key: cord-281081-rifr5uub
authors: Deng, Junhua; Jin, Yipeng; Liu, Yuxiu; Sun, Jie; Hao, Liying; Bai, Jingjing; Huang, Tian; Lin, Degui; Jin, Yaping; Tian, Kegong
title: Serological survey of SARS‐CoV‐2 for experimental, domestic, companion and wild animals excludes intermediate hosts of 35 different species of animals
date: 2020-05-07
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.13577
sha: 
doc_id: 281081
cord_uid: rifr5uub

The pandemic SARS‐CoV‐2 has been reported in 123 countries with more than 5,000 patients died from it. However, the original and intermediate hosts of the virus remain unknown. In this study, 1,914 serum samples from 35 animal species were used for detection of SARS‐CoV‐2‐specific antibodies using double‐antigen sandwich ELISA after validating its specificity and sensitivity. The results showed that no SARS‐CoV‐2‐specific antibodies were detected in above samples which excluded the possibility of 35 animal species as intermediate host for SARS‐CoV‐2. More importantly, companion animals including pet dogs (including one dog the SARS‐CoV‐2 patient kept and two dogs which had close contact with it) and cats, street dogs and cats also showed serological negative to SARS‐CoV‐2, which relieved the public concerns for the pets as SARS‐CoV‐2 carriers.

DENG Et al. giant panda, masked civet, porcupine, bear, yellow-throated marten, weasel, red pandas and wild boar). The results showed that no SARS-CoV-2-specific antibodies were detected in above species of animals including pangolin which has been reported as an intermediate host of SARS-CoV-2 (Kangpeng Xiao, 2020) . More importantly, we found companion animals including dogs and cats were serologically negative to SARS-CoV-2 including one dog kept by the SARS-CoV-2 patient and two dogs with close contact with it during the quarantine.

The SARS-CoV-2 double-antigen sandwich ELISA was purchased from Luoyang Putai Biotechnology Co., Ltd. The coating was based on S1 protein of SARS-CoV-2. The same antigen was linked to horseradish peroxidase (HRP) to function as conjugate. The serum samples were tested according to the manufacture manual instructions.

Briefly, 100 µl serum sample was added into each well of ELISA plate and incubated at 37°C for 30 min. After washing the plate with washing buffer for five times, HRP-labelled antigen was added into the wells at 37°C for 30 min before 100 μl of the substrate solution was added to each well and incubated at 37°C for 10 min to stop the reaction. The optical density (OD) was measured at 450 nm. 

To test the specificity of ELISA kit, the serum samples of SPF chicken (28), duck (25), mouse (23), rat (20) and pig (20) were applied. The final value of OD 450 of samples ranged from 0.005 to 0.103 (median 0.007), 0.004 to 0.008 (median 0.006), 0.005 to 0.190 (median 0.007), 0.004 to 0.050 (median 0.007) and 0.005 to 0.134 (median 0.007) for chicken, duck, mouse, rat and pig, respectively. Serum samples from other species of experimental animals including guinea pig (30), rabbits (34), beagle dogs (130) and rhesus monkeys (38) were also tested.

There were no SARS-CoV-2 antibodies detected in above animals (data not shown). Next, the potential cross-reaction with other coronavirus including IBV (26) We next tested the sensitivity of ELISA kit. The SARS-CoV-2 experimental-infected ferret positive sera were tested. As shown in Table 1 , the neutralizing antibody titres of 5 infected ferret (F1-F5) were between 1:128 and 1:256 at 22 days post-infection (dpi). By contrast, the neutralizing antibody titres of 5 placebo ferrets (C1-C5) were all negative at 22 dpi. In the line with the results of neutralizing antibodies, the final OD 450 of 5 positive sera detected by ELISA was all above 3, which indicated strongly serological positive to SARS-CoV-2. To further test the dynamic changes of ELISA titre of infected ferret, serum samples from one ferret were collected from 0, 7, 12, 17 and 22 dpi, respectively. The positive ELISA results were shown at 7 dpi and lasted until 22 dpi when the ferrets were humanely euthanized (Table 1) . The above results showed that the ELISA has good specificity and sensitivity and suitable for different species of animals.

