Serosurvey of hantavirus infection in humans in the border region between Brazil and Argentina

Introduction: According to reports by the Ministry of Health, in the far western region of the State of Santa Catarina, there have been no reports of hantavirus pulmonary syndrome, a zoonotic disease transmitted by feces of infected rodents. A seroepidemiological study of residents of this region, was conducted, with the aim of determining the presence of hantavirus infections. A total of 340 volunteers of both genus, from the towns of Belmonte and Paraíso, were studied. Methods: The serum of these patients was collected and used to detect IgG antibodies against recombinant N protein of Araraquara hantavirus, by ELISA assay. The positive samples were then titrated and confirmed by immunofluorescence assay. Results: This study demonstrated the presence of IgG antibodies against hantavirus N protein in 3.5% of the population. The most frequent occupation was farm worker, 81% had direct and indirect contact with rodents, 91.7% of positive cases were farm workers, indicating that the probable cause of infection occurred during barn cleaning. These antibodies are noteworthy, given that the levels of antibodies were verified in individuals whose contact with hantavirus may have occurred many years ago. Conclusions: This study shows the circulation of hantavirus in the region, a fact that until now, had not reported. All the serum reagents had contact with the pathogen, but did not develop pulmonary and cardiovascular syndrome. It is important to remain alert, because hantavirus is a serious and emerging disease of some relevance.


INTRODUCTION
Viruses of the genus hantavirus can cause two serious illnesses when transmitted from rodents to humans: hemorrhagic fever with renal syndrome (HFSR) or hantavirus pulmonary syndrome (HPS), characterized by respiratory failure, shock and high mortality, making it an important public health problem 1 .
Hantavirus is an enveloped virus with trisegmented negative sense RNA genome, defined as small, medium and large.Human infection is acquired by inhalation of aerosols containing excreta of rodents infected with hantavirus; i.e., infected by a virus of the family Bunyaviridae 2 , which includes than 20 hantaviruses identified throughout the American continent 3 .
Interpersonal transmission of hantavirus has been reported only in Argentina and Chile, but further investigation indicated that this possibility is very unlikely [4][5][6] .The contact with hantavirus in and of itself does not cause HPS, since infection is dependent on the quantity of viral particles, such that lower concentrations of particles could lead to subclinical infection.However, another possibility is infection by a nonpathogenic strain that induces immune response activation with antibody production 3,7 .
The pathogenesis of HPS in humans is hypothesized to be mediated by immune activation and excess inflammatory responses, especially macrophages and CD8 + T cells 3 .Excess cytokines IL-1-α, IL-1-β, IL-6 and TNF-α interferon-γ, IL-2, IL-4 and TNF-β produced by activated macrophages in hantaviruses, specific T cells in recognition antigen infected pulmonary endothelial cells, are thought to be critical due to the increased permeability in the pathogenesis of HPS 8 .
Despite the severity of HPS, cases of human infection with mild forms of hantavirus without respiratory failure do occur; however the causes and factors involved in clinical behavior remain unknown 9 .

Souza WM et al -Human hantavirus serosurvey on the Brazil Argentina border
Numerous diagnostic methods for these viruses have been produced, among these, the production of recombinant proteins, which are used as antigen in ELISA to enable detection of specific antibodies against this virus, should be highlighted 10,11 .
In South America, hantaviruses have been reported in Argentina, Chile, Uruguay, Paraguay, Bolivia and Venezuela 12 .In Brazil, the first evidence that this virus was circulating was observed in the study of three individuals living in a rural area of Juquitiba, São Paulo, in 1993 13 .Today, over 1,200 cases of hantavirus pulmonary syndrome have occurred in Brazil since 1993, fatalities were reported at a rate of 39%, according to geographic and ethnic differences 14 .Five hantaviruses associated with HPS are currently known in Brazil: the Juquitiba, Araraquara, Laguna Negra-like, Castelo dos Sonhos and Anajatuba viruses 15 .
The first confirmed case of hantavirus in Santa Catarina was in 1999 and up to 2009, 205 cases have been confirmed.The first death from hantavirus in the state was reported in 2001 and 48 deaths have been reported up to the present, making this the state with the second highest death rate 16 .HPS is an emerging public health problem in Brazil due to the overlap of urban and agricultural areas, livestock and the disorderly growth of human population in areas where imbalance occurs, which increases human contact with several species of rodents that are reservoirs of hantavirus 15 .This study was conducted to investigate the presence of antibodies against hantavirus in the far western region of the State of Santa Catarina and the southern border of Brazil and northern Argentina and correlate this presence through interviews to elucidate the medical history and morbidity of the participants.
The study region has an economic base that comes from agricultural activities and accordingly, the degrading of native forest has occurred throughout the region, which is favorable to colonization by wild rodents.The study revealed the presence of Necromys lasiurus and Oryzomys nigripes in the region, which are known rodent reservoirs of hantavirus, suggesting there is a chance of viral circulation and, therefore, risk of human infection 17 .
Although Santa Catarina is the state with the second d highest number of HPS cases, no cases of the disease have been recorded in the far west of the state 16 or in municipalities in the study area that borders northeastern Argentina; however, previous studies have shown that the region has high numbers of HPS 18 .Thus, it is unclear whether the lack of notification of hantavirus disease in this region is due, in fact, the absence of local disease or that infections occur and diagnosis is negligent.So far, no accurate survey of the presence of hantavirus has been conducted in the far west of Santa Catarina.Diagnostic tests to detect hantaviruses are only performed following clinical suspicion during hospital attendance, which is not common in the region.
The serological survey aimed to assess, for the first time, current results and scientific evidence of the absence or circulation of hantavirus in the far west of the state by helping to clarify the real situation of the virus in the region.

