Acta Tropica 131 (2014) 1–10

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Ecological study of hantavirus infection in wild rodents in an endemic area in Brazil Renata Carvalho Oliveira a,∗ , Rosana Gentile b , Alexandro Guterres a , Jorlan Fernandes a , Bernardo Rodrigues Teixeira b , Vanderson Vaz b , Fernanda Pedone Valdez b,c , Luciana Helena Bassan Vicente a , Sócrates Fraga da Costa-Neto b , Cibele Bonvicino b,c , Paulo Sergio D’Andrea b , Elba R.S. Lemos a a Laboratório de Hantaviroses e Rickettsioses, Instituto Oswaldo Cruz, Fundac¸ão Oswaldo Cruz, FIOCRUZ-IOC, Avenida Brasil 4365, Manguinhos, Rio de Janeiro, Brazil b Laboratório de Biologia e Parasitologia de Mamíferos Silvestres Reservatórios, Instituto Oswaldo Cruz, Fundac¸ão Oswaldo Cruz, FIOCRUZ-IOC, Avenida Brasil 4365, Manguinhos, Rio de Janeiro, Brazil c Departamento de Genética, Instituto Nacional de Câncer, INCA, Rio de Janeiro, Brazil

a r t i c l e

i n f o

Article history: Received 14 August 2013 Received in revised form 13 November 2013 Accepted 21 November 2013 Available online 28 November 2013 Keywords: Rodents Population ecology Hantavirus Jabora virus Juquitiba virus

a b s t r a c t A 3-year ecological study of small mammals was carried out in an endemic area for hantavirus pulmonary syndrome in the state of Santa Catarina in Southern Brazil. A total of 994 rodents of 14 different species corresponding to the subfamilies of Sigmodontinae, Murinae, Eumysopinae, and Caviinae were captured during 2004–2006. Oligoryzomys nigripes and Akodon montensis were the most abundant species and showed a clear seasonal pattern with higher population sizes during the winter. Rodent population outbreaks, associated within bamboo mast seeding events, were detected predominantly in areas where hantavirus pulmonary syndrome cases were notified in the state. Antibody reactivity to Hantavirus was detected in five sigmodontine species: O. nigripes (39/435), A. montensis (15/318), Akodon paranaensis (4/37), Thaptomys nigrita (1/86) and Sooretamys angouya (1/12). The highest hantavirus antibody prevalence occurred during the period of highest population size in A. montensis. For O. nigripes, hantavirus prevalence was higher in late spring, when reproduction was more frequent. Co-circulation of Juquitiba (JUQV) and Jabora (JABV) viruses was observed – JABV in A. paranaensis and A. montensis; JUQV in O. nigripes and T. nigrita. JABV occurrence was associated to gender and population size of the rodent while JUQV was related to gender, season, temperature, and locality. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The genus Hantavirus (family Bunyaviridae), a group of rodent/insectivore-borne RNA viruses, is widely distributed in the world and includes a variety of strains recognized as human pathogens (Hjelle and Torres-Pérez, 2010; Jonsson et al., 2010; Schmaljohn and Hjelle, 1997). Rodents of the families Muridae and Cricetidae were described as the primary zoonotic reservoirs of these viruses, but distinct hantaviruses have also been discovered in several species of shrews and moles (Arai et al., 2007; Kang et al., 2009; Klempa et al., 2007; Yadav et al., 2007). Transmission is assumed to occur through human inhalation of aerosolized virus from rodents’ urine and/or feces, direct (agonistic encounters),

∗ Corresponding author at: Pavilhão Hélio e Peggy Pereira, sala B115, Instituto Oswaldo Cruz, FIOCRUZ, Avenida Brasil 4365, Manguinhos, 21040-900 Rio de Janeiro, RJ, Brazil. Tel.: +55 21 2562 1727; fax: +55 21 2562 1897. E-mail address: reoliveira@ioc.fiocruz.br (R.C. Oliveira). 0001-706X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.actatropica.2013.11.016

or indirect (contaminated food or environment) contact among rodents (Sauvage et al., 2003). Hantavirus cardiopulmonary syndrome (HCPS) is considered as one of the major emerging diseases in Brazil, mainly owing to its high mortality rate (∼40%). Since 1993, over 1600 cases of HCPS have been reported and at present, there are eight hantavirus described in Brazil related to Sigmodontinae rodents: Juquitiba/Araucaria, Araraquara, Castelo dos Sonhos, Anajatuba, Laguna Negra, Rio Mearim, Jabora, and Rio Mamore viruses carried by Oligoryzomys nigripes, Necromys lasiurus, Oligoryzomys utiaritensis, Oligoryzomys fornesi, Calomys callidus, Holochilus sciureus, Akodon montensis, and Oligoryzomys microtis, respectively (Oliveira et al., 2011; Firth et al., 2012; Johnson et al., 1999; Raboni et al., 2009, 2005; Rosa et al., 2005; Suzuki et al., 2004; Travassos Da Rosa et al., 2012; Travassos da Rosa et al., 2011, 2010), the latest three were not described as causing human HCPS. Outbreaks of different genotypes of Hantavirus in Brazil have often been related to periods of high population sizes of sigmodontine rodents that are commonly associated to

