Seroprevalence of arboviruses in Nasua nasua (Mammalia, Carnivora, Procyonidae) of synanthropic habitats in the Iguaçu National Park, Brazilian Atlantic Forest

ABSTRACT: Arboviruses are agents transmitted by arthropods and represent a considerable threat to public health worldwide, causing diseases in humans and animals. A serological investigation was carried out to detect total antibodies against different types of arboviruses in free-living coatis (Nasua nasua) from the Iguaçu National Park (INP) through the Hemagglutination Inhibition test. Serum samples were tested using antigens from 33 arboviruses belonging to the genera Alphavirus, Flavivirus, Orthobunyavirus, and Phlebovirus. The data showed that 23.6% (17/72) of coatis were seropositive for at least one of the tested antigens, including monotypic and heterotypic reactions. Seropositivity was detected for Alphavirus (5.9%, 1/17; WEEV), Flavivirus (64.7%, 11/17; YFV, ILHV, SLEV, BSQV, ROCV, WNV, DENV-1, DENV-2, DENV-3, DENV-4, and NJLV), Phlebovirus (88.2%, 15/17; ICOV and BUJV) and Orthobunyavirus (5.9%, 1/17; ORIV). The presence of antibodies to these viruses in coatis from INP indicated an apparent silent circulation of arbovirus, implying N. nasua to be a possible amplifying host of these arboviruses in the studied area. The data reported also serve as a warning about the possible risk of establishing an arbovirus transmission cycle involving vector arthropods and coatis, or even other wild animals, consequently, including humans in this transmission chain.


INTRODUCTION
The Iguaçu National Park (INP) is one of the main centers of ecotourism convergence in Brazil and is part of the official list of Natural Heritage of Humanity by the United Nations Educational, Scientific and Cultural Organization (UNESCO) since 1986 (GUIMARÃES et al., 2003).The INP has an abundant Ciência Rural, v.53, n.2, 2023.Rodrigues et al. faunal diversity of mammals, such as jaguars, tapirs, and coatis (FERNANDES & GARCIA, 2011).
Coatis (Nasua nasua) are omnivorous animals that have a rich diet characterized mainly by insects and wild fruits (GOMPPER & DECKER, 1998).Populations of coatis are quite tolerant of anthropogenic disorders, which has facilitated the synanthropic process, and thus, the risk of transmission of zoonotic pathogens, such as those causing rabies, arboviruses, and leishmaniasis (ALVES- COSTA & ETEROVICK, 2007;GUIMARÃES et al., 2012).
Arboviruses are agents transmitted by arthropods and represent a considerable threat to public health worldwide (WEAVER & REISEN, 2010), causing diseases in humans and animals (ICTV, 2020).In infected humans, arbovirus can range from undifferentiated, moderate, or severe febrile illnesses, skin rashes, and arthralgia to neurological and hemorrhagic syndromes (CLETON et al., 2012).Therefore, this study investigated the seroprevalence of arbovirus in coatis of synanthropic habits in the INP, Paraná State, Brazil.

Study animals and sample collection
Serum samples were collected between 2014 and 2015 from 72 coatis, males and females, of synanthropic habits in the INP, Paraná State, Southern Brazil, using three sampling locations (Figure 1).Coatis were attracted to peanut baits, captured with a hand net, and anesthetized with a combination of tiletamine-zolazepan (Zoletil 50, Virbac ® , Carros/ France, 7 mg/kg), and xylazine hydrochloride 2% (Xilazin, Syntec ® , São Paulo/Brazil, 2 mg/kg), administered intramuscularly.After recovery, all animals were reintroduced into their original habitat.Blood samples obtained through the jugular vein were centrifuged (2000 rpm, 5 minutes) to obtain serum samples, which were identified and stored at -70 ºC until further analysis.

