Susceptibility of raccoon dogs for experimental SARS-CoV-2 infection
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Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China at the end of 2019, and became pandemic. The zoonotic virus most likely originated from bats, but definite intermediate hosts have not yet been identified. Raccoon dogs ( Nyctereutes procyonoides ) are kept for fur production, in particular in China, and were suspected as potential intermediate host for both SARS-CoV6 and SARS-CoV2. Here we demonstrate susceptibility of raccoon dogs for SARS-CoV-2 infection after intranasal inoculation and transmission to direct contact animals. Rapid, high level virus shedding, in combination with minor clinical signs and pathohistological changes, seroconversion and absence of viral adaptation highlight the role of raccoon dogs as a potential intermediate host. The results are highly relevant for control strategies and emphasize the risk that raccoon dogs may represent a potential SARS-CoV-2 reservoir. Our results support the establishment of adequate surveillance and risk mitigation strategies for kept and wild raccoon dogs.
Article Summary Line
Raccoon dogs are susceptible to and efficiently transmit SARS-CoV2 and may serve as intermediate host
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SciScore for 10.1101/2020.08.19.256800: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: The animal experiments were evaluated and approved by the ethics committee of the State Office of Agriculture, Food Safety, and Fishery in Mecklenburg – Western Pomerania (LALLF M-V: LVL MV/TSD/7221.3-2-010/18-12) Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Study design: Fourteen adult, male (n=4) and female (n=10) raccoon dogs originating from a commercial farm were used. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources After the incubation of the sera or saliva and the following washing, dog-specific, horseradish-peroxidase (HRP) labelled Ig antibodies … SciScore for 10.1101/2020.08.19.256800: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: The animal experiments were evaluated and approved by the ethics committee of the State Office of Agriculture, Food Safety, and Fishery in Mecklenburg – Western Pomerania (LALLF M-V: LVL MV/TSD/7221.3-2-010/18-12) Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Study design: Fourteen adult, male (n=4) and female (n=10) raccoon dogs originating from a commercial farm were used. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources After the incubation of the sera or saliva and the following washing, dog-specific, horseradish-peroxidase (HRP) labelled Ig antibodies (goat-α-dog-IgA 1:1,000 for saliva and 1:5,000 for serum; goat-α-dog-IgM1 1:15,000; goat-α-dog-IgG, goat-α-dog-IgG1, goat-α-dog-IgG2 all 1:20,000; Bethyl Laboratories INC) were added and incubated for 1h at RT. HRP ) labelled Ig antibodiessuggested: None1:15,000; goat-α-dog-IgG , goat-α-dog-IgG1 , goat-α-dog-IgG2suggested: Nonegoat-α-dog-IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources Virus titration: Virus titer used for infection experiments was confirmed by titration on Vero E6 cells (Biobank Friedrich-Loeffler-Institut, catalogue N° 0929) and evaluation of CPE after 5 days. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources Statistical information: All data were analyzed and visualized using GraphPad Prism Version 7.0 GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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Our take
In a preprint that has not yet been peer reviewed, the authors demonstrate that raccoon dogs (a canid indigenous to East Aisa) are susceptible to SARS-CoV-2 infection in an experimental setting, and can transmit to other animals in close contact. Animals shed virus starting two days after exposure and produced neutralizing antibodies but showed only minor clinical signs and tissue pathology. Large aggregations of this species on fur farms in China may present an elevated risk for outbreaks of infection seeded by workers and should be a target of more intense surveillance.
Study design
prospective-cohort;ecological;other
Study population and setting
The study describes an experimental infection of fourteen raccoon dogs (Nyctereutes procyonoides) from a commercial fur farm with SARS-CoV-2. Nine …
Our take
In a preprint that has not yet been peer reviewed, the authors demonstrate that raccoon dogs (a canid indigenous to East Aisa) are susceptible to SARS-CoV-2 infection in an experimental setting, and can transmit to other animals in close contact. Animals shed virus starting two days after exposure and produced neutralizing antibodies but showed only minor clinical signs and tissue pathology. Large aggregations of this species on fur farms in China may present an elevated risk for outbreaks of infection seeded by workers and should be a target of more intense surveillance.
Study design
prospective-cohort;ecological;other
Study population and setting
The study describes an experimental infection of fourteen raccoon dogs (Nyctereutes procyonoides) from a commercial fur farm with SARS-CoV-2. Nine animals (three males, six females) were inoculated intranasally with 10^5 median tissue culture infectious dose (TCID50). Three contact animals (all females) were added in adjacent cages 24 hours after inoculation of the nine other animals. Two other animals (one male, one female) were used as controls to assess pathological changes in infected animals. Swabs from the upper respiratory tract and rectum and fecal samples were taken to test for viral shedding (via RT-PCR), and blood was taken to test for antibodies (reactive and neutralizing) regularly for 28 days post infection. To check for pathological signs in infected tissues, two inoculated animals each were sacrificed on day 4, 8, and 12, and the remaining inoculated animals and direct contact animals were sacrificed on day 28 and subject to autopsy.
Summary of main findings
Six of the nine inoculated animals became infected and shed infectious virus, detectable in nasal and oropharyngeal swabs starting at 2 days post infection (dpi). Shedding was detectable up to 16 dpi in nasal swabs, to 12 dpi in oropharyngeal swabs, and to 4 dpi in rectal swabs. Two of three direct contact animals became infected while the third did not become infected because its inoculated neighbors never shed virus. Viral shedding was detected at 8 dpi (7 days post contact, dpc) and lasted until 16 dpi (15 dpc), mainly in nasal secretions. Antibodies against SARS-CoV-2 (reactive and neutralizing) were found in all inoculated animals at 8 dpi and infected contact animals at 12 dpi. Antibody titers increased up to 28 dpi. The only clinical signs of infection were reduced activity in three animals at 4 dpi and mild tissue pathology (rhinitis) at 4, 8, and 12 dpi. There was no change in body temperature or weight loss.
Study strengths
The authors tested for the presence of neutralizing antibodies (not just reactive antibodies), performed assays that detected shedding of live virus and isolated viruses, and confirmed that no viral evolution had occurred in the animals by sequencing isolated viruses and comparing to the original virus inoculum.
Limitations
Only one inoculation route was tested (intranasal). Other routes may result in a higher or lower likelihood of infection and potentially different clinical manifestations. The lack of pulmonary signs in infected animals indicates that this species would not be a good animal model for pulmonary symptoms of COVID-19 in human patients.
Value added
This is the first study to confirm raccoon dogs as potential hosts of SARS-CoV-2. These findings are important because this species was found naturally infected with SARS-CoV in 2003 and may have been an intermediate host in that pandemic. China has a large fur industry for raccoon dogs (millions of animals), so there may be a risk for outbreaks of the virus on fur farms similar to outbreaks seen on mink farms in Europe and the US. Additionally, this species is invasive in Europe, presenting a possible risk of reverse zoonotic transmission from humans into wild populations.
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