Serological evidence of hantavirus infection in neotropical bats in an urban area of São Paulo State, Brazil

ABSTRACT

Background

Although hantaviruses have long been associated with rodents, they are also described in other mammalian hosts, such as shrews, moles and bats. Hantaviruses associated with bats have been described in Asian, European and Brazilian species of bats. As these mammals represent the second major mammalian order, and they are the major mammals that inhabit urban areas, it is extremely important to maintain a viral surveillance in these animals. Our aim was to conduct serosurveillance in bats in an urban area in the city of Ribeirão Preto, São Paulo State, Brazil, to contribute to the information about hantaviruses circulation in bats.

Methods

We analyzed samples from 778 neotropical bat specimens classified into 21 bat species and four different families collected in the urban area of Ribeirão Preto city, from 2014 to 2019 by an ELISA for the detection of IgG antibodies against orthohantavirus.

Results

We detected IgG-specific antibodies against the nucleoprotein of orthohantavirus in 0.9% (7/778) bats tested, including four Molossus molossus (Pallas' Free-tailed Bat), two Glossophaga soricina (Pallas's Long-tongued Bat) and one Eumops glaucinus (Wagner's mastiff bat).

Conclusions

Overall, our results show the first serological evidence of hantavirus infection in three common bat species in urban areas.

Introduction

Hantaviruses (Hantaviridae family) are enveloped with three unique segments of single-strained RNA,¹ and these viruses can cause two distinct diseases, the hemorrhagic fever with renal syndrome in Asia and Europe, and the hantavirus cardiopulmonary syndrome (HCPS) in the Americas.² Both are transmitted by inhaling aerosolized excretions or direct contact with secretions of wild rodent species, and also some person-to-person transmission cases have been described.^3,4

From 1993 to 2018, 2117 HCPS cases were reported in Brazil,⁵ mainly associated with six pathogenic hantaviruses, namely, Anajatuba, Castelo dos Sonhos, Laguna Negra, Araraquara, Juquitiba and Rio Mamoré.^2,6,7 Previous studies have described hantaviruses in several species of bats in Asia and Europe,^3,8 but bat-borne hantaviruses have not yet been reported in neotropical bats. However, some molecular and serological evidence has been reported in neotropical bats, such as Anoura caudifer, Artibeus gnomus, Artibeus lituratus, Artibeus obscurus, Artibeus planirostris, Carollia perspicillata, Chiroderma villosum, Chrotopterus auritus, Desmodus rotundus, Diphylla ecaudata and Phyllostomus hastatus.^9–11 Most studies have described hantavirus in bats captured in the natural environment, and the circulation of hantaviruses in bats in urban settings remains poorly studied. Thus, we aimed to investigate hantavirus circulation in neotropical bats in an urban area in the city of Ribeirão Preto, São Paulo State, Brazil.

Materials and Methods

From 14 November 2014 to 25 July 2019, blood and anal swab samples were collected by the Zoonoses Control Center of Ribeirão Preto city from bats found in residential buildings, as part of a rabies virus surveillance program that was authorized by the Ethics Committee on the Use of Animals of the University of São Paulo (0173/2018). All the animals were identified based on the external morphology.¹² We tested all blood samples (dilution 1:100) in duplicate by an ELISA for detection of IgG antibodies against orthohantavirus, using the nucleoprotein recombinant protein (rN) of Araraquara virus (classified into Andes orthohantavirus) as antigen and an anti-Bat (Bethyl Laboratories, Inc., USA, catalog number: A140-118P) as secondary antibody (1:2000), as previously described.¹³ This ELISA has a sensitivity of 97.2% and a specificity of 100%.¹³ The cutoff value was defined as the mean plus three SDs. We also tested anal swab samples for all ELISA-positive samples for detection of hantavirus RNA. The RNA was extracted using QIAamp viral RNA Extraction Kit (Qiagen, Germany) following the manufacturer’s instructions. Then cDNA synthesis was performed using the M-MLV enzyme (Thermo Fisher Scientific, USA), with random primers as recommended by the manufacturer. For the detection of hantaviruses’ RNA, we performed a nested-PCR targeting a conserved region of the Orthohantavirus L gene, as previously described.¹⁴ Rio Mamore HTN-007 strain was used as positive control.

Results

We analyzed biological samples from 778 neotropical bat specimens captured during almost 5 years that were classified into 21 bat species of four different families, namely, Noctilionidae (n=1 species), Vespertilionidae (n=6 species), Phyllostomidae (n=6 species) and Molossidae (n=8 species). The molossid bats (free-tailed bats) constituted 68.8% (535/778) of bat specimens collected in this study, with Molossus molossus, the Pallas' Free-tailed Bat (42.3%, 329/778), and Nyctinomops laticaudatus, the broad-eared bat (18.6%, 145/778), the most frequent bat species. We detected hantavirus-specific IgG antibodies against the nucleoprotein of orthohantavirus in 0.9% (7/778) of the tested bats, of which four of the seven were detected in Molossus molossus, representing 1.2% (4/329) of positive IgG antibodies in this bat species (Figure 1A). In addition, we found IgG specific for hantaviruses in two Glossophaga soricina, the Pallas's Long-tongued Bat, and one Eumops glaucinus, the Wagner's mastiff bat, which represented 9.5% (2/21) and 1.3% (1/76) of IgG antibodies positive for hantaviruses in each bat species, respectively (Figure 1A). The anal swabs tested for the presence of RNA hantavirus were all negative.

