Pathogenicity of SARS-CoV-2 and MERS-CoV in Beagle Dogs


 The coronavirus disease 19 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in unprecedented challenges to healthcare worldwide. In particular, the anthroponotic transmission of human coronaviruses has become a common concern among pet owners. Here, we experimentally inoculated beagle dogs with SARS-CoV-2 or Middle East respiratory syndrome (MERS)-CoV to compare the viral susceptibility and pathogenicity. The dogs exhibited weight loss and increased body temperature and shed the viruses in nasal secretion, faeces, and urine. Mild interstitial pneumonia lesions were observed in the lung tissues of infected dogs. Additionally, clinical characteristics of SARS-CoV-2 infection, such as increased lactate dehydrogenase levels was observed in the current study.


Introduction
Coronavirus disease-2019 (COVID-19) first emerged in China and quickly became a worldwide pandemic. Although most studies have focused on focused on the pathogenesis of COVID-19 in humans, the zoonotic aspects of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) have raised public health concerns worldwide. In particular, pet dogs living with patients affected by COVID-19 have become infected and shown seroconversion in Hong Kong; these initial cases of human-to-animal transmission of SARS-CoV-2 have suggested potential anthroponotic issues related to COVID-19 1 . Middle East respiratory syndrome (MERS)-CoV, first identified in Saudi Arabia in 2012, belongs to the beta coronavirus genus, as dose SARS-CoV-2, and has a fatality rate of 34.5% owing to severe respiratory illness 2 . Non-human primates, hDPP4-expressing transgenic mice, and dromedary camels, the natural host of the virus, have been shown to be appropriate animal models for MERS-CoV infection through experimental studies using a variety of animal species 3 . However, no studies have assessed whether MERS-CoV can infect dogs. For SARS-CoV-2, several attempts have been made to select appropriate animal models that accurately recapitulate clinical manifestations of COVID-19 4 . Dogs have also been inoculated with SARS-CoV-2, and limited pathogenesis, such as viral RNA detection in rectal swabs, was observed 5,6 . Because pet dogs share living space with humans and are a major companion animal as well as an important large animal model for drug development, further analyses are needed to fully establish whether SARS-CoV-2 and MERS-CoV can infect dogs. Accordingly, in this study, we assessed the susceptibility of dogs to SARS-CoV-2 and MERS-CoV following experimental inoculation.

Results
Beagle dogs (9 months old) were experimentally inoculated with SARS-CoV-2 or MERS-CoV, and their susceptibility to these human CoVs was assessed. Most dogs, except for 1 dog inoculated with SARS-CoV-2, showed elevated body temperature compared with the noninfected dog ( Figure. 1, A). Weight loss was also observed in most dogs until 7 days-postinoculation (dpi), whereas the weight of 1 dog infected with SARS-CoV-2 was recovered after 6 dpi ( Figure. 1, B).
LDH is a biomarker that is present in most body tissues and is elevated following tissue damage. Recently, several clinical studies demonstrated that increased LDH levels were associated with disease severity in patients with COVID-19, suggesting that this parameter may be a useful biomarker for disease progression [10][11][12] . In the current study of experimental infection, LDH levels were significantly increased in dogs infected with SARS-CoV-2 or MERS-CoV; indeed, LDH levels were increased by 1.7-fold at 3 dpi, 4.2-fold at 5 dpi, and 5.5-fold at 6 dpi in three SARS-CoV-2 infected dogs and 2.4-, 4.9-, and 4.1-fold higher at 3 dpi in three MERS-CoV infected dogs compared with those in the non-infected dog (normal range: 40-400 U/L; Figure. (Table 1). In the three dogs infected with SARS-CoV-2, viral RNA was detected in nasal swabs, rectal swabs, and urethral swabs at 3, 5, 6, and 7 dpi, whereas MERS-CoV RNA was partially detected in nasal swabs (3 of 3 dogs at 3 dpi, 2 of 3 dogs at 5 dpi, and 2 of 3 dogs at 6 dpi), rectal swabs (2 of 3 dogs at 3 dpi), and urethral swabs (2 of 3 dogs at 3 dpi).
All samples were inoculated into Vero E6 cells for analysis of viral viability. SARS-CoV-2 was cultivated from some nasal swabs (3 of 3 dogs at 3 dpi, 2 of 3 dogs at 5 dpi, and 2 of 3 dogs at 6 dpi) and urethral swabs (1 of 3 dogs at 3, 5, and 6 dpi and 2 of 3 dogs at 7 dpi), but not from rectal swabs. MERS-CoV was not cultivated from any samples. A neutralizing antibody was measured using serum samples, and only one dog infected with SARS-CoV-2 showed an antibody response at 6 dpi (2 Log2) and 7 dpi (3 Log2).
Pathological examinations were performed on euthanized dogs at the end of the experiment.
On autopsy of dogs infected with SARS-CoV-2, pulmonary consolidation was observed on each lung surface (Figure 2A and 2B), however the other organs were normal. There were no pathologic changes on the organs of MERS-CoV infected dogs. Histopathology from the lung, pharynx, lymph node, spleen, and kidney from all dogs revealed pathologic changes only in lung tissues. Dogs infected with SARS-CoV-2 or MERS-CoV showed similar interstitial pneumonia with mild multifocal peribronchial and perivascular infiltration by inflammatory cells (Figure 2C -2F). Immunohistochemistry revealed the presence of the SARS-CoV-2 antigen, following viral infection, in the lung and alveolar wall ( Figure 2G and 2H), however the MERS-CoV antigen was not detected ( Figure 2I).