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agricultural and peridomestic rural environments (Mills and Childs, 1998; Suzuki et al., 2004). These rodents have predominantly granivorous feeding habits and opportunistic reproductive strategies (Gentile et al., 2000). These features allow them to reach high population sizes in certain periods, mainly in productive cropland areas responding quickly to environmental changes. Both the abundance of hantavirus reservoir species and the serostatus within the reservoir population are associated with a suite of biotic and abiotic environmental variables, such as precipitation, habitat quality, and food availability (Mills, 2005). Ecological studies of hantaviruses are still lacking in Brazil. Little is known about the ecology and temporal dynamics of hantavirus infection in host populations, since most of the studies were punctual in space and time (Oliveira et al., 2009; Raboni et al., 2009; Suzuki et al., 2004; Travassos da Rosa et al., 2010). In the Municipality of Jaborá, an endemic area of HCPS in Southern Brazil, we observed the co-circulation of Juquitiba and Jabora viruses in O. nigripes and A. montensis and recently described the genetic characterization of these two hantaviruses in this region (Oliveira et al., 2011). The present paper reports a population dynamic study of the hantavirus rodents’ reservoirs in this area, investigating the factors related to the hantavirus infection and transmission in the rodent populations during three years.

2. Materials and methods 2.1. Study area and sampling An ecological study of small mammals was carried out in the municipality of Jaborá located in the mid-western region of Santa Catarina State, in Southern Brazil (Fig. 1). Jaborá has 4041 inhabitants (IBGE – Brazilian Institute of Geography and Statistics) and most of them (60.28%) live in rural areas. The economic development of this region is based on farming and cattle raising (agricultural activities). The study area presents a mixed ombrophilous forest that is a vegetation type in the Brazilian Atlantic Rainforest (Rizzini, 1997). Nowadays, native vegetation is being replaced by cultivated areas (mainly cornfields), secondary forests, and shrublands. The climate is subtropical humid mesothermal (Cfb, Köppen) with an annual temperature average of around 18.7 ◦ C, and a total accumulated rainfall of 1930 mm (mainly in summer). The mean altitude is about 689 m (Nimer, 1989). The National Institute of Meteorology of Brazil (INMET) provided the monthly data on air temperature (average) and rainfall (accumulated) obtained from the Campos Novos Station in Santa Catarina for 2004–2006. This was the closest and most representative weather station around Jaborá municipality. During the study period (March 2004 to December 2006), the average temperature in the warm months (November to April) ranged from 17.5 ◦ C to 22.1 ◦ C, and in the cold months (May to October) the corresponding range was 11.2–18.2 ◦ C. The mean monthly rainfall was 143.1 mm (range: 32.1–381.4 mm) and 165.6 mm (29.4–323.1 mm) in the warm and cold months, respectively (Fig. 2). Captures were conducted in four small rural properties formed by patches of secondary vegetation intermixed with subsistence plantations under the same climatic conditions and the same vegetation domain (mixed ombrophilous forest). (1) Avelino Vieira Farm (AV): small forest fragment with disturbed understory, sparse trees and shrubs, grasslands, surrounded by pastureland and cornfields. (2) Avelino Cumerlato Farm (AC): shrub vegetation with brushwood, grass, and understory of bamboo thickets along a stream near dwellings and cornfields. (3) Domingos Vieira Farm (DV): small forest fragments with sparse high trees and dense bamboo

