SARS-CoV-2 in fruit bats, ferrets, pigs, and chickens: an experimental transmission study

Abstract

Background: In December, 2019, a novel zoonotic severe acute respiratory syndrome-related coronavirus emerged in China. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became pandemic within weeks and the number of human infections and severe cases is increasing. We aimed to investigate the susceptibilty of potential animal hosts and the risk of anthropozoonotic spill-over infections.

Methods: We intranasally inoculated nine fruit bats (Rousettus aegyptiacus), ferrets (Mustela putorius), pigs (Sus scrofa domesticus), and 17 chickens (Gallus gallus domesticus) with 10⁵ TCID₅₀ of a SARS-CoV-2 isolate per animal. Direct contact animals (n=3) were included 24 h after inoculation to test viral transmission. Animals were monitored for clinical signs and for virus shedding by nucleic acid extraction from nasal washes and rectal swabs (ferrets), oral swabs and pooled faeces samples (fruit bats), nasal and rectal swabs (pigs), or oropharyngeal and cloacal swabs (chickens) on days 2, 4, 8, 12, 16, and 21 after infection by quantitative RT-PCR (RT-qPCR). On days 4, 8, and 12, two inoculated animals (or three in the case of chickens) of each species were euthanised, and all remaining animals, including the contacts, were euthanised at day 21. All animals were subjected to autopsy and various tissues were collected for virus detection by RT-qPCR, histopathology immunohistochemistry, and in situ hybridisation. Presence of SARS-CoV-2 reactive antibodies was tested by indirect immunofluorescence assay and virus neutralisation test in samples collected before inoculation and at autopsy.

Findings: Pigs and chickens were not susceptible to SARS-CoV-2. All swabs, organ samples, and contact animals were negative for viral RNA, and none of the pigs or chickens seroconverted. Seven (78%) of nine fruit bats had a transient infection, with virus detectable by RT-qPCR, immunohistochemistry, and in situ hybridisation in the nasal cavity, associated with rhinitis. Viral RNA was also identified in the trachea, lung, and lung-associated lymphatic tissue in two animals euthanised at day 4. One of three contact bats became infected. More efficient virus replication but no clinical signs were observed in ferrets, with transmission to all three direct contact animals. Mild rhinitis was associated with viral antigen detection in the respiratory and olfactory epithelium. Prominent viral RNA loads of 0-10⁴ viral genome copies per mL were detected in the upper respiratory tract of fruit bats and ferrets, and both species developed SARS-CoV-2-reactive antibodies reaching neutralising titres of up to 1/1024 after 21 days.

Interpretation: Pigs and chickens could not be infected intranasally by SARS-CoV-2, whereas fruit bats showed characteristics of a reservoir host. Virus replication in ferrets resembled a subclinical human infection with efficient spread. Ferrets might serve as a useful model for further studies-eg, testing vaccines or antivirals.

Funding: German Federal Ministry of Food and Agriculture.

Figure 1

Outline of the in vivo experiments On days 4, 8, and 12, two fruit bats, ferrets, and domestic pigs were euthanised. The same schedule was applied to three chickens at each timepoint. All remaining animals, including the contacts, were euthanised on day 21. Black animals (n=9 for bats, ferrets, and pigs; n=17 for chickens) were inoculated intranasally (or oculo-oronasally for chickens) with 10⁵ TCID₅₀. Grey animals (n=3 for each species) indicates direct contact animals included 1 day after inoculation. On the right-hand side, black and grey animals were not susceptible; red animals became infected and showed strong viral shedding; and purple animals were infected but displayed only little virus shedding.

Figure 2

SARS-CoV-2 viral genome loads during the study period Oral swabs of fruits bats (A), nasal washes of ferrets (B), tissues collected from fruit bats (C), and tissues collected from ferrets (D) that were experimentally infected with SARS-CoV-2 and the contact animals. Genome copies per μL RNA eluate were calculated on the basis of a quantified standard RNA. Each extracted sample was eluted in 100 μL. Limit of detection was 1 genome copy per μL RNA. Contact fruit bats were infected but displayed negligible shedding of the virus, whereas contact ferrets became infected and showed strong viral shedding. Viral genome was undetectable in fruit bats 6 (day 12), 11 and 12 (day 21), and ferrets 5 (day 12) and 8 (day 21). SARS-CoV-2=severe acute respiratory syndrome coronavirus 2.

Figure 3

SARS-CoV-2-associated rhinitis and antigen detection at day 4 (A) Rhinitis in a bat, with intraluminal debris (arrowhead), slight mucosal oedema, and minimal inflammation (arrow). (B) Nasal respiratory epithelium in a bat, showing intralesional viral antigen mainly within intraluminal debris. (C) Non-respiratory epithelium in a bat, with single antigen positive cells and no inflammation. (D) Rhinitis in a ferret, with degeneration and necrosis of the respiratory epithelium (arrowhead), slight mucosal oedema, and numerous infiltrates (arrow). (E) Nasal respiratory epithelium in a ferret, showing intralesional, abundant viral antigen. (F) Olfactory epithelium in a ferret, showing multifocal, intralesional viral antigen. Parts A and D show histopathology, haematoxylin and eosin stain; bar 20 μm. Parts B, C, E, and F show immunohistochemistry, Avidin-Biotin Complex method, aminoethyl carbazole chromogen (red-brown), Mayer's haematoxylin counter stain (blue); bar 20 μm. SARS-CoV-2=severe acute respiratory syndrome coronavirus 2.