Antibodies Against Henipa-Like Viruses in Brazilian Bats

Abstract

Bats are reservoir hosts for many paramyxoviruses, some of which cause human and zoonotic diseases of public health importance. We developed a Nipah virus nucleoprotein enzyme-linked immunosorbent assay to detect cross-reactive antibodies in serum samples from several bat species in Brazil. Our results warrant further investigation of henipa-like virus reservoirs in the Western hemisphere.

Paramyxoviruses have been responsible for outbreaks of zoonotic diseases of public health importance. Pteropid bats found throughout Southeast Asia and Australia are reservoir hosts of highly pathogenic viruses in the genus Henipavirus (Halpin et al. 2000, Chua et al. 2002). However, a landmark study identifying bats as reservoir hosts of paramyxoviruses included molecular evidence of henipa-like virus infection in African bats, as well as of morbilli-like virus infection in Brazilian bats (Drexler et al. 2012). Considering the diversity of flora and fauna in Brazil, we explored the possibility of henipa-like virus infection in Brazilian bats. In October 2010, we nonselectively captured, identified (to species whenever possible), and sampled 76 bats of various species in the Diamantina municipality of Minas Gerais state (GPS: 18 14′ 24" S 43 35′ 40" W), which falls within the diverse tropical savannah biome known as the Cerrado.

Bats were sampled for blood and swabs (oral/fecal) according to protocols approved by the Ethics Committee on Animal Experimentation of the Institute of Biomedical Sciences, University of São Paulo and with the permission of the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA, process number 16575-3), and were released on completion of sample collection. All samples were stored in liquid nitrogen in the field and maintained at −80°C. RNA extracted from all swab samples tested negative for the presence of NiV N gene RNA by quantitative reverse-transcription polymerase chain reaction (RT-PCR) (Lo et al. 2012). Aliquoted serum samples were shipped on dry ice to the U.S. Centers for Disease Control and Prevention and were inactivated by gamma-irradiation (5 × 10⁶ rad) before being tested.

We developed an enzyme-linked immunosorbent assay (ELISA) using recombinant NiV nucleoprotein (N) antigen from human embryonic kidney cell lysate (HEK293T) transfected with a eukaryotic expression plasmid expressing NiV N gene (pC-NiVN). Our negative control antigen was cell lysate from HEK293T cells transfected with empty vector plasmid (pC-293T). We validated our NiV N ELISA using both NiV seropositive human and bat sera (Fig. 1A, B), and also confirmed the specificity of our NiV N ELISA by showing a lack of significant cross-reactivity against NiV N antigen by antisera specific for other paramyxoviruses (Fig. 1C).

FIG. 1.

Development and validation of NiV N ELISA. (A) Absorbance values from NiV seropositive human serum incubated with serially diluted NiV N antigen (pC-NiV N) and negative control antigen (pC-293T). (B) Validation of NiV N ELISA for bat serum. Adjusted absorbance values from both NiV seronegative and seropositive Pteropid bat sera along with NiV seropositive human serum incubated with serially diluted pC-NiVN and pC-293T antigens. (C) Confirmation of specificity of NiV N ELISA. Adjusted absorbance values from seropositive human (Hu), bat (Pteropus NiV+), and mouse HMAF compared to NiV-seronegative human (Hu) and goat (Gt) sera specific for other paramyxoviruses as indicated. bRSV, bovine respiratory syncytial virus; CDV, canine distemper virus; ELISA, enzyme-linked immunosorbent assay; HMAF, hyperimmune ascites fluid; hPIV3, human parainfluenza virus-3; MeV, measles virus; MuV, mumps virus; NiV N, Nipah virus nucleoprotein.

We then used the ELISA to query our collection of Brazilian bat sera. Bat serum was considered positive if its adjusted absorbance ([absorbance against pC-NiVN]–[absorbance against pC-293T]) readout was higher than the cutoff value, which was defined as the average absorbance value of the NiV-positive control human serum against negative control antigen (performed in triplicate) plus three times the standard deviation of the average absorbance value. Of the 11 species of bats tested, 5 species tested positive by NiV N ELISA, with 9 of 76 total serum samples testing positive (∼12%) (Table 1). To confirm our results, we assayed these sera by indirect immunofluorescence assay (IFA) on formalin-fixed NiV-infected African green monkey kidney (Vero) cells (Fig. 2 and Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/vbz). Limited amounts of sera precluded us from conducting additional virus neutralization assays. All serum samples that tested positive by NiV N ELISA also tested positive by IFA. There were four additional serum samples that tested positive by IFA that did not test positive in the NiV N ELISA. This could be due to the presence of cross-reactive antibodies against other NiV proteins apart from the nucleoprotein, such as matrix or envelope glycoproteins. By using the IFA results as the indicator for true positive and negative results, the sensitivity of the NiV N ELISA was ∼76.5% and the specificity was 100%.

FIG. 2.

Immunofluorescence assay. NiV seropositive pooled Pteropus serum, Glossophaginae sp3 serum (1:40 final dilution), and NiV seronegative Pteropus poliocephalus serum were incubated with both mock-infected and NiV-infected African green monkey kidney (Vero) cells. A goat anti-bat antibody and a donkey anti-goat antibody conjugated with DyLight 550 (Bethyl) were used, respectively, to detect bat serum reactivity against NiV antigen. DAPI was used as a nuclear counterstain. Representative images were taken at 40× magnification using a Nikon Eclipse Ti inverted fluorescence microscope. Scale bar indicates length of 50 μm. DAPI, diamidino-2-phenylindole dye.