After confirming the specificity, sensitivity and suitability of SARS-CoV-2 ELISA kit for different species of experimental animals, clinical serum samples from domestic livestock (pig, cow, sheep, horse), poultry (chicken, duck, goose), experimental animal (mice, rat and rhesus monkey), companion animal (dog and cat) and wild animals (camel, fox, mink, alpaca, ferret, bamboo rat, peacock, eagle, tiger rhinoceros, pangolin, leopard cat, jackal, giant panda, masked civet, porcupine, bear, yellow-throated marten, weasel, red pandas and wild boar) were used for antibody detection. As shown in Table 2 unknown. Since SARS-CoV-2 is genetically close to SARS-CoV, it has been proposed that bat could be the natural host (Phan, 2020) . Snake is also presumed as wildlife animal reservoir for SARS-CoV-2 based on the virus relative synonymous codon usage (RSCU) bias (Ji, Wang, Zhao, Zai, & Li, 2020) . However, there is no report of SARS-CoV-2 isolation or molecular and serological confirmation of infection from snake samples. Pangolins recently was suggested to be direct animal source of SARS-CoV-2 for humans since the SARS-CoV-2-related coronaviruses were isolated from Malayan pangolins which shared 97.4% similarity with SARS-CoV-2 in virus receptor-binding domain in S gene (Kangpeng Xiao, 2020) . In our study, we did not detect SARS-CoV-2 antibodies in 17 pangolin serum samples. Consistent with our results, Li et al., (2020) reported the coronavirus carried by pangolins did not have the RRAR motif, a unique peptide insertion in the human SARS-CoV-2 virus. The RRAR motif may be involved in the proteolytic cleavage of spike protein and host range and transmissibility which suggests human SARS-CoV-2 virus did not come directly from pangolins . Masked civet and camel were confirmed to be natural hosts for SARS-CoV and MERS-CoV, and no specific SARS-CoV-2 antibodies were detected in 10 masked civets and 31 camels in this study.

to the SARS-CoV-2 has been major concern for the public. One pet dog was reported to be SARS-CoV-2-positive detected by RT-PCR in Hongkong (https://www.news.gov.hk/eng/2020/02/20200 228/20200 228_093205_796.html). Later, the serological result of the dog showed negative after quarantine of 14 days. In our study, 87 cats including 66 pet cats and 21 street cats showed serological negative to SARS-CoV-2 (Table 2) . At the same time, 487 dogs including 90 beagle dogs, 147 pet dogs and 250 street dogs during the outbreak of SARS-CoV-2 were also tested serological negative. Among them, 15 pet dog and 99 street dog sera were collected from Wuhan City. It should be noted that one pet dog from confirmed SARS-CoV-2-infected patient showed serologically negative, and other two dogs which had close contact with this dog also tested to be negative. However, we cannot rule out of susceptibility of cats and dogs to SARS-CoV-2, which need to be tested by experimental infections.

Molecular techniques such as reverse-transcriptase PCR tests and viral genome sequencing are widely used for the confirmation of human infection. These techniques are also used to explore the potential hosts of SARS-CoV-2 (Pfefferle, Reucher, Norz, & Lutgehetmann, 2020) . Compared to these molecular methods, serological test such as ELISA has several advantages. First, the host generates SARS-CoV-2-specific antibodies after infection which could last longer than the viraemia. It provides a wider detection window for ELISA than RT-PCR. Second, RNA extraction from susceptive infected samples has to be performed in a BSL-3 laboratory. By 

Abbreviation: dpi, days post-infection.

*The neutralizing antibody titre of positive samples was ≥4. contrast, ELISA can be performed in a safety level 2 laboratory and does not require high containment facilities after the serum samples were inactivated at 56°C for 30 min. Third, double-antigen sandwich ELISA based on recombinant S1 protein could detect both IgM and IgG antibodies and is not limited to species. To find the host of SARS-CoV-2, the screening of other wild animals using ELISA is undergoing in our laboratory.

We want to thank Dr. Zhigao Bu, Director of Harbin Veterinary

Research Institute, Chinese Academy of Agricultural Sciences for providing inactivated SARS-CoV-2-negative and SARS-CoV-2positive ferret serum samples. This study was supported by Luoyang

Heluo Talent Plan (Kegong Tian).

We declare that ethical statement is not applicable.

There was no conflict of interest with others.

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

Kegong Tian https://orcid.org/0000-0001-5362-1415

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Serological survey of SARS-CoV-2 for experimental, domestic, companion and wild animals excludes intermediate hosts of 35 different species of animals