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Experimental challenge of a North American bat species, big brown bat (Eptesicus fuscus), with SARS-CoV-2

Jeffrey S. Hall,

Corresponding Author

U.S. Geological Survey National Wildlife Health Center, Madison, WI, USA

Correspondence

Jeffrey S. Hall, U.S. Geological Survey National Wildlife Health Center, Madison, Wisconsin, USA.

Email: jshall@usgs.gov

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Susan Knowles,

U.S. Geological Survey National Wildlife Health Center, Madison, WI, USA

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Sean W. Nashold,

U.S. Geological Survey National Wildlife Health Center, Madison, WI, USA

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Hon S. Ip,

U.S. Geological Survey National Wildlife Health Center, Madison, WI, USA

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Ariel E. Leon,

U.S. Geological Survey National Wildlife Health Center, Madison, WI, USA

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Tonie Rocke,

U.S. Geological Survey National Wildlife Health Center, Madison, WI, USA

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Saskia Keller,

U.S. Geological Survey National Wildlife Health Center, Madison, WI, USA

Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, USA

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Mariano Carossino,

Louisiana National Animal Disease Diagnostic Laboratory and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA

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Udeni Balasuriya,

Louisiana National Animal Disease Diagnostic Laboratory and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA

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Erik Hofmeister,

U.S. Geological Survey National Wildlife Health Center, Madison, WI, USA

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First published: 09 December 2020

Abstract

The recently emerged novel coronavirus, SARS-CoV-2, is phylogenetically related to bat coronaviruses (CoVs), specifically SARS-related CoVs from the Eurasian bat family Rhinolophidae. As this human pandemic virus has spread across the world, the potential impacts of SARS-CoV-2 on native North American bat populations are unknown, as is the ability of North American bats to serve as reservoirs or intermediate hosts able to transmit the virus to humans or to other animal species. To help determine the impacts of the pandemic virus on North American bat populations, we experimentally challenged big brown bats (Eptesicus fuscus) with SARS-CoV-2 under BSL-3 conditions. We inoculated the bats both oropharyngeally and nasally, and over the ensuing three weeks, we measured infectivity, pathology, virus concentrations in tissues, oral and rectal virus excretion, virus transmission, and clinical signs of disease. We found no evidence of SARS-CoV-2 infection in any examined bat, including no viral excretion, no transmission, no detectable virus in tissues, and no signs of disease or pathology. Based on our findings, it appears that big brown bats are resistant to infection with the SARS-CoV-2. The potential susceptibility of other North American bat species to SARS-CoV-2 remains to be investigated.

1 INTRODUCTION

In December 2019, a novel coronavirus of likely zoonotic origin, SARS-CoV-2, was discovered following detection of an outbreak of acute respiratory disease in people from Wuhan, China (Zhou et al., 2020). The virus has since spread across the world and in early March 2020 the WHO declared COVID-19, the disease caused by the virus, a pandemic (https://www.who.int/dg/speeches/detail/who-director-general-s-opening-remarks-at-the-media-briefing-on-covid-19---11-march-2020). As of November 2020, worldwide mortality from COVID-19 was over 1,360,000 deaths (https://coronavirus.jhu.edu/map.html; Accessed November 20, 2020).

Coronaviruses have large (~30 kb), positive-sense, RNA genomes and are known to infect a variety of mammals and birds. Their genomes are also known to have a high propensity for recombination with other coronaviruses. Coronaviruses are classified as alpha-, beta-, gamma- or deltacoronaviruses. Both alpha- and betacoronaviruses infect mammals, and while worldwide bats are known to be infected by both types of viruses (Decaro & Larusso, 2020; Falcon et al., 2011), only alphacoronaviruses have been found to date in North American bats (Olival et al., 2020).

Recent human outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) were caused by betacoronaviruses that likely originated from Eurasian bats, although intermediate hosts such as masked palm civets (Paguma larvata) and dromedary camels (Camelus dromedarius), respectively, were involved in zoonotic transmission to humans. SARS-CoV-2 is a betacoronavirus related to SARS and MERS, and multiple lines of evidence indicate that it also originated from bats, specifically from horseshoe bats (Rhinolophus sp.) (Boni et al., 2020; Zhou et al., 2020).