tickets surrounded by grasslands and dirt paths. (4) Linha Castelhano (LC): pasturelands mixed with short trees along a dirt road. On each rural property, transects of traplines were placed in peridomestic environments, cornfields, shrub, bamboo thickets, and forest borders. The rodents were captured during three consecutive years, in fall, winter and spring, totaling ten capture sessions between 2004 and 2006. In 2006, two capture sessions were carried out during the winter owing to bamboo mast-seeding, which occurred in November and December of 2005 (approximately 6 months before). Each capture station was sampled with Sherman® (7.62 cm × 9.53 cm × 30.48 cm) and Tomahawk® (40.64 cm × 12.70 cm × 12.70 cm) live traps, placed 10 m apart, in linear ground transects of 20 capture stations. The bait was a mixture of bacon, peanut butter, banana, and oatmeal. The traps were set in the late afternoon and inspected in the early morning on five consecutive days. Each animal was anesthetized and euthanized in accordance with the Guidelines for the Care and Use of Laboratory Animals of Oswaldo Cruz Foundation, Brazil. Blood was collected by heart puncture from each anesthetized rodent. Tissue samples of liver, spleen, kidney, lung, and heart were collected and stored immediately in liquid nitrogen for further processing. All the animals collected were deposited as voucher specimens at the National Museum of the Federal University of Rio de Janeiro. Capture of small mammals were authorized by the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA)/Chico Mendes Institute for Biodiversity and Conservation, and it was carried out and handled according to recommended safety procedures (Gannon and Sikes, 2007; Mills et al., 1995). 2.2. Small mammal taxonomy Rodents and marsupial species were identified by external morphology and cranial characteristics. Taxonomy of cryptic rodent species was confirmed by karyotypical analysis according to Bonvicino et al. (1996). Analyses of DNA sequence data of the mitochondrial cytochrome b gene were also carried out to confirm species identification for specimens without cell material for karyotypical analysis and to estimate the phylogenetic relationship of hantavirus-positive specimens (specimens with and without karyotyping data) following the procedures previously described in Oliveira et al. (2011). 2.3. Rodent hantavirus infection diagnostics 2.3.1. Serology and molecular analysis Rodent serum samples were examined for IgG antibodies against the recombinant Andes nucleocapsid (N-ANDV) protein used as the specific antigen by enzyme-linked immunosorbent assay (Padula et al., 2000) as described by Oliveira et al. (2011). Total RNA was extracted from lung and kidney tissues of antibody positive rodents by using the PureLink Micro-to-Midi Total RNA Purification System kit (Invitrogen, San Diego, CA) following the manufacturer’s protocol. Nested reverse transcription-PCR (RTPCR) of genome partial S-segment was performed as described previously (Oliveira et al., 2011). For RNA purification, the Wizard® SV Gel and PCR Clean-Up System kit (Promega, Corp., Madison, WI, USA) was used according to the manufacturer’s recommendations and the strands were directly sequenced. Direct nucleotide sequencing of amplicons was performed using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer’s recommendations, and the reaction was run in an ABI Prism 3130x (Applied Biosystems). Nucleotide sequences were analyzed by using MEGA5 software,

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and a consensus sequence for each viral genome was derived from contiguous sequences assembled with even software. The sequence generated was then compared with high similar sequences of other hantavirus available in GenBank by using the BLASTN tool (Altschul et al., 1990).

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2.3.2. Data analysis We investigated changes in the rodent’s population size and prevalence of hantavirus during the study period. Population size was estimated as the absolute number of captured specimens divided by a corrected constant trapping effort. Population data

Fig. 1. Map of the state of Santa Catarina, southern Brazil, showing the municipality of Jaborá located in the mid-western part of the state. Satellite image of rodents collection sites (below); Avelino Vieira Farm (AV), Avelino Cumerlato Farm (AC), Domingos Vieira Farm (DV) and Linha Castelhano (LC).

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Fig. 2. Climatic diagram of the study area, west of Santa Catarina, Brazil, from January 2004 to December 2006 (Rainfall (mm) = gray bars; Mean temperature (◦ C) = dark line.)

were combined for all localities in order to analyze the dynamics in a metapopulation scale. The reproductive condition of female rodents was assessed by the presence of embryos (pregnant) or visible nipples indicating recent weaning (lactating). The frequency of reproducing females was determined by the proportion between the numbers of females in reproductive status compared to the total of adult females in each capture session. Age structures of the most abundant species A. montensis and O. nigripes were analyzed by dividing animals in three classes with approximately the same number of animals, based on body weight, according to Mills et al. (2007). The 2 -square test was used to test the association between the hantavirus antibody status with gender and age class. Data were analyzed by using PASW Statistics 18 software. Prevalences were estimated by dividing the number of seropositive animals with the total number of animals analyzed to the infection per species. We performed multiple linear regressions by using the stepwise backward method to identify ecologic and environmental variables that influence the population sizes of A. montensis and O. nigripes. Frequency of reproducing females, rodent species richness (S), mean monthly temperature, and monthly rainfall for one and two capture sessions of time lag were the variables used in the analyses. The influence of rodent population size on seroprevalence was investigated by using simple linear regressions for the current month and for 1-capture session time lag for A. montensis and O. nigripes. These data were analyzed by using PASW statistics software 18. We performed generalized linear model analysis in order to investigate the influence of environmental variables on rodents’ infection rates. We first analyzed the individual effects of independent variables on the response variable (Hantavirus infection by Jabora virus or Juquitiba virus) by using logistic regression in order to minimize the number of variables in the final model. All independent variables with p ≤ 0.05 in this first stage were selected for their inclusion in the final model. The independent variables were: (i) species, (ii) gender, (iii) age class, (iv) locality, (v) season, (vi) precipitation, (vii) temperature, (viii) rodent species richness, (ix) A. montensis population size, and (x) O. nigripes population size. Variables (vi) and (vii) were computed per month and variables (viii), (ix), and (x) were computed per month and trap locality. Subsequently, generalized linear models with binomial distribution and the logit link function were used to analyze

