key: cord-0885589-q1qkve7o
authors: Lee, Hansaem; Lee, Tae-Young; Jeon, Pyeonghwa; Kim, Nayoung; Kim, Jun-Won; Yang, Jeong-Sun; Kim, Kyung-Chang; Lee, Joo-Yeon
title: J2N-k hamster model simulates severe infection caused by severe acute respiratory syndrome coronavirus 2 in patients with cardiovascular diseases
date: 2021-09-30
journal: J Virol Methods
DOI: 10.1016/j.jviromet.2021.114306
sha: 4b88692349a4e0cd1dbd06888588d63f0315c0db
doc_id: 885589
cord_uid: q1qkve7o

Considering the global impact of the coronavirus disease 2019 (COVID-19) pandemic, generating suitable experimental models is imperative. For pre-clinical studies, researchers require animal models displaying pathological features similar to those observed in patients; therefore, establishing animal models for COVID-19 is crucial. The golden Syrian hamster model mimics conditions observed in humans with mild severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, a golden Syrian hamster model of severe infection has not been reported. J2N-k hamsters are utilized as a cardiomyopathy model; therefore, we used cardiomyopathic J2N-k hamsters showing conditions similar to those of severe COVID-19 complicated with cardiovascular diseases, as patients with cardiovascular diseases exhibit a higher risk of morbidity and mortality due to COVID-19 than patients without cardiovascular diseases. Unlike that in golden Syrian hamsters, SARS-CoV-2 infection was lethal in J2N-k hamsters, with a median lethal dose of 10(4.75) plaque-forming units for the S clade of SARS-CoV-2 (A, GenBank: MW466791.1). High viral titers and viral genomes were detected in the lungs of J2N-k and golden Syrian hamster models harvested 3 days after infection. Pathological features of SARS-CoV-2-associated lung injury were observed in both models. The J2N-k hamster model can aid in developing vaccines or therapeutics against COVID-19.

3  Abbreviations: SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; COVID-19, coronavirus disease 2019; CVD, cardiovascular disease; dpi, days post-infection; PFU, plaque-forming unit; LD50, median lethal dose; TCID50, median tissue culture infectious dose Keywords: SARS-CoV-2, golden Syrian hamster, J2N-k hamster, The golden Syrian hamster model was established in 2005 for studying severe acute respiratory syndrome (SARS) (Roberts et al., 2005) . Recently, this model was deemed suitable for studying SARS-coronavirus 2 (SARS-CoV-2; family Coronaviridae, order Nidovirales, subgenus Sarbecovirus), the etiological agent for the ongoing coronavirus disease 2019 pandemic (Chan et al., 2020; Sia et al., 2020) . The human and hamster angiotensin-converting enzyme 2 (ACE2) sequences share a high similarity (82.8%) (Chan et al., 2020) , and hamsters infected with SARS-CoV-2 exhibit loss in body weight similar to that observed in patients with SARS-CoV-2 infection. So far, lethal outcomes have not been observed in the golden Syrian hamster model following SARS-CoV-2 infection. Thus, this model effectively mimics the conditions of patients experiencing mild COVID-19 symptoms. However, lethal outcomes have been reported in dwarf hamster (Trimpert et al., 2020) , STAT-knockout hamster (Boudewijns et al., 2020) , and human ACE2-transgenic mouse models (Winkler et al., 2020) with SARS-CoV-2 infection.

Higher risk of morbidity and mortality (10%-15%) due to SARS-CoV-2 infection has been reported in patients with cardiovascular diseases (CVDs) than in those without cardiovascular diseases (~5%) (Clerkin et al., 2020; Zheng et al., 2020) . Therefore, an animal model for severe J o u r n a l P r e -p r o o f SARS-CoV-2 infection complicated with CVDs is urgently required. As J2N-k hamsters have previously been utilized as a cardiomyopathic animal model harboring abnormalities in the ADP/ATP carrier protein and a defective delta-sarcoglycan encoding gene (Kato et al., 1993; Mitsuhashi et al., 2003) , we investigated the susceptibility of the J2N-k hamster model to SARS-CoV-2 infection in this study and compared it with that of the golden Syrian hamster model. All Seven-week-old male golden Syrian hamsters and J2N-k hamsters were purchased from Japan SLC, Inc. (Shizuoka, Japan). They were anesthetized with tiletamine/zolazepam combined with xylazine injected intraperitoneally before infection. Subsequently, serially 10-fold diluted SARS-CoV-2 was administered intranasally (from 10 6 to 10 3 plaque-forming units (PFU)/100 µL) to the hamsters (golden Syrian (n = 6) and J2N-k (n = 4) per group). The infected hamsters were monitored daily for weight loss and mortality for 14 days. The median lethal dose (LD50) was determined by assessing the number of dead and live hamsters on day 14, following methods previously described (Ramakrishnan, 2016) .

To measure the viral load in the tissues of SARS-CoV-2-infected hamsters, the lung or tracheal tissues were weighed and homogenized in 1 mL of DPBS using bead-beating homogenizer. After centrifugation at 13,000 rpm for 20 min, the clarified supernatant was harvested. Aliquots of the supernatant were incubated with confluent Vero-E6 cells in 96-well plates for the median tissue Intranasal administration of 10 6 PFU/100 μL SARS-CoV-2 in 7-week-old male golden Syrian hamsters resulted in disease symptoms similar to those reported previously ( Figure 1A ) (Chan et al., 2020; Sia et al., 2020) . A 20% decline in body weight was noted, and recovery was observed 7 days post-infection (dpi). However, 7-week-old male J2N-k hamsters infected with 10 6 PFU SARS-CoV-2 exhibited 25.7% weight loss. Additionally, lethality was observed in all infected J2N-k hamsters at 9 dpi ( Figure 1 ). The LD50 was calculated as 10 4.75 PFU for the S clade of SARS-CoV-2. In contrast, no lethality was detected in the six golden Syrian hamsters infected with SARS-CoV-2.