As SARS-CoV-2 continues to circulate in human populations across the world, there is increasing risk of reverse zoonotic transmission of the virus to wild animals by infected humans. The potential impacts of this novel coronavirus on native North American bat populations are unknown, as are the abilities of North American bats to serve as reservoirs or as intermediate hosts able to transmit the virus to humans or to other animal species. Currently, many North American bat species are severely threatened by other pathogens, principally Pseudogymnoascus destructans, the fungal cause of white-nose syndrome (Blehert et al., 2009). Consequently, wildlife biologists have raised additional concerns that SARS-CoV-2 could be transmitted to bat colonies by scientists conducting research, by human recreational activity, or by other means with unknown consequences on North American bat populations (Olival et al., 2020).

To assess the potential impact of SARS-CoV-2 on North American bat populations, we used big brown bats (Eptesicus fuscus) as a model species. Big brown bats are a common bat species in North America that often live in close proximity to humans, both roosting and hibernating in manmade structures. Therefore, this species is at elevated risk for exposure to the virus from infected humans. We challenged big brown bats with SARS-CoV-2 under Biosafety Level-3 conditions and measured oral and rectal virus excretion, clinical signs of disease, morbidity and mortality, virus transmission, virus concentrations in tissues, and pathology. These data can be used to estimate risks of SARS-CoV-2 to this wild bat species.

2 MATERIALS AND METHODS

2.1 Virus acquisition and propagation

We obtained the SARS-CoV-2 virus (2019-nCoV/USA-WA1/2020) (BEI Resources, Manassas, VA). The virus was isolated from the first confirmed patient with COVID-19 in the United States (Harcourt et al., 2020). We propagated and quantified the virus stock in Vero E6 cell culture using standard techniques.

2.2 Animal acquisition and husbandry

During the winter of 2019–2020, hibernating big brown bats (Eptesicus fuscus) were removed from local human residences in Waushara County, Wisconsin by a wildlife removal specialist under permit #1012 issued by the Wisconsin Department of Natural Resources (WDNR) and transferred to the USGS National Wildlife Health Center (NWHC) under WNDR permit #922. At the NWHC, bats were examined, weighed, wing banded and placed in mesh cages within environmental chambers for the duration of a natural cycle of hibernation. Chambers were maintained at 7.0°C (±0.5°C) and 95% (±1.0%) relative humidity. In the spring, the bats were moved from the environmental chambers to mesh flight cages within a greenhouse tent with increased humidity provided by warm mist humidifiers. Temperature was maintained at 21–24°C. Bats were given physical examinations by a veterinarian and treated topically with selamectin for parasites (Zoetis, Florham Park, NJ). The bats were fed mealworms (Tenebrio molito) supplemented with a vitamin and mineral mixture, and water was provided ad libitum. Bats underwent a quarantine and acclimatization period of 30 days prior to commencement of this study. All husbandry and experimental protocols for live bats were approved by the National Wildlife Health Center Institutional Animal Care and Use Committee.

2.3 Pre-inoculation faecal sampling and coronavirus analysis

During acclimatization, we collected faecal samples from a subset of the bats to determine the presence of other coronaviruses in these subjects. Each faecal sample was suspended in a 10% (w/v) solution with viral transport medium. Viral RNA was extracted using the MagMax Pathogen RNA/DNA kit (Applied Biosystems, Forest City, CA) on a Kingfisher Flex magnetic particle processor according to the manufacturer's instructions. The presence of alpha and betacoronaviruses was determined using the nested RT-PCR methods of (Decaro & Larusso, 2020).

2.4 Bat inoculation

We acquired 16 male big brown bats for this study. We cohoused pairs of bats in mesh cages. One bat from each of seven bat pairs was inoculated with SARS-CoV-2, and its cage mate was left uninoculated to determine whether the virus could be horizontally transmitted between bats. The SARS-CoV-2 inoculum was 105 TCID50 /bat diluted in 50 µl brain heart infusion (BHI) broth and administered nasally (20 µl) and orally (30 µl) using a micropipette. One bat pair was sham inoculated with comparable volumes of BHI broth. This technique has been used to inoculate other species with SARS-CoV-2 (Munster et al. 2020; Schlottau et al. 2020; Shi et al., 2020). The inoculum titre was verified by qRT-PCR (see below for procedure) and virus viability confirmed in cell culture using Vero E6 cells.