Hemagglutination inhibition test
The serum samples were subjected to the hemagglutination inhibition (HI) test according to CLARKE & CASALS (1958), with adaptation to microplate by Shope (1963).The study was carried out at the Section of Arbovirology and Hemorrhagic Fevers of the Evandro Chagas Institute (SAARB/ IEC), municipality of Ananindeua, Pará State, Brazil.
Samples that showed antibody titers during the screening step were tested in the titration step.Positive serum (25 µL) was added and diluted using 0.4 % bovine albumin serum at pH 9.0.Next, 25 µL of the antigen was added and incubated for 12 h at 4 ºC.The result was considered positive when the erythrocyte sedimentation and the titer were greater than or equal to 1:20.
Of the seropositive animals, 58.82% (10/17) presented a heterotypic reaction for Flavivirus, Phlebovirus, and/or Orthobunyavirus.Phlebovirus was the most prevalent genus, occurring in 88.23% of seropositive animals (15/17), and Flavivirus exhibited the highest cross-reactivity, due to the high sensitivity of the HI test, it is common for there to be a high rate of cross-reactions between viruses belonging to the same genus, as they are similar to each other (VASCONCELOS, 2003).
This study is the first to specifically investigate the role of coatis as possible hosts of arbovirus and shows the distribution and diversity of the arboviruses who belong to the genera Alphavirus, Flavivirus, Phlebovirus, and Orthobunyavirus, in the INP.In Brazil, remnants of the Atlantic Forest and the Amazon rainforest are known to be the largest reservoirs of arbovirus (SILVA & ANGERAMI, 2008), and the serological findings of the present study suggested the same trend in the INP.
The Foz de Iguaçu region brings together the factors considered ideal for the proliferation of vectors: it has a large circulation of people because it is a tourist city, and it provides a variety of food options (animals and humans), which facilitates pathogen transmission, thereby making it a city prone to the emergence or reemergence of diseases (GÓIS, 2017).According to the municipality's Epidemiological Bulletin (BRASIL, 2018), the case records for Chikungunya and Zika started in 2015.In this study, antibodies were detected for the Alphavirus and Flavivirus genera (the genera to which the Chikungunya and Zika viruses belong to), suggesting that coatis may play a role as host of arbovirus.
Although, mosquitoes have low host specificity, feeding on several wild species (GÓIS, 2017), references confirming the importance of coatis as a food source for mosquitoes are scarce.However, these animals are known hosts of other ectoparasites, such as ticks and lice, which even Conversely, monotypic reactions were observed for Alphavirus (WEEV), Flavivirus (DENV-4), and Phlebovirus (BUJV), which suggested the circulation of these or other viruses related to them in the studied population.Paraná State, among other states in Brazil and countries in South America, is part of the migratory route for wild birds, which may constitute natural reservoirs of viruses that cause encephalomyelitis (FERNáNDEZ et al., 2000).Wild birds and horses from a region of the Brazilian Pantanal were seroreactive for EEEV, WEEV, Venezuelan equine encephalitis virus (VEEV), ILHV, ROCV, SLEV, and TCMV (IVERSSON et al., 1993), which is similar to the observation of the current study.The INP has a great diversity of wild birds  (FERNANDES & GARCIA, 2011), which, among other free-living species in the park, may be potential transmitters of these arboviruses to coatis, and further studies are needed to clarify this role.
The dengue virus has four serotypes (1, 2, 3, and 4).In wild transmission, primates are the main hosts, while humans are accidental hosts.There is no reported cross-immunity, so infection by one of the serotypes only confers immunity to that serotype (CHEN & WILSON, 2010;KORSMAN et al., 2012;TAUIL, 2001).BATISTA (2019) reported cases of dengue in Foz do Iguaçu, noting that in 2010, 2011, 2013, and 2015, the highest numbers of cases of the disease were experienced.Among the monotypic reactions observed in this study, antibodies against DENV-4 were observed in 8.3% of the samples.
The genus Phlebovirus has ten species (ICTV, 2020), which cause a variety of syndromes ranging from a mild febrile illness to infections of the central nervous system (BRAITO et al., 1998;DEPAQUIT et al., 2010;PAPA et al., 2011).In this study, the Phlebovirus species that showed the most monotypic reaction in coatis was the Bujaru virus, suggesting that the INP may be a new area of circulation for this virus.Bujaru virus is an arbovirus whose information is still scarce.It was isolated in Amazonas and Pará (Brazilian Amazon) in rodents Proechimys guyannensis, being serologically detected also in other rodents and marsupials.Arthropod vector and its occurrence in humans are still poorly understood (CDC, 2021).
The monotypic reactions observed suggested the occurrence of infections by at least three of the investigated arbovirus or other antigenically related ones.The Bujaru, western equine encephalitis, and dengue 4 viruses were the only ones that showed a monotypic reaction.In addition to wild cycle viruses, arbovirus with predominantly urban cycles have been detected, such as DENV, which are responsible for outbreaks and epidemics in Brazil (LEÃO et al., 2013;BRASIL, 2018).

CONCLUSION
The presence of viral antibodies in coatis from INP indicate diversity, distribution and an apparent silent circulation of arbovirus of the genera Flavivirus, Alphavirus, Orthobunyavirus, and Phlebovirus, suggesting N. nasua to be a possible amplifying host of these arbovirus in the studied area.The data reported also serve as a warning about the possible risk of establishing an arbovirus transmission cycle involving arthropods vectors and coatis, or even other wild animals, and consequently, may include man as an accidental host in this transmission chain.

Figure 1 -
Figure 1 -Geographic location of the study area.A) Brazil, with emphasis on the Paraná State; B) The municipality of Foz do Iguaçu (west region) is highlighted in the State; C) Iguaçu National Park area, highlighting the tourist area (point); D) Sampling locations in the tourist area.Source: A, B: present study; C, D: Google Earth TM software (georeferenced).

Table 1 -
Individual serological result for Arbovirus in coatis according to sex, age, and type of antibodies.