Table 1.

Nipah Virus Nucleoprotein ELISA and IFA Results for Brazilian Bats, 2010

Bat species	No. of positive/total no. of sera (% positive by ELISA)	No. of positive/total no. of sera (% positive by IFA)
Artibeus lituratus	0/2 (0)	1/2 (50)
Artibeus planirostris	1/4 (25)	2/4 (50)
Carollia brevicauda	0/1 (0)	0/1 (0)
Carollia perspicillata	2/4 (50)	2/4 (50)
Desmodus rotundus	1/2 (50)	1/2 (50)
Glossophaginae sp1	1/17 (6)	1/17 (6)
Glossophaginae sp3	4/28 (14)	6/28 (21)
Glossophaga soricina	0/10 (0)	0/10 (0)
Lophostoma brasiliense	0/1 (0)	0/1 (0)
Molossideo sp1	0/1 (0)	0/1 (0)
Platyrrhinus lineatus	0/2 (0)	0/2 (0)
Pygoderma bilabiatum	0/1 (0)	0/1 (0)
Vespertilionideo sp1	0/3 (0)	0/3 (0)
Total	9/76 (12)	13/76 (17)

ELISA, enzyme-linked immunosorbent assay; IFA, immunofluorescence assay.

In summary, 13 of 76 total serum samples (17%) tested positive by IFA. These positive sera were from Glossophaginae sp3, (n = 6), Artibeus planirostris (n = 2), Carollia perspicillata (n = 2), Glossophaginae sp1 (n = 1), Artibeus lituratus (n = 1), and Desmodus rotundus (n = 1). Our study provides serological evidence of henipa-like virus infection in five Brazilian bat species (Glossophaginae sp1 and sp3 are combined as one species due to the inability to identify to species level). The overall henipa-like virus seroprevalence in our study falls within the range documented from prior bat henipavirus seroprevalence studies (from 2% to up to 63%) (Iehle et al. 2007, Jonathan et al. 2008, Li et al. 2008, Andrew et al. 2010, Peel et al. 2012, Hume et al. 2013).

Although our study lacked molecular evidence of henipa-like virus infection, our results support existing molecular evidence available for bat henipa-like virus infection in the Western hemisphere (Drexler et al. 2012). While only pteropodidae bats have been identified as reservoirs of henipaviruses to date, all of the bat species tested in this study were from the diverse family Phyllostomidae. Hollow trees, fruit groves, caves, as well as man-made buildings and tunnels can all serve as dwellings for phyllostomid bats. Although the majority of bats sampled in this study were frugivorous and/or insectivorous, there also were two common vampire (D. rotundus) bats sampled, one of which tested positive by both NiV N ELISA and IFA.

The bats captured for this study were at a location directly adjacent to human development, which highlights the potential of human–bat interactions leading to disease spillover events by zoonotic pathogens due to increasing human encroachment into undeveloped areas. Similar to a study that provided evidence of anti-henipavirus antibodies in insectivorous bats in China (Li et al. 2008), the results of our study expand the host range for henipa-like viruses and provide impetus for future efforts to identify new henipa-like viruses as well as their potential host reservoirs in the Western hemisphere.

Footnotes

Acknowledgments

Financial support for this research came from CDC core funding and grant sponsor: Fundação de Amparo a Pesquisa do Estado de São Paulo-FAPESP, grant number: 2006/00572-0. We thank Marcel Soares for identification of the bats. This research has been approved by the animal subjects research review board at University of Sao Paulo.

Disclaimer

The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the CDC, U.S. Department of Health and Human Services.

Author Disclosure Statement

No competing financial interests exist.

References

Andrew

, Meng

, Jennifer

, Gary

, et al. Prevalence of henipavirus and rubulavirus antibodies in Pteropid bats, Papua New Guinea. Emerg Infect Dis J, 2010; 16:1997.

Chua

, Koh

, Hooi

, Wee

, et al. Isolation of Nipah virus from Malaysian Island flying-foxes. Microbes Infect, 2002; 4:145–151.

Drexler

, Corman

, Muller

, Maganga

, et al. Bats host major mammalian paramyxoviruses. Nat Commun, 2012; 3:796.

Halpin

, Young

, Field

, Mackenzie

. Isolation of Hendra virus from pteropid bats: A natural reservoir of Hendra virus. J Gen Virol, 2000; 81:1927–1932.

Hume

, Carol

EdJ

, Kim

, Craig

. Henipaviruses and fruit bats, Papua New Guinea. Emerg Infect Dis J, 2013; 19:670.

Iehle

, Razafitrimo

, Razainirina

, Andriaholinirina

, et al. Henipavirus and Tioman virus antibodies in pteropodid bats, Madagascar. Emerg Infect Dis, 2007; 13:159–161.

Jonathan

, Vibhu

, Craig

, Peter

, et al. Henipavirus infection in fruit bats (Pteropus giganteus), India. Emerg Infect Dis J, 2008; 14:1309.

, Wang

, Hickey

, Zhang

, et al. Antibodies to Nipah or Nipah-like viruses in bats, China. Emerg Infect Dis, 2008; 14:1974–1976.

, Lowe

, Hummel

, Sazzad

, et al. Characterization of Nipah virus from outbreaks in Bangladesh, 2008–2010. Emerg Infect Dis, 2012; 18:248–255.

10.

Peel

, Baker

, Crameri

, Barr

, et al. Henipavirus neutralising antibodies in an isolated island population of African fruit bats. PLoS One, 2012; 7:e30346.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.60 MB

0.00 MB