2.5 Animal monitoring and sampling

After inoculation, bats were observed at least twice daily to document development of clinical signs and to monitor health status. Prior to inoculation and every other day thereafter, each bat was weighed, and oropharyngeal and rectal swabs (Puritan Medical Products, Guilford, ME) were collected. On day post-inoculation (DPI) 6 and on DPI 12, bats from one cage (one inoculated bat, one uninoculated) were euthanized, a postmortem examination was conducted, and tissues and blood collected (for details, see ‘necropsy and histopathology’ section below). At the end of the study (DPI 20), all remaining bats were euthanized, and the control bats and an additional cage pair had postmortem examinations conducted with tissues and blood collected for histopathological, serological and virological analyses.

2.6 Quantitative (q) RT-PCR analyses

RNA extractions of swab material were performed in 96-well plates using MagMax-96 AI/ND Viral RNA Isolation Kit (Applied Biosystems, Foster City, CA) following the manufacturer's instructions. A positive control sample consisting of a 1:100 dilution of the SARS-CoV-2 inoculum used in the study was included with each extraction series to validate successful RNA extraction. Quantitative RT-PCR (qRT-PCR) analyses of swab samples, virus inoculum and tissues were conducted utilizing the Centers for Disease Control 2019-nCoV N1 primers and probe (https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html) and AgPath-ID One-Step RT-PCR reagents (Ambion/ThermoFisher, Waltham, MA). We included a standard curve of RNA extracted from 10-fold serially diluted SARS-CoV-2 virus stock (107 TCID50/ml) in each qRT-PCR assay to quantify viral amounts.

2.7 Necropsy and histopathology

Two bats (inoculated and uninoculated cage mates) were euthanized at 6 DPI, 12 DPI and 20 DPI using an overdose of isoflurane with subsequent decapitation; two control animals were also euthanized at 20 DPI. Postmortem examinations were immediately conducted with body condition and gross observations recorded. Portions of the nares, lung (cranial and caudal lobes), heart, liver, spleen, colon and brain were collected and frozen at −80°C for virological analysis. Additional tissue samples were fixed in 10% neutral buffered formalin for histological analysis. Fixed tissues for pathological analyses were embedded in paraffin, sectioned at 5 µm and stained with haematoxylin and eosin at the Wisconsin Veterinary Diagnostic Laboratory (Madison, WI).

2.8 SARS-CoV-2-specific RNAscope® in situ hybridization (ISH)

For RNAscope® ISH, an anti-sense probe targeting the spike protein (nt 21,563–25,384) of SARS-CoV-2, WA1 strain (GenBank accession number MN985325.1) was designed (Advanced Cell Diagnostics [ACD], Newark, CA) and used as previously validated and described (Carossino et al., 2020). 4-µm sections of formalin-fixed paraffin-embedded tissues were mounted on positively charged Superfrost® Plus Slides (Fisher Scientific, Pittsburgh, PA). The ISH assay was performed using the RNAscope 2.5 HD Red Detection Kit (ACD) as previously described (Carossino et al., 2019). Sections from mock- and SARS-CoV-2-infected Vero cell pellets were used as negative and positive assay controls.

2.9 Virus RNA extraction and RT-PCR from bat tissues

Approximately 10 mg of each tissue were macerated in 200 µl of extraction buffer and RNA extracted using the ZYMO Research Quick DNA/RNA Pathogen Miniprep kit (ZYMO Research, Irvine, CA). qRT-PCR analyses were performed as described above.

2.10 Antibody detection

To detect IgM or IgG antibodies to SARS-CoV-2, indirect ELISAs were performed. Briefly, in separate assays, 96-well high sorbency flat-bottomed plates (Corning-Costar, Cambridge, MA) were coated with 50 µl of 2 µg/ml dilution of recombinant SARS-CoV-2 receptor-binding domain (RBD) or Spike S1 proteins (USBiologicals, Salem, MA) in carbonate–bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. Alternate columns on the plates were coated with 50 µl of negative control antigen produced from non-transfected HEK293 cells (Fisher Scientific, Waltham, MA) and purified by Ni NTA columns (Millipore-Sigma, St. louis, MO). After incubation, the coating buffer was discarded, and the plate was blocked for 1 hr at 37°C using wash buffer with 5% non-fat dry milk (blocking buffer). Afterwards, the plates were washed four times with PBS/Tween20 (wash buffer) and 50 µl of each test serum, diluted to 1:100 in blocking buffer, was applied to duplicate wells coated with SARS-CoV-2 antigen and to two wells coated with negative control antigen and allowed to incubate for 1 hr at 37°C. The plates were washed four times, and 50 µl of a 1:2,000 dilution of mouse anti-bat IgG (Novus Biologicals, Centennial, CO) was applied to each well. Following incubation for 1 hr at 37°C and washing, 50 µl of goat anti-mouse antibody conjugated with horseradish peroxidase was applied to each well. Following incubation for 1 hr at 37°C and washing, 75 µl of ABTS peroxidase substrate (Seracare Life Sciences, Milford, MA) was applied and incubated at room temperature for up to 30 min before being inactivated with an equal volume of 1% sodium dodecyl sulphate solution. Absorbance at 405 nm was read using a microtitre absorbance plate reader.