Discussion
Dogs were first used as a model animal for SARS-CoV-2 by a Chinese research group.
Their results showed that dogs exhibited low susceptibility to intranasal inoculation with the virus, as demonstrated by partial virus shedding and no viral detection in tissues 5 . However, we speculated that their data could have been somewhat limited with regard to determining the susceptibility of dogs to SARS-CoV-2. In addition, most pet owners care for their pets a lot and are concerned with the potential for their pets to be infected 13,14 .
Therefore, in this carefully designed study, we aimed to demonstrate the pathogenicity of SARS-CoV-2 in dogs based on evaluation of multiple parameters, including clinical signs and blood parameters, and to compare the results with those from another human coronavirus, MERS-CoV. Interestingly, our results were not consistent with the previous study and showed that the dogs exhibited clinical signs of infection after inoculation with SARS-CoV-2 or MERS-CoV. Pathological examinations showed that both human CoVs developed mild interstitial pneumoniae in dogs. SARS-CoV-2 RNA was detected in all nasal and rectal swab samples in this study, and viral viability was even observed in some nasal swabs, whereas viral RNA was not detected in any of the oropharyngeal swabs and in only a few rectal swabs (2 of 5 dogs at 2 dpi, 1 of 4 dogs at 6 dpi, respectively) in a previous experiment 5, 6 . Furthermore, SARS-CoV-2 RNA was detected all urethral swabs, and some of the collected viruses could be cultivated.
SARS-CoV-2 viability was also detected in urine from a patient with COVID-19 and was confirmed in a clinical study 15 . Among the blood parameters, LDH levels were markedly altered in the dogs in our study. In a recent report, a predictive model using machine learning algorithms and abundant epidemiological, clinical, and laboratory information was established to identify prognostic biomarkers for patients with COVID-19 16 . The model identified three key features, including LDH levels, as important factors for prognostic prediction in patients with COVID-19 16 . Consistent with this, in our study, LDH levels were the most prominent parameter affected by respiratory viral infection.

Histopathology
All dogs were euthanized using an intravenous injection of 0.1 mg/kg pancuronium bromide and 0.1 M KCl at the end of the experiment (7 dpi). At necropsy, gross lesions were observed seen mostly in the lung, pharynx, lymph nodes, spleen, and kidney, and then tissues were Colour development was performed using 3, 3'-diamino-benzidine tetrahydrochloride (DAB; K5007, Dako, Denmark) followed by counterstaining with haematoxylin. Light microscopic examination was performed using a BX53 microscope (Olympus, Japan). Tissue samples from all dogs were placed into soft tissue homogenizing CK14 tubes (Precellys, Betin Technologies) prefilled with ceramic beads and DMEM and then homogenized using a Bead blaster 24 (Benchmark Scientific, NJ, USA). Viral RNA was extracted from the homogenized tissues using the QIAamp viral RNA Mini Kit (Qiagen) according to the manufacturer's protocol. Real-time PCR for each virus was conducted using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA).

Lung Tonsil
Serum Nasal Anal Urethra dpi a dpi dpi dpi