2 recognized clinical forms, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome (HPS) (2). The respiratory form of the disease was described in June 1993 during an epidemic of severe respiratory disease caused by Sin Nombre virus in the United States (3). A few months later, 3 HPS cases were identifi ed in 3 siblings in Juquitiba, São Paulo State, Brazil (4). During 1993-2009, a total of 1,246 HPS cases (264 in the Amazon region) were reported in Brazil, and new hantaviruses were identifi ed (Juquitiba virus, Castelo dos Sonhos virus, Araraquara virus, Anajatuba virus, and Rio Mearim (5).
During 2003-2005, an ecoepidemiologic study was conducted in the municipality of Anajatuba, Maranhão, Brazil, to identify reservoirs of hantaviruses after identifi cation of 3 HPS cases (6). Two new hantaviruses, Anajatuba virus and Rio Mearim virus, were isolated from Oligoryzomys fornesi (rice rat) rodents and Holochilus sciureus (marsh rat) rodents, respectively, and genetically characterized (5). To confi rm circulation of Anajatuba virus in Maranhão, Brazil, we conducted a serologic survey (immunoglobulin [Ig] G ELISA) and phylogenetic studies (nucleocapsid gene sequences) of hantaviruses obtained from wild rodents and persons with HPS.

The Study
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 fl oodplain of the Maranhão River in Maranhão State, Brazil (Figure 1, panel A). The region has chains of lakes with extensive swamps and fl ooded fi elds, forest areas, and rice fi elds 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. Fiftyfour 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 fi ndings 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 fi shing; 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 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 amplifi cation of partial nucleocapsid gene sequences from human and rodent samples, respectively, by using primers described (11). Purifi ed amplicons were obtained by using the GFX PCR DNA and Gel Band Purifi cation 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 confi dence 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.
All phylogenetic methods showed similar topologies, and the ML maximum-likelihood construction was select- Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 12, December 2010 ed for representing the fi nal 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%) (online Technical Appendix, www.cdc.gov/EID/content/16/12/1952-Techapp.pdf).
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.

Conclusions
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.