Study area
The towns of Belmonte and Paraíso have respective populations of 2,681 and 4,195 inhabitants, who are mostly descended from Italians, Germans and Poles.They have a mesothermal humid climate, with hot summers and an average temperature of 18.3°C, with the main economic activity based on agriculture, particularly the cultivation of monocultures of corn, wheat, tobacco and, consequently, a large part of the native forest has suffered degradation (Figure 1).

A n t i r g e n t i n a
Paraguay

Human samples
Prior statistical analysis determined the calculation of a proportional stratified sample of the population; thus, a total of 340 volunteers of both sexes, aged between 18 and 90 years-old in December (summer) 2009, permitted the collection of blood samples.

Immunoassay
The blood samples were subjected to centrifugation to extract the serum, which were subjected to enzyme linked immunosorbent assay (ELISA) using as antigen a recombinant N protein of Araraquara hantavirus produced in E. coli.The samples were tested in duplicate, using a known sample positive for hantavirus Araraquara as the positive control and as the negative control, a protein extract of E. coli not containing the plasmid encoding the recombinant protein, according to the protocol developed by Figueiredo 2009 (Evaluation of an ELISA using rNucleoprotein of Araquarara).Following the test, the samples considered positive were then titrated.To achieve this, the sera were subjected to serial dilution of 1:100 to 1:12,800.These dilutions were retested in ELISA to determine the concentration of IgG anti-hantavirus in each of the serum.

Immunofluorescence
As a confirmatory test, positive sera in ELISA and titrated by the same method were submitted to indirect immunofluorescence assay (IFA) using cells infected with Rio Mamoré hantavirus and prefixed in spot slides for IFA, as described by Leduc 19 .The sera were then added to each of the spots.To determine the presence or absence of IgG anti-hantavirus, the sera were tested in a second antibody antihuman IgG labeled with the fluorochrome FITC and then observed under a fluorescent microscope.Positive and negative controls were performed using sera with or without anti-hantavirus antibodies.

Ethical considerations
The study was reviewed and approved by the Research Ethics Committee, in accordance with Ministry of Health protocol n o .073/2009.Free informed consent was obtained from all patients, and the information was kept confidential.
A total of 340 volunteers with no prior history of hantaviruses were studied, residents of the microregion of the far west of the State of Santa Catarina, from the towns of Belmonte and Paraíso, on the Brazilian border with the Province of Misiones of the Republic of Argentina.Of these, 52.7% were men and 47.3% were women.Regarding the occupation of the respondents, 96% were directly related to the agricultural system and the occupation of farm worker was the most frequent with 85%.
Among those interviewed, 80% reported storing food in barns and sheds (grain and tobacco) and at these sites and in the surrounding areas, 81% reported visual or physical contact with rodents.The procedure for cleaning these sites included sweeping and raking in 75.9%.In houses, 64.3% said there was no garbage collection, such that this accumulated and was then incinerated, a practice that could attract rodents, thus increasing possible contact with hantavirus.
In enzyme immunoassays, 12 samples were seroreactive for the presence of IgG antibodies against N protein of hantaviruses, determining a prevalence of 3.5% of these antibodies in the population studied, while quantitative analysis, involving titration of positive sera, showed evidence of variation of up to 1:200 1:6,400.Sera positive for hantavirus by ELISA were confirmed by IFA.
Regarding medical history of morbidity presented by individuals who were positive for hantavirus, these patients reported the following symptoms: fever over 38°C for several days (33.3%), dyspnea (25%) and nasal bleeding (16.6%), though the symptoms evolved to cure in a few days without medical intervention or hospitalization (Table 1).Although these symptoms are common in HPS, they are not very specific and the events cited by respondents cannot be directly linked to any disease or infection, since few responded with certainty regarding the moment of contact and any subsequent production of IgG antibodies against hantavirus.
Evidence of the presence of hantavirus in Brazil have been observed since the 1970's, initially isolated in Brazil in the State of Pará in Rattus norvegicus in the 1980's 19 .At this time showed, the presence of antibodies to Hantaan virus was also observed, the prototype of the genus hantavirus in rodent sera in the cities of Belém, São Paulo and Recife 19 .Seroepidemiology in patients of northern Brazil identified Hantaan IgG and IgM in 8.4% to 1.9%.In São Paulo, in 1976, 1.2% of patients admitted presented Hantaan IgM 19 .In 1993, between November and December, three individuals living in rural area of Juquitiba, SP 7 , became ill and from these cases, the importance of intensive epidemiological studies of hantaviruses in Brazil became apparent.Since then, HPS has been observed in all regions of the country; however, most known cases of HPS occurred in the south and southeast of Brazil, where studies conducted in different periods showed volunteer prevalence in these regions.
The seroprevalence in this study falls within the expected profile of areas with confirmed cases of hantavirus infection, as shown by previous studies 7, [19][20][21][22] .However, this finding contradicts the epidemiological situation for hantaviruses, which according to the Brazilian Ministry of Health, is considered harmless for hantavirus.In the region studied, the overall prevalence of anti-hantavirus antibodies among rural and peri-urban residents was 3.5%, with higher prevalence among women, who represented 58.3% of the total number of positive cases.Another study showing seroprevalence of

FIGURE 1 -
FIGURE 1 -Localization of study area Belmonte and Paraíso County.

TABLE 1 -Cases of hantavirus serology reagents according to gender, occupation, city, title and symptoms.
*Fever over 38°C lasting for several days.