High viral titers and viral genomes were detected in the lungs of both animal models sacrificed 3 dpi but not after 7 dpi, as evidenced by the TCID50 assay and real-time RT-PCR assay, respectively ( Figure 2A ). However, comparable viral titers and viral genomes were detected in the lung tissues when both models were administered with different viral doses ( Figure 2B ).

Additionally, the lungs of both models infected with SARS-CoV-2 exhibited severe chronic J o u r n a l P r e -p r o o f active pneumonia in the pulmonary parenchyma. Furthermore, their lungs showed hyperplastic type II alveolar epithelial cells with infiltration of inflammatory cells and neutrophils ( Figure   2C ).

Next, the possibility of using the J2N-k hamster model for developing vaccines or therapeutic drugs against SARS-CoV-2 was considered. Seven J2N-k hamsters that survived SARS-CoV-2 infection (10 3 and 10 4 PFU) were assessed for resistance to viral re-infection. The exposed J2N-k hamsters tested positive for the presence of neutralizing antibodies prior to re-infection ( Figure   3A ). Seven J2N-k hamsters were re-infected with 10 6 PFU of the homologous virus on day 25 after the primary infection ( Figure 3B ). The body weight and the survival rate were monitored daily, and the lung and trachea tissues were collected 3 dpi for virus titration. Six naïve J2N-k hamsters showed the presence of infection and succumbed, thus proving that survival was associated with body weight loss, as predicted. In contrast, all previously infected hamsters survived re-infection without any significant weight loss. Additionally, the surviving hamsters presented with markedly lower viral titers and viral genomes (unpaired t-test, with p-value < 0.05) in their lung and trachea tissues homogenates 3 dpi than the naïve hamsters ( Figure 3C ). In conclusion, pre-exposure to SARS-CoV-2 elicited neutralizing antibodies that helped in protecting the J2N-k hamsters against lethal SARS-CoV-2 re-infection. Therefore, J2N-k hamsters are a potential model for testing vaccines and antiviral drugs to treat SARS-CoV-2 infection.

Although this study had a few limitations, including a low number of hamsters per group, not having used female J2N-k hamsters, and performing viral titration only using samples from the J o u r n a l P r e -p r o o f lungs and trachea, the results suggested that the J2N-k hamster model could successfully simulate severe COVID-19 infection in patients with CVDs. In addition, male hamsters have been reported to be more susceptible to SARS-CoV-2 infection than females (Dhakal et al., 2021; Yuan et al., 2021) . It is assumed that male J2N-k hamsters would be more suitable as SARS-CoV-2 models owing to their higher susceptibility to SARS-CoV-2 than females. Notably, other hamster models are also useful for studying SARS-CoV-2. For instance, an STAT2knockout hamster model lacking STAT2-dependent interferon response may be used for systemic immune-compromised COVID-19 studies (Boudewijns et al., 2020) . Additionally, the susceptibility of Chinese hamsters to SARS-CoV-2 is comparable to that of normal golden Syrian hamsters (Bertzbach et al., 2021) . Dwarf hamsters can be utilized as models for severe COVID-19; however, factors contributing to the pathology of severe SARS-CoV-2 infection in this model remain unclear (Trimpert et al., 2020) . Several strategies, including repurposing of drugs, and use of monoclonal therapeutic antibodies, anti-inflammatory agents, and vaccines have been used to treat patients with severe COVID-19 or to prevent SARS-CoV-2 infection.

Therapeutic and immunization strategies for COVID-19 patients with CVDs are crucial, considering that mortality due to COVID-19 is significantly attributed to CVDs (Yi et al., 2021) .

To evaluate the potential efficacy of cardiovascular or antiviral drugs in these patients, the theoretical basis of pre-clinical animal models should be established before clinical treatment.

This study may help guide the preclinical evaluation of treatment strategies and the effects of vaccination for severe COVID-19 in patients with CVDs. Additionally, the J2N-k hamster model could be used in studies pertaining to CVDs and SARS-CoV-2 infection and viral transmission. were monitored after re-infection for 14 days. (C) Infectious viral load (log10 TCID50/mL) and viral genome copies (log10 Genome copies/mL) detected in the lungs and trachea of hamsters rechallenged with SARS-CoV-2. A selected number of hamsters from the re-challenged group (n = 3) and from the infected-survival group, which included animals that had survived infection with 10 3 PFU (n = 2) and 10 4 PFU (n = 1) of SARS-CoV-2, were sacrificed at 3 dpi. The infectedsurvival group is indicated with orange bars and dots. The dotted line represents the detection limit. Blue bars and dots indicate the virus titers and viral genomes, respectively, of the previously uninfected hamsters infected with 10 6 PFU of SARS-CoV-2. The SARS-CoV-2 live virus titers in the lungs and trachea were not detected in the surviving hamsters re-infected with SARS-CoV-2. The remaining hamsters were maintained alive to monitor the changes in body J o u r n a l P r e -p r o o f

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