key: cord-0831107-sabnutbq
authors: Chu, Hin; Fuk-Woo Chan, Jasper; Wang, Yixin; Tsz-Tai Yuen, Terrence; Chai, Yue; Shuai, Huiping; Yang, Dong; Hu, Bingjie; Huang, Xiner; Zhang, Xi; Hou, Yuxin; Cai, Jian-Piao; Zhang, Anna Jinxia; Zhou, Jie; Yuan, Shuofeng; Kai-Wang To, Kelvin; Fan-Ngai Hung, Ivan; Cheung, Tan To; Tsui-Lin Ng, Ada; Hau-Yee Chan, Ivy; Yu-Hong Wong, Ian; Ying-Kit Law, Simon; Chi-Chung Foo, Dominic; Leung, Wai-Keung; Yuen, Kwok-Yung
title: SARS-CoV-2 induces a more robust innate immune response and replicates less efficiently than SARS-CoV in the human intestines: an ex vivo study with implications on pathogenesis of COVID-19
date: 2020-10-01
journal: Cell Mol Gastroenterol Hepatol
DOI: 10.1016/j.jcmgh.2020.09.017
sha: 6d7150c3c524f7be4526a590eed91f4e7c48a160
doc_id: 831107
cord_uid: sabnutbq

Background And Aims Besides prominent respiratory involvement, gastrointestinal manifestations are commonly reported in Coronavirus Disease 2019 (COVID-19) patients. We compared infection of ex vivo human intestinal tissues by SARS-CoV-2 and SARS-CoV with respect to their replication kinetics and immune activation profile. Methods Human intestinal tissues were obtained from patients while undergoing surgical operations at the Queen Mary Hospital, Hong Kong. Upon surgical removal, the tissues were immediately processed and infected with SARS-CoV-2 or SARS-CoV. Replication kinetics were determined with immunohistochemistry, qRT-PCR, and plaque assays. Immune activation in the infected intestinal tissues was assessed by detecting the gene expression of interferons and representative pro-inflammatory cytokines and chemokines. Results SARS-CoV-2 could infect and productively replicate in the ex vivo human intestinal tissues with the release of infectious virus particles, but not in ex vivo human liver and kidney tissues. Importantly, SARS-CoV-2 replicated less efficiently than SARS-CoV, induced less cytopathology in the human intestinal epithelium, and induced a more robust innate immune response including the activation of both type I and type III interferons, than SARS-CoV in human intestinal tissues. Conclusion Using the ex vivo human intestinal tissues as a physiologically relevant model, our data indicated that SARS-CoV-2 could productively replicate in the human guts, suggesting the gastrointestinal tract might serve as an alternative route of virus dissemination. SARS-CoV-2 replicated less efficiently and induced less cytopathology than SARS-CoV in keeping with the clinical observations reported for SARS-2003 and COVID-19, which might be a result of the more robust immune activation by SARS-CoV-2 than SARS-CoV in the human intestine.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly among 146 the human population, 1, 2 resulting in more than 20 million laboratory-confirmed cases and 147 over 750,000 deaths within eight months. In addition to systemic and respiratory 148 manifestations, gastrointestinal symptoms including diarrhoea, vomiting, and abdominal 149 pain, were also commonly reported in patients with Coronavirus Disease 2019 . 3-150 9 Viral RNA was persistently detected in the stool specimens of 28.8-70.3% of COVID-19 151 patients 10 and viral protein expression or virus particles were also observed in intestinal 152 tissues on biopsy. 11, 12 These findings suggested that SARS-CoV-2 might be able to infect 153 and replicate in the human gastrointestinal tract, which might serve as an alternative route of 154 virus dissemination. 155

In addition to SARS-CoV-2, gastrointestinal involvement was also reported in SARS-156

Approximately 20% to 70% of SARS 13-16 and 26% of MERS 17 patients had diarrhea during 158 the course of illness, respectively. In comparison, we have recently estimated the pooled 159 prevalence of diarrhoea in COVID-19 to be 12.5% (95% CI, 9.6-16.0) 10 , which is 160 considerably lower than that of SARS and MERS. In line with this observation, faecal 161

shedding of viral RNA was detected in 28.8% to 70.3% of COVID-19 patients, which was 162 substantially lower than the 86% to 100% reported in SARS patients. 10 These findings 163 indicated a differential degree of gastrointestinal tract involvement in 164 which is intriguing because both of these lineage B betacoronaviruses use angiotensin 165 converting enzyme 2 (ACE2) as the entry receptor 18 and have similar tissue tropism. 19 166

To physiologically simulate SARS-CoV-2 infection in the human intestinal tract, we 167 investigated the infection of SARS-CoV-2 in ex vivo human intestinal tissue explants, which 168 authentically represent the cell type composition and protein expression profile of the human 169 J o u r n a l P r e -p r o o f intestine as they were directly extracted and immediately infected upon resection from patient 170 donors. We further performed side by side comparison of SARS-CoV-2 and SARS-CoV 171 infection and investigated the replication kinetics and host innate immune activation of the ex 172 vivo human intestinal tissue explants in response to the two coronaviruses. Our study 173 revealed a number of intriguing findings. First, we demonstrated that SARS-CoV-2 could 174 infect and productively replicate in the human intestinal tissues. Second, SARS-CoV-2 175 replicated less efficiently than SARS-CoV and induced less cytopathology in the human 176 intestinal epithelium. Third, SARS-CoV-2 induced a more robust innate immune response, 177

including the activation of both type I and type III interferons, than SARS-CoV in the human 178 intestinal tissues. Overall, our study revealed that SARS-CoV-2 and SARS-CoV exhibited 179 different replication efficiencies and induced differential immune activation profiles in 180 human intestinal tissues, thus providing novel insights into our understanding on the 181 relatively less severe and frequent gastrointestinal involvement reported in COVID-19 than in 182

A total of 14 patients with large intestine tumors who underwent surgery joined the study. 187 There were 7 females and 7 males and their median age was 68.1 years (range, 54-81 years). 188

Additionally, 3 patients with renal tumors and 3 patients with hepatocellular carcinoma who 189 underwent surgery donated their kidney and liver tissues, respectively. control, viral N antigen or cytopathic effects were not detected in mock-infected intestinal 206 tissues ( Figure 1M-1P ). In addition to the human intestinal tissues, we also obtained human 207 liver tissues from three donors and human kidney tissues from three different donors. The 208 liver tissues and kidney tissues were similarly processed and challenged with SARS-CoV-2. 209

Interestingly, immunohistochemistry staining did not identify any SARS-CoV-2 antigen from 210 the liver or kidney tissues from all evaluated donors, suggesting that SARS-CoV-2 did not 211 intestinal tissues. To this end, the intestinal tissues were infected with SARS-CoV-2 or 220 SARS-CoV with an inoculum of 1×10 6 PFU/ml. The supernatant samples were harvested at 2 221 hpi, 12 hpi, and 24 hpi. SARS-CoV replicated more efficiently than that of