Sera were collected from experimentally inoculated bats on DPI 6 and DPI 12, and were tested in the same manner; however, mouse anti-bat IgG and IgM (Novus Biologicals, Centennial, CO) was used as a detection antibody. The antibody was incubated for 1 hr at 37°C and washed four times. Bound anti-bat antibodies were detected with goat anti-mouse IgG conjugated with horse radish peroxidase for 1 hr at 37oC, followed by a final washing and development with ABTS solution. Absorbance was read as in the IgG assay.

To determine the positive threshold if an experimentally exposed bat had produced specific antibodies to either the SARS-CoV-2 RBD or S1 protein, 10 serum samples were obtained from big brown bats that had previously been used at NWHC in prior experimental studies. These negative control bats were captured, held and sampled at the NWHC prior to the fall of 2019. The absorbance values of duplicate wells for these samples were obtained, and the mean and standard deviation determined. Rabbit polyclonal antibody to either SARS-CoV-2 RBD or Spike S1antigens (ProSciInc., Poway, CA) served as positive control, and negative control was commercial rabbit serum (Invitrogen, Carlsbad, CA), which were detected using goat anti-rabbit IgG conjugated to horse radish peroxidase (SouthernBiotech, Birmingham, AL).

3 RESULTS

3.1 Presence of coronaviruses in big brown bats prior to inoculation

Prior to initiating this study, we collected faecal material from the bats and tested them for the presence of alpha- and betacoronaviruses. Five of the bats were positive for coronavirus. Sequence analyses of the PCR products showed that all five were alphacoronaviruses, with no indication of the presence of any betacoronavirus in the samples tested (Table 1). All of the virus sequences were identical indicating that the bats were likely transmitting the virus amongst themselves during hibernation.

TABLE 1. The presence of alphacoronaviruses in big brown bats prior to experimental challenge
Bat ID PCR % Homologyaa Percent homology to GenBank Accession JX537914 (Alphacoronavirus Eptesicus fuscus strain ARCoV.1).
442 Positive 96%
444 Negative
449 Negative
852 Negative
853 Negative
854 Positive 96%
856 Positive 96%
858 Negative
860 Positive 96%
866 Negative
867 Negative
868 Negative
870 Negative
883 Negative
885 Positive 96%
888 Negative
  • a Percent homology to GenBank Accession JX537914 (Alphacoronavirus Eptesicus fuscus strain ARCoV.1).

3.2 Clinical signs of SARS-CoV-2 infection

Before and after inoculation with SARS-CoV-2, no bat in our study exhibited any overt clinical signs of disease. All bats maintained or gained body weight (Table 2) and appeared healthy over the course of this study. They ate and drank normally (compared with controls) and no indications of respiratory, neurologic, digestive, or any other health issues associated with exposure to the virus were evident.

TABLE 2. Body weights (g) of big brown bats experimentally inoculated with SARS-CoV-2