the effect of independent variables on the response variable. All analyses were performed in R statistical software (v. 2.13.1). Following this, as a second stage, a forward–backward stepwise procedure was used and the corrected Akaike Information Criterion (AIC) value was considered to select the most parsimonious risk factors model. Selected models were validated by Hosmer–Lemeshow test (p > 0.05). 3. Results 3.1. Population dynamics From March 2004 to December 2006, we captured 994 individuals of 14 rodent species: A. montensis (n = 323), Akodon paranaensis (n = 39), Brucepattersonius iheringi (n = 18), Delomys dorsalis (n = 1), N. lasiurus (n = 8), Nectomys squamipes (n = 17), O. nigripes (n = 437), Oxymycterus judex (n = 21), Sooretamys angouya (n = 12), Thaptomys nigrita (n = 89) (Rodentia, Sigmodontinae), Mus musculus (n = 15), Rattus rattus (n = 10) (Rodentia, Murinae), Euryzygomatomys spinosus (n = 3) (Rodentia, Echimyidae), Cavia aperea (n = 1) (Rodentia, Cavidae); and 39 marsupials of five different species: Cryptonanus chacoensis (n = 1), Didelphis albiventris (n = 10), Didelphis aurita (n = 4), Micoureus demerarae (n = 1), and Monodelphis dimidiata (n = 23) (Didelphimorphia, Didelphinae). The richness of rodent species ranged from six to ten per capture session. The overall capture success was 8%. The most abundant species were O. nigripes (n = 437) and A. montensis (n = 323). The other rodent species had low population sizes. A. montensis and O. nigripes were captured at the four rural localities in each capture session throughout the entire period. The species T. nigrita (n = 89) were present in almost every capture session and in all the localities except from the most disturbed one (LC locality). Population dynamics of the two most frequently captured species followed similar patterns of annual fluctuation (Fig. 3). Population sizes of these species showed the highest values during the winter, declining during the subsequent spring, remaining at low levels through the summer, and beginning to increase in early autumn (Fig. 3). Population sizes of T. nigrita were relatively low during the first two years (2004 and 2005), but began to increase in early fall of 2006 reaching the highest peak during the winter months of 2006, when we observed an abrupt increase in their population size. Reproductive activity in O. nigripes and A. montensis occurred more frequently during the summer and fall resulting in increases

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Fig. 3. (a) Population sizes, prevalences of hantavirus infection, and proportion of reproducing females for Oligoryzomys nigripes in Jaborá, Santa Catarina, along the years; (b) population sizes, prevalences of hantavirus infection, and proportion of reproducing females for Akodon montensis in Jaborá, Santa Catarina, along the years.

in population sizes during the winter. Reproducing females of these species were found only in fall and spring (Fig. 3). Age classes were divided as follows: O. nigripes: males (Class I = 5–19 g, Class II = 20–25 g, Class III = 26–50 g), females (Class I = 6–15 g, Class II = 16–20 g, Class III = 21–38 g); A. montensis: males (Class I = 10–23 g, Class II = 24–30 g, and Class III = 31–55 g), females (Class I = 12–20 g, Class II = 21–26 g, Class III = 27–46 g). Individuals of class-I age were more frequent during the winter and age class III during the spring in both rodent species (Fig. 4). These results indicate a larger recruitment of young after the fall, resulting in higher population sizes during the winter and an aging process from winter to the following summer. The population size of A. montensis was inversely related with the frequency of reproducing females and with rainfall of the current month (r = 0.921, ANOVA of the best model: F = 19.490, p = 0.01) and directly with the frequency of reproducing females with two capture sessions time lag (r = 0.861, ANOVA of the best model: F = 17.187, p = 0.006). The population size of O. nigripes was inversely related only with the frequency of reproducing females of the current month (r = 0.681, ANOVA of the best model: F = 6.913, p = 0.03).