Figure 1.

The bat species analyzed in this study and the geographic distribution of those that were positive. (A) Bar chart representing the number of individuals for each species analyzed for the ELISA (blue bars), and the percentage of positive individuals (red bars). (B) Geographical distribution of the positive species Molossus molossus, Eumops glaucinus and Glossophaga soricina. The geographic range for the three bat species was obtained from the International Union for Conservation of Nature Red List.²⁵

Discussion

In this study, we identified IgG antibodies specific for orthohantavirus in three species of neotropical bats in the urban area of Ribeirão Preto city, a large city within São Paulo State, with >710 000 inhabitants. In contrast to previous studies, where evidence of hantavirus infection in bats was described in bats captured in the forest and natural environment,^9–11,¹⁵ the current study shows the hantavirus circulation in bats in an urban setting. Bats are described as the main diversity mammal order in urban areas,¹⁶ and one consequence of the adaption of some bat species in urban areas is frequent contact with humans and domestic animals, including some species inhabiting the roofs of residential buildings, such as the Pallas' Free-tailed Bat (M. molossus).¹⁷

Our study shows the first serological evidence of orthohantavirus infection in molossid bats, the Pallas' Free-tailed Bat and the Wagner's mastiff bat (M. molossus and E. glaucinus, respectively); both species are insectivorous and present a broad distribution throughout Brazil, and they are very common in the urban environment (Figure 1B).¹⁸Molossus molossus represented around 42.3% of our sampled bats during almost 5 consecutive years in Ribeirão Preto city, indicating that this species is highly abundant in the urban area of this city. In addition, we found antibodies against orthohantavirus in a species from the Phyllostomidae family, the Pallas's Long-tongued Bat (G. soricina), a nectarivorous species with broad distribution throughout Brazil (Figure 1B).¹⁸ Previous studies have reported the detection of RNA of Araraquara virus (Andes orthohantavirus) in the Tailed tailless bat (Anoura caudifer), another nectarivorous species of the Phyllostomid family.¹¹ Currently, 10 different species of bat-borne hantavirus have been described in Old World bats, of which nine were described in insectivorous bats and only one in a frugivorous bat.³ The feeding behavior of bats could influence the transmission of hantaviruses among bat species if transmission was related to the same or similar diets, or by using the same foraging sites.¹¹

Here, we did not detect hantaviruses’ RNA in the ELISA-positive individuals. However, we could only investigate the presence of genomes by RT-PCR in anal swab samples due to the limited volume of blood samples available, and also because of the absence of tissue samples from these animals. Several factors could explain the absence of hantavirus RNA in our anal swab samples. First, tissue samples (e.g. lungs) seem to be the most reliable samples for detecting hantavirus RNA due to the higher viral load.¹⁹ Second, some orthohantaviruses, such as Andes orthohantavirus and Puumala orthohantavirus, are not commonly found in urine and feces samples because of the inhibitors that are present in feces that can degrade the viral RNA.^19–21 Third, it might be possible that the hantavirus infecting the bats in our study could be a divergent virus that cannot be detected by the primer sets used in our nested PCR.¹⁴ Fourth, some rodent reservoirs can be persistently infected by hantaviruses throughout their lifespan,²² however, the general aspects of the immune system and clearance during the hantavirus infection in bats is poorly investigated.^23,24 Thus, according to our results, we recommend, if available, a screening for viral RNA in tissue samples, mainly lungs and kidneys, and also by using different methodologies, such as metagenomics, or even different PCR assays.

In addition, we highlight that bats play several critical ecological roles, such as pollination, seed dispersion and insect and plague controls, and our study does not intend to encourage any villainization or culling of bats and the fact that bats can harbor several viruses should be treated as another factor for conservation of the natural areas that these animals inhabit.

Conclusions

In conclusion, our results show hantavirus infection in three species of neotropical bats in the urban area. The role of bats in the hantavirus transmission cycle remains poorly understood. However, viral surveillance in these mammals is critical for monitoring and understanding the viral diversity present in these animals that share common areas with humans and domestic animals.

Authors’ contributions

Data collection was performed by RDA. Analysis and interpretation of data were performed by LMB and DMM. The manuscript was written by LMB and WMS. The review of the manuscript was carried out by LMB, DMM, WMS, RDA and LTMF. All the authors approved the final version.

Funding

This work was financed by the São Paulo Research Foundation—FAPESP [process no. 2014/02438–6]. LMB was a recipient of a FAPESP scholarship [process no. 2019/07443–1] and DMM is a recipient of a Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—CAPES scholarship [process no. 88882.328424/2019–01]. WMS is supported by the Global Virus Network fellowship and the NIH [AI12094].

Competing interests

The authors declare no conflicts of interest.

Ethical approval

The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. The Guidelines of the American Society of Mammalogists for the use of wild mammals in research and education were followed.

Data availability

Aditional data underlying this article will be shared on reasonable request to the corresponding author.