Bat ID

Treatment DPIbb Day Post-inoculation.
0

DPI

2

DPI

4

DPI

6

DPI

8

DPI 10 DPI 12 DPI 14 DPI 16 DPI 18 DPI 20
888 Control 17.2 18.3 18.4 18.9 18.8 19.5 19.7 19.0 19.5 19.1 19.5cc Bat euthanized, tissues and sera collected, necropsy performed.
885 Control 17.2 16.6 17.2 17.2 17.4 18.0 18.0 18.4 17.8 18.4 18.5 cc Bat euthanized, tissues and sera collected, necropsy performed.
870 Inoculated 17.4 17.6 17.6 18.0 cc Bat euthanized, tissues and sera collected, necropsy performed.
867 a a Uninoculated bat cohoused with inoculated bat.
24.1 24.0 23.6 23.0 cc Bat euthanized, tissues and sera collected, necropsy performed.
444 Inoculated 20.0 20.3 20.3 20.2 21.0 21.5 18.8 cc Bat euthanized, tissues and sera collected, necropsy performed.
442 a a Uninoculated bat cohoused with inoculated bat.
15.9 15.8 15.7 15.6 15.5 15.9 16.2 cc Bat euthanized, tissues and sera collected, necropsy performed.
852 Inoculated 15.9 15.7 16.0 15.5 16.0 15.7 16.2 15.9 16.5 16.5 16.9 cc Bat euthanized, tissues and sera collected, necropsy performed.
854 a a Uninoculated bat cohoused with inoculated bat.
20.5 20.2 20.3 20.0 20.1 19.9 19.8 19.5 19.9 19.9 19.9 cc Bat euthanized, tissues and sera collected, necropsy performed.
449 Inoculated 20.6 21.2 21.1 21.2 21.3 22.6 23.3 25.7 25.1 26.5 25.2
883 a a Uninoculated bat cohoused with inoculated bat.
16.2 16.0 16.4 15.9 16.3 15.9 16.0 14.9 15.3 15.3 15.0
858 Inoculated 22.4 22.3 22.9 22.5 22.8 22.5 22.5 23.2 23.6 23.0 23.9
853 a a Uninoculated bat cohoused with inoculated bat.
15.6 16.2 15.9 15.1 14.8 15.8 15.5 15.3 15.9 15.8 15.3
860 Inoculated 16.8 17.5 17.5 16.5 16.2 16.9 17.3 17.7 18.4 18.6 19.3
866 a a Uninoculated bat cohoused with inoculated bat.
15.2 16.0 16.3 16.3 17.3 16.8 17.0 16.5 17.1 17.3 16.9
856 Inoculated 19.6 18.6 18.7 19.4 19.4 19.3 19.4 19.7 19.9 20.2 19.8
868 a a Uninoculated bat cohoused with inoculated bat.
18.5 18.2 17.7 16.7 16.1 16.8 17.1 16.6 16.4 16.6 16.8
  • a Uninoculated bat cohoused with inoculated bat.
  • b Day Post-inoculation.
  • c Bat euthanized, tissues and sera collected, necropsy performed.

3.3 SARS-CoV-2 excretion

qRT-PCR analysis of oropharyngeal swabs taken from inoculated and transmission subjects revealed that no bat, either inoculated or uninoculated subjects, excreted detectable SARS-CoV-2 through 20 days post-inoculation (Table 3). Five of 16 bats showed low levels of virus in oropharyngeal swabs collected on day 0 (Ct values > 34). However, all bats in which SARS-CoV-2 was detected on day 0 were tested after their cage mate had been inoculated with the virus and thus likely represented cross-contamination instead of a previous SARS-CoV-2 infection. Analyses of rectal swabs indicated that no bat, whether directly inoculated or uninoculated, excreted any detectable virus by this route for the duration of this study (Table 4). Further, all rectal swabs collected on day 0 tested negative for SARS-CoV-2.

TABLE 3. Quantitative RT-PCR analysis of SARS-CoV-2 oral excretion by experimentally challenged big brown bats
Bat ID Treatment DPIbb Day Post-inoculation.
0