3.2. Hantavirus antibody prevalence and viral characterization Antibody reactivity to ANDV was detected in five sigmodontine species with different prevalences: O. nigripes (39/435; 9.0%), A. montensis (15/318; 4.7%), A. paranaensis (4/37; 10.8%), T. nigrita (1/86; 1.2%), and S. angouya (1/12; 8.3%). Antibodies to hantavirus antigens were more often found in males than in females (2 = 21.39, df = 1, p < 0.0001). Among the 994 captured rodents, 977 were tested for hantavirus infection (559 males and 418 females), 52 (9.3%) males and 8 (1.9%) females showed seropositivity. Among the 252 O. nigripes males tested, 35 (13.9%) were antibody positive for hantavirus, whereas 4 (2.2%) of the 183 females were positive indicating a significant difference in seropositivity between genders (2 = 16.39, df = 1, p = 0.0001). Of the 184 A. montensis males, 13 (7.1%) were antibody positive, whereas 2 (1.5%) of the 134 females tested were positive, also indicating significantly more males infected (2 = 4.19, df = 1, p = 0.0407). Seropositivity was found in 4 (21.1%) males and none in females of A. paranaensis specimens among a total of 19 males and 18 females analyzed, still indicating a significant difference between the two, despite the small sample size.

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Fig. 4. (a) Age structure of Oligoryzomys nigripes in Jaborá, Santa Catarina, along the years (Class I = black; Class II = dark gray; Class III = light gray); (b) Age structure of Akodon montensis in Jaborá, Santa Catarina, along the years (Class I = black; Class II = dark gray; Class III = light gray).

Table 1 Virus identification (ID) and trap sites of positive rodents specimens collected in four localities of the study area, Jaborá, Santa Catarina State, Brazil (2004–2006). Trap sessions

Akodon montensis Number antibody positive

March 04 June 04 November 04 April 05 August 05 December 05 April 06 July 06 September 06 December 06 Total a b c d e f

Oligoryzomys nigripes Number RNA positive

1 3 2

1 3 2

1

0

2 6

2 5

15

13

Jabora virus. Juquitiba virus. Avelino Vieira Farm. Avelino Cumerlato Farm. Domingos Vieira Farm. Not processed by RT-PCR.

ID/virus

Locality

Number antibody positive

JABVa JABV JABV

AVc ACd /DVe AC/AV

1 2f 10

10

JUQV

AV AC/DV AC/DV

4 3

3 3

JUQV JUQV

AC AC/DV

7 9 3 39

7 9 3 35

JUQV JUQV JUQV

AC/DV AC/DV AC

AC

JABV JABV

AV/DV AC/DV

Number RNA positive

ID/virus

0 b

Locality

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Table 2 Prevalence of hantavirus infection, richness, evenness and diversity for wild rodents collected in four localities of the study area, Jaborá, Santa Catarina, Brazil (2004–2006). Locality

Richness (S)

Total prevalence of infection

Prevalence of infection A. montesis

Prevalence of infection O. nigripes

Avelino Cumerlato Farm Avelino Vieira Farm Domingos Vieira Farm Linha Castelhano

13 7 13 4

8.5 0.9 6.2 3.7

6.8 1.9 5.6 0.0

12.2 0.0 8.5 0.0

Table 3 Stepwise model selection procedure based on the corrected Akaike Information Criteria (AIC). AIC represents the difference between the AIC value of a model and that of the best model (the lowest AIC value). Virus genotype

AIC

AIC

Model

Hosmer–Lemeshow (HL)

JABV

13.01 0

188.19 175.18

Null model Gender + Akodon montensis abundance

HL = 3.268285 p = 0.91641

JUQV

45.88 0.31 0

327.25 281.68 281.37

Null model Gender + locality + season + temperature + Richness Gender + locality + season + temperature