DPI

2

DPI

4

DPI

6

DPI

8

DPI 10 DPI 12 DPI 14 DPI 16 DPI 18

DPI

20

888 Control No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ctcc Bat euthanized, tissues and sera collected, postmortem analysis performed.
885 Control No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ctcc Bat euthanized, tissues and sera collected, postmortem analysis performed.
870 Inoculated No Ct No Ct No Ct No Ctcc Bat euthanized, tissues and sera collected, postmortem analysis performed.
867 a a Uninoculated bat cohoused with inoculated bat.
No Ct No Ct No Ct No Ctcc Bat euthanized, tissues and sera collected, postmortem analysis performed.
444 Inoculated No Ct No Ct No Ct No Ct No Ct No Ct No Ctcc Bat euthanized, tissues and sera collected, postmortem analysis performed.
442 a a Uninoculated bat cohoused with inoculated bat.
35.57 No Ct No Ct No Ct No Ct No Ct No Ctcc Bat euthanized, tissues and sera collected, postmortem analysis performed.
852 Inoculated No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ctcc Bat euthanized, tissues and sera collected, postmortem analysis performed.
854 a a Uninoculated bat cohoused with inoculated bat.
36.23 No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ctcc Bat euthanized, tissues and sera collected, postmortem analysis performed.
449 Inoculated No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
883 a a Uninoculated bat cohoused with inoculated bat.
No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
858 Inoculated 38.33 No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
853 a a Uninoculated bat cohoused with inoculated bat.
No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
860 Inoculated No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
866 a a Uninoculated bat cohoused with inoculated bat.
34.24 No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
856 Inoculated No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
868 a a Uninoculated bat cohoused with inoculated bat.
38.19 No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
  • a Uninoculated bat cohoused with inoculated bat.
  • b Day Post-inoculation.
  • c Bat euthanized, tissues and sera collected, postmortem analysis performed.
TABLE 4. Quantitative RT-PCR analysis of SARS-CoV-2 rectal excretion by experimentally challenged big brown bats
Bat ID Treatment DPIbb Day Post-inoculation.
0

DPI

2

DPI

4

DPI

6

DPI

8

DPI 10 DPI 12 DPI 14 DPI 16 DPI 18

DPI

20

888 Control No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ctcc Bat euthanized, tissues and sera collected, postmortem analysis performed.
885 Control No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ctcc Bat euthanized, tissues and sera collected, postmortem analysis performed.
870 Inoculated No Ct No Ct No Ct No Ctcc Bat euthanized, tissues and sera collected, postmortem analysis performed.
867 a a Uninoculated bat cohoused with inoculated bat.
No Ct No Ct No Ct No Ctcc Bat euthanized, tissues and sera collected, postmortem analysis performed.
444 Inoculated No Ct No Ct No Ct No Ct No Ct No Ct No Ctcc Bat euthanized, tissues and sera collected, postmortem analysis performed.
442 a a Uninoculated bat cohoused with inoculated bat.
No Ct No Ct No Ct No Ct No Ct No Ct No Ctcc Bat euthanized, tissues and sera collected, postmortem analysis performed.
852 Inoculated No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ctcc Bat euthanized, tissues and sera collected, postmortem analysis performed.
854 a a Uninoculated bat cohoused with inoculated bat.
No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ctcc Bat euthanized, tissues and sera collected, postmortem analysis performed.
449 Inoculated No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
883 a a Uninoculated bat cohoused with inoculated bat.
No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
858 Inoculated No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
853 a a Uninoculated bat cohoused with inoculated bat.
No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
860 Inoculated No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
866 a a Uninoculated bat cohoused with inoculated bat.
No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
856 Inoculated No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
868 a a Uninoculated bat cohoused with inoculated bat.
No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
  • a Uninoculated bat cohoused with inoculated bat.
  • b Day Post-inoculation.
  • c Bat euthanized, tissues and sera collected, postmortem analysis performed.

3.4 Postmortem examination and histopathology

All bats were in excellent body condition evidenced by abundant fat stores. Gross findings included variably-sized red foci or mottling in the lungs (Bat ID 870, 444, 442, 885, 888, 852, 854; note that bat IDs 885 and 888 were mock-inoculated controls), mild splenomegaly with nodular hyperplasia (867), mild splenomegaly (442, 885, 852), unilateral cerebral reddening (444) and mild mesenteric lymphadenopathy (444). Histopathologic findings included pulmonary congestion, haemorrhage, oedema and alveolar collapse (all bats), multinucleated cells in pulmonary bronchioles, alveoli or vasculature (867, 442, 885, 888, 852, 854), mild cerebral haemorrhage (all bats examined), mild focal cerebral gliosis (854), active splenic germinal centers (all bats examined), multifocal hepatic sinusoidal (all bats examined) or perivascular inflammation (885), demodex in the adnexa of the nares (870, 885, 852, 854), mild dermal perivascular or periadnexal inflammation (867, 870, 444, 442, 885, 852, 854), mild submucosal intestinal haemorrhage (867), intestinal trematodiasis (444, 442, 885, 854), and eosinophilic enteritis (867, 888, 854). These histopathologic findings were consistent with the euthanasia procedures utilized and/or parasitism and were not consistent with findings observed in other animals experimentally infected with SARS-CoV-2 (Munster et al. 2020; Schlottau et al. 2020; Shi et al., 2020).

3.5 RNAscope® in situ hybridization (ISH)

SARS-CoV-2-specific ISH was used to examine formalin-fixed paraffin-embedded tissues (brain, nares, cranial and caudal lobes of lung, colon, spleen, liver and heart) from eight bats, including three inoculated animals and two controls. No SARS-CoV-2 spike protein RNA was detected in any tissue section from any subject examined. These results additionally indicate absence of SARS-CoV-2 virus.

3.6 SARS-CoV-2 in bat tissues

qRT-PCR analyses of eight tissues (brain, nares, cranial and caudal lobes of lung, colon, spleen, liver and heart) taken from inoculated bats euthanized on DPI6, DPI12 and DPI20, as well as from the two control subjects, showed that none of these tissues from any bat analysed contained detectable SARS-CoV-2 genetic material (Table 5). Although we did not have any positive bat infection samples that served as inoculation controls, positive- and negative-assay controls performed as expected.

TABLE 5. Quantitative RT-PCR analysis of tissues collected from SARS-CoV-2 inoculated big brown bats
Bat ID
Tissue 888aa Control.
885 aa Control.
870 867 444 442 852 854
Brain No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
Nares No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
Heart No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
Lung, cranial lobe No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
Lung, caudal lobe No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
Spleen No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
Liver No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
Colon No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct
  • a Control.

3.7 Serological Analyses

Our assays to detect IgG and IgM generated by SARS-CoV-2 exposed bats did not to detect antibodies in any experimental bat (Table 6). The optical densities from the exposed bats were similar to the experimental control bats and to sera from big brown bats sampled prior to the pandemic. There were no statistically significant differences in group means in IgG RBD, IgG S1, or IgG, IgM RBD serological assays as determined by one-way ANOVA, (F (2,24)= 0.250, p = .78), (F (2,24)= 1.55, p = .23), (F (2,13)= 0.301, p = .75), respectively.

TABLE 6. Enzyme-linked immunosorbent assay for antibody development in SARS-CoV-2 experimentally exposed big brown bats compared to big brown bats sampled prior to the pandemic
Immunoglobulin Class Antigen SARS-CoV−2 Exposed Experimental Bats Control Experimental Bats Big Brown Bats sampled prior to outbreak
IgG RBDbb RBD- SARS-CoV-2 receptor-binding domain.
0.034 ± 0.022aa Optical density, Mean ± SD (Number of bats tested).
(13)
0.034 ± 0.034 (2) 0.027 ± 0.022 (12)
IgG S1cc S1- SARS-CoV-2 Spike protein.
0.088 ± 0.054 (13) 0.085 ± 0.094 (2)

0.053 ± 0.041 (12)

IgG, IgM RBD 0.058 ± 0.025 (4) 0.071 ± 0.024 (2) 0.054 ± 0.031 (10)
  • a Optical density, Mean ± SD (Number of bats tested).
  • b RBD- SARS-CoV-2 receptor-binding domain.
  • c S1- SARS-CoV-2 Spike protein.

These data indicate that none of the big brown bats experimentally exposed to SARS-CoV-2 developed a detectable serological response to the virus.

4 DISCUSSION

SARS-CoV-2 is known to infect a variety of mammals other than humans. Natural infections, including human-animal transmission, have been found in felines including captive Bengal tigers (Panthera tigris), lions (Panthera leo), domestic cats (Felis catus), domestic dogs (Canis lupus familiaris), Malayan pangolins (Manis javanica) and farmed mink (Neovison vison) (Bartlett et al., 2020; Schlottau et al., 2020; Shi et al., 2020; Sit et al., 2020; Zhang et al., 2020; Oreshkova et al. 2020). Experimental work has shown that a variety of non-human primates are susceptible to the virus including rhesus macaques (Macaca mulatta), cynomolgus macaques (Macaca fascicularis), marmosets and African green monkeys (Chlorocebus aethiops) (Lu et al., 2020; Munster et al., 2020). Ferrets (Mustela putorius), Egyptian fruit bats (Rousettus aegyptiacus), deer mice (Peromyscus maniculatus) and Syrian golden hamsters (Mesocricetus auratus) have also been shown to be susceptible (Griffin et al., 2020; Schlottau et al., 2020; Shi et al., 2020; Sia et al., 2020). Other species, including swine (Sus scrofa) and various domestic poultry, are not susceptible to the virus (Schlottau et al., 2020; Suarez et al., 2020). Thus, susceptibility to SARS-CoV-2 varies and determination is needed for each species or for each phylogenetic lineage in question. In our study, inoculated big brown bats showed no virus excretion, no clinical signs of disease, no disease-associated pathology, no virus in any tissues examined and no transmission. While we had no available positive control bat samples, our consistent findings showed that big brown bats, by all measured parameters, were resistant to infection by SARS-CoV-2.

Old-world chiropterans (bats) are the evolutionary source of the human betacoronaviruses SARS, MERS and now SARS-CoV-2. North American bats frequently have contact with humans, often residing and hibernating in human habitations and agricultural structures. Additionally, bats are encountered by wildlife biologists conducting studies and by humans during recreational activities. This has led to concerns that SARS-CoV-2 could be transmitted from infected humans to North American bat species, many of whose populations are severely declining. Several models and risk analyses of this reverse zoonotic scenario have been published based on the assumption that North American bats are susceptible to infection by SARS-CoV-2; however, no experimental or natural evidence has shown that these species are susceptible (Franklin & Bevins, 2020; Runge et al., 2020). Certainly, it is prudent to take all appropriate precautions to safeguard these populations; however, our data indicate that more work is needed to accurately understand the potential impacts of SARS-CoV-2 to our native wildlife species and populations.

Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor that the SARS-CoV-2 spike protein binds during the infection process. A survey of ACE2 sequences from 410 vertebrate species predicted that chiropterans, including big brown bats, have low to very low likelihood to be potential hosts of the virus, especially in comparison with humans, apes and primates, which have very high host potential (Damas et al., 2020). Based on the low host potential of chiropterans, taken together with the established connections of SARS-CoV-2 and old-world bats, there are likely factors other than ACE-2 binding that modulate SARS-CoV-2 infection. However, Becker et al. 2020, in a computational analysis of a variety of models, determined that four North American bat species, including Eptesicus fuscus, are likely betacoronavirus hosts. These contrasting findings underscore the importance of experimental challenge studies to definitively determine the susceptibility of host species and the impacts of emerging diseases on those species.

It is interesting that a significant portion of our bats (5/16) were positive for the presence of an alphacoronavirus, even after being in hibernation for months. Other researchers have also found a persistent alphacoronavirus in the intestines and lungs of hibernating little brown bats (Myotis lucifugus), so our findings are not surprising (Subuhi et al. 2017). We do not know how this prevalence of an indigenous alphacoronavirus influences the ability of the novel SARS-CoV-2 betacoronavirus to infect big brown bats; however, the long-term persistence of a presumably bat-adapted alphacoronavirus in these subjects and the immunological implications of its presence warrant further investigation.

While this study determined that big brown bats were not susceptible to infection by SARS-CoV-2, the risks of this novel coronavirus infecting other species of North American bats remain uncharacterized. If other species of North American bats are susceptible to SARS-CoV-2 infection, viral infection could adversely impact these species’ populations or wild, free-ranging bats could serve as reservoir hosts for SARS-CoV-2 and subsequently transmit the virus to humans. Thus, SARS-CoV-2 infections in wild bats may pose unknown risks to human health, as well as infected humans presenting potential risks to wildlife. There are 45 species of bats known to occur in the continental United States and Canada, and this study provides evidence indicating that big brown bats are at low to no risk for infection by SARS-CoV-2.

ACKNOWLEDGEMENTS

The authors express their sincere gratitude to Michael Stumpf of the Midwest Bat Specialists LLC, for providing the bats. We also thank Katy Griffin, Jeffrey Messer, Melissa Lund, Dana Calhoun, Harrison Lamb, Lauren Dycee-Holtz, Rachel Lambert, Carrie Allison Smith, Casey Hall and Jenna Motz for technical contributions. We thank Kendra Shultz, Kimberly Harper and other members of the histopathology and immunohistochemistry section at the Louisiana Animal Disease Diagnostic Laboratory for assistance in slide preparation for RNAscope® assays. We are particularly indebted to the NWHC Animal Services staff, without whose assistance, diligence and hard work this study could not have been accomplished. The use of trade, firm or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government.

    CONFLICTS OF INTEREST

    The authors acknowledge they have no personal financial interests or conflicts of interest with this research.

    DATA AVAILABILITY STATEMENT

    The data that support the findings of this study are available from the corresponding author upon reasonable request.

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