HL = 1.863353 p = 0.98492

Jabora virus (JABV) was detected in two rodent species, A. montensis (13) and A. paranaensis (3), whereas Juquitiba virus (JUQV) appeared in O. nigripes (35) and T. nigrita (1), the latter for the first time. We did not detect any viral RNA in S. angouya. These two viruses, JUQV and JABV, were identified in close proximity (collected on the same transect in the same capture session) of two rural localities (AV and DV) (Table 1). According to age classes, in O. nigripes only two individuals of age class I were found infected, six of age class II, and 31 of age class III indicating a clear predominance of older individuals infected (2 = 53.05, p = 0.00001, df = 2). For A. montensis, infected animals were found only in class II (three individuals) and in class III (12 individuals), also indicating a predominance of infection in older animals (2 = 15.865, p = 0.00036, df = 2). The only S. angouya and T. nigrita antibody-positive individuals were females captured in winter 2006 and fall 2006, respectively. Prevalence also varied among localities. Positive rodent individuals were found in all study localities (AV, AC, DV, and CH). However, the rodent species richness was higher for AC and DV localities, where we observed the highest prevalence of infection by considering all species (Table 2). Antibody-positive A. montensis were found in Avelino Vieira (AV), Avelino Cumerlato (AC), and Domingos Vieira (DV) farms (Table 1). Prevalences were higher (5.6–6.8%) at the AC and DV study sites, but were less than 2% at AV, where only two antibodypositive individuals were captured and a relatively high number of A. montensis was tested (n = 101). Antibody-positive O. nigripes were found in AC and DV, with prevalences varying from 8.5% to 12.2%. Infection in this species was apparently absent in the other two localities. Antibody-positive A. paranaensis were captured only in DV and LC but with the highest prevalences (21–33%), especially considering the low number of individuals tested. A. paranaensis was the only antibody-positive species that was captured in LC, the most disturbed area. Only one antibody-positive individual each of T. nigrita and S. angouya was captured at the same locality (DV). Antibody prevalence was higher at AC, followed by DV, AV, and LC. The unique exception occurred in 2006, when the highest antibody prevalence was observed in DV for A. montensis. The highest hantavirus antibody prevalences for A. montensis occurred during the periods of highest population size in 2005 and 2006, except for spring 2004 when we observed the opposite trend (Fig. 3b). For O. nigripes, antibody prevalence was lower when the population size was higher throughout the entire study. The highest prevalences for this species were observed in spring 2004, spring

2005, and spring 2006, during the late spring/early summer when the population size decreased (Fig. 3a). We also noticed an apparent absence of infection in both species in early fall 2005 and 2006, and for A. montensis also in late spring 2005 and 2006. As mentioned before, both species presented a different pattern of prevalence throughout the year (except for 2004, when they showed the same seasonal pattern). The influence of population sizes on prevalence was observed only in A. montesis for the current month (r = 0.690, p = 0.027). No significant relation was observed in A. montensis for one capture session time lag (r = 0.380, p = 0.313) neither for O. nigripes (for the current month r = 0.371, p = 0.291; for one capture session time lag r = 0.469, p = 0.354). 3.3. Generalized linear models: variables related to hantavirus infection According to logistic univariate analysis, two independent variables were considered for the model of JABV: gender and A. montensis population size. After the forward–backward stepwise procedure, both variables were retained in the final model (Table 3) indicating that infection will more probably occur in males and will be higher in A. montensis population size. For the JUQV model, logistic univariate analysis considered five variables: gender, locality, season, temperature, and rodent species richness. Forward–backward stepwise procedure retained four variables in the final model (sex, locality, season, and temperature) (Table 3) indicating that infection is related to males, AC and DV localities, spring/summer seasons, and higher mean temperatures (virus-positive animals occurred during months with higher mean temperature than virus-negative animals). 4. Discussion More than 18 years have passed since HCPS was first described in the American continent. Since then, hantavirus studies, predominantly ecological studies on wild reservoirs, were conducted in many regions (Mills et al., 2010; Palma et al., 2012). In Brazil, most of the studies concerning hantavirus reservoirs were either virus descriptions or short-term local investigations (Oliveira et al., 2009; Raboni et al., 2009; Suzuki et al., 2004; Travassos da Rosa et al., 2010). In this study, we thus present the first population ecology study of rodent reservoir population dynamics and hantavirus prevalence in an endemic area of HCPS in Brazil.

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O. nigripes and A. montensis, which were the most abundant rodent species in this study, accounted for more than 90% of hantavirus antibody positive specimens indicating their important role in the dynamics of the hantavirus cycle in the study area. The two distinct hantaviruses observed, JUQV in O. nigripes and the JABV in A. montensis, were previously reported in Brazil and in Paraguay (Chu et al., 2009; Oliveira et al., 2011; Suzuki et al., 2004). The total antibody prevalence found (6.14%) during the three years was in agreement with a similar estimate (5.6%) reported by Mills et al. (2007) in Argentina, which was also in a three-year study. The population dynamics of O. nigripes and A. montensis rodents showed a seasonal pattern with population increases during the winter. The marked pattern of high population sizes of both species at the end of the mild seasons and beginning of the cold seasons could be owing to the recruitment of young individuals born in the previous summer and early fall. The inverse relation observed between population sizes and the frequency of reproducing females during the same months in both rodents as well as the direct relation with two capture sessions time lag corroborates this pattern. According to Mills (2005), increases in reproductive success owing to environmental factors, such as rainfall, habitat quality and food availability, influence hantavirus transmission rates within rodent populations. In our study, O. nigripes and A. montensis were captured in anthropic environments, especially in agroecosystem borders and cornfields, where they could have extra offerings of food derived from grain crops, and which may have caused the increase in the observed population sizes. Both species are considered habitat generalists (Bonvicino et al., 2002; Weksler and Bonvicino, 2005) and they were observed in all study localities, in areas of forest edges and the interface between sylvatic and peridomestic environments being a potential risk for hantavirus transmission to humans. These increases in rodent populations, especially for O. nigripes, may also be a consequence from mast seeding of the taquara lixa bamboos, Merostachys fistulosa, observed in the study area during 2006. O. nigripes as well as A. montensis are considered as granivorous/frugivorous species, eating different kinds of seeds and fruits (Robinson and Redford, 1986; Vieira et al., 2006). This bamboo species blooms synchronically over a region after a long period of vegetative reproduction producing a large amount of seeds on the ground. This phenomenon has been associated with rodent outbreaks in South America called “ratadas” (Jaksic and Lima, 2003; Miller and Rottmann, 1976; Pereira, 1941). Sage et al. (2007) reported an increase in populations of another Oligoryzomys species (O. longicaudatus) in Southwestern Argentina that is associated to bamboo blooming, with an extending breeding season and larger litter sizes suggesting that other species which eat invertebrates were not affected. In Brazil, Giovannoni et al. (1946) also reported an outbreak of Akodon nigrita (= T. nigrita) and Oligoryzomys in the State of Paraná that was associated with such phenomena. A. montensis and O. nigripes presented different patterns of hantavirus infection dynamics resulting in different descriptive models. The positive correlation found between prevalence and population sizes in A. montensis and the results of GLM analysis for JABV showed a positive relation between A. montensis population sizes and positive seroprevalence, which occurred during the winter/spring seasons indicating that transmission is a function of density or population size (Dobson and Hudson, 1995) that corroborates with other studies (Engelthaler et al., 1999; Gubler et al., 2001; Suzán et al., 2009; Yates et al., 2002). Given the horizontal transmission of hantavirus within rodent populations, increasing population sizes should result in increased rodent-to-rodent contact, opportunities for virus transmission (to susceptible rodents), and overall incidence and cumulative prevalence of infection within host populations (Mills et al., 1999). The low temperature and humid environmental conditions could favor

survival of the virus outside the host (Kallio et al., 2006). Furthermore, in A. montensis, the winter could adversely affect rodent condition, increase susceptibility to hantavirus infection, and thus create higher environmental contamination owing to increased virus shedding by hosts with low virus resistance, facilitating indirect transmission during winter months as observed for hantavirus in Europe (Beldomenico et al., 2008; Kallio et al., 2006; Tersago et al., 2011). In O. nigripes, which was infected with JUQV, the prevalence pattern of hantavirus infection increased in spring/summer, periods of higher reproductive frequency and higher temperatures. Although we did not observe a significant correlation between prevalence and population sizes for this rodent, the GLM analysis showed that seasons and temperatures were related to positive JUQV seroprevalence in this rodent. The lack of a relation between host population size and antibody prevalence occurred also in others studies (Mills et al., 1997; Owen et al., 2010) and the observation of the highest antibody prevalence during periods of low population densities has also been observed in North American (Abbott et al., 1999; Douglass et al., 2001) and, recently, in South American hantavirus host populations (Mills et al., 2007). Gender was significant in both univaritate and multivariate models for A. montensis and O. nigripes hantavirus infection. Univariate analysis also indicated that older animals were more prone to infection. These results indicate a life history component in the hantavirus transmission for both species and are consistent with the hypotheses that transmission of hantavirus occurs mainly with breeding activity and agonistic behavior of adult males during the breeding season by horizontal transmission (Douglass et al., 2001; Mills et al., 1999). Although both males and females are aggressive during the breeding season, as documented for genus Peromyscus and Akodon, females spend more time in the core of their territories, whereas males spend more time on the periphery and have larger home ranges than females (Bonaventura et al., 1992; Gentile et al., 1997; Ostfeld, 1990; Wolff, 1989), thus males come in contact with other individuals more frequently than females, resulting in a higher number of aggressive interactions. Recent laboratory studies suggested that the cause-and-effect relationship between aggression and infection might be complex. For example, Norway rats infected with Seoul virus were more aggressive than uninfected males, and aggressive males had more virus in their tissues than did less aggressive males (Klein et al., 2004). Differences in host immune response to infection might also affect the likelihood that infected individuals remain infectious. Lehmer et al. (2007) reported experimental evidence that male deer mice were less immunocompetent than females and suggested that this condition helps explain the commonly observed higher infection prevalence in males compared to females. Considering the population turnover, we observed for both species, that the highest host population sizes occurred after the breeding season (early winter), when the population has a high proportion of young and therefore, uninfected individuals. Therefore, the highest prevalence of infection might occur in the spring (earlier in A. montensis and later in O. nigripes) when the population age structure is older. Once infection is greatest in older animals, prevalences should rise when populations move toward an older age structure (Madhav et al., 2007; Mills et al., 2010, 1999). O. nigripes showed a delay in this pattern in relation to A. montesis, what can be owing to differences in the dynamic transmission patterns of the two hantavirus genotypes. The sporadic infection observed in T. nigrita and A. paranaensis may have been owing to the spillover from O. nigripes and A. montensis. Only one T. nigrita antibody-positive individual was found during the late winter of 2006, harboring the JUQV, when the O. nigripes population size was the highest, increasing interspecies contact chances. The same was observed for A. paranaensis in relation to JABV, in which the four antibody-positive individuals

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were detected during the period of highest population sizes of A. montensis. Studies of sympatric host–virus systems have indicated that sympatric hantaviruses tend to maintain their host specificity. Significant spillover only occurs during periods of high rodent population sizes and increased interspecific contact (Childs et al., 1994; Mills et al., 1997; Nichol et al., 1993; Suárez et al., 2003). Both JUQV and JABV were detected in the same localities, almost in every capture session, near enough to interact (with their rodent reservoirs), that is, the viruses circulate in the same rodent reservoir communities. Similar results were observed by Chu et al. (2009), where these two distinct hantaviruses appear to be maintaining a sympatric status across a considerable expanse of landscape, rather than reflecting a temporary or localized phenomenon. Hantaviral RNA were not detected in five antibody positive rodent specimens – 2 O. nigripes from class III (1 male and 1 female) and 2 A. montensis (1 male and 1 female) from class II, and also one S. angoya (female, 120 g) suggesting that antibodies could be a result of a nonproductive persistent infection, especially in the case of females that are considered more immunocompetent than males. On the other hand, despite their weight classes, the possibility of the transmission (detection) of maternal antibodies cannot be excluded, since recent studies have shown that these antibodies can play a protective role against hantavirus infection during the first 2 months of life in various rodent reservoirs (Easterbrook and Klein, 2008; Kallio et al., 2010, 2006). Locality was also an important variable in the infection transmission model described for JUQV, which is until now the only hantavirus that causes HCPS in humans in this area. Although all the studied areas are disturbed agroecosystems, the AC and DV localities favored the occurrence of hantavirus infection. The most important characteristic of these areas, in relation to the others, is the wide abundance of bamboo thickets. As mentioned before, the bamboo blooming during the studied period may have favored the increases in population sizes of O. nigripes and T. nigrita, in 2006, the same year of the highest incidence of HCPS in the Santa Catarina State, mainly in the western region. Approximately 50% of these cases were in municipalities where rodent outbreaks were reported after a widespread bamboo mast event (Epidemiologic Surveillance of Santa Catarina State, unpublished data). The probability of transmission to humans varies according to human risk behaviors and their associated land uses. Some activities are associated with a close proximity to host habitat and thus, increase the likelihood of human–host contacts. Harvesting and storing grains increase human exposure to rodents and probably is the major risk factor of human infection in this area. Increases in JUQV transmission in rodents were modulated by gender, season, and environmental characteristics of the local habitats, while in JABV they were modulated by gender and rodents’ population size. This indicates that the causes of HCPS outbreaks are multifactorial and vary with the virus, once they are reservoir specific. Conflict of interest statement No competing financial interests exist. Acknowledgments We thank the Jaborá Municipal Health Secretary, the Santa Catarina Health Secretary, and the Secretary of Health Surveillance for assistance during the development of our studies in Jaborá. We are grateful to the field team of the Laboratory of Biology and Parasitology of Wild Mammal Reservoirs, IOC/FIOCRUZ, who participated in the Jaborá expeditions. We thank Dr. Helena Bergallo (UERJ/RJ) and Dr. Sotiris Missailidis for time spent on reviewing the manuscript. We thank Raphael Gomes for technical assistance, Ms.

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Maria Angélica Mares-Guia for image production and treatment assistance. We thank Dr. Fabiana Caramaschi and Dr. Danielle Dias for assistance specific identification of small mammals. This study was supported financially by CNPq Project 39/2004 A – “Animais Reservatórios e Controle Vetorial,” Brazil and partially supported by Grant No. DPC/CD/133/06 awarded by the Pan American Health Organization. All the procedures involving the animals were previously approved by the institutional Ethics Committee on Animal Research, Process number CEUA P-0336-07/CNPq Process No. 403050/2004-9. Permits for field collection were granted by IBAMA (Brazilian Institute of Environment and Renewable Natural Resources) under process number IBAMA No. 02001.005266/200315. References Abbott, K.D., Ksiazek, T.G., Mills, J.N., 1999. Long-term hantavirus persistence in rodent populations in central Arizona. Emerg. Infect. Dis. 5, 102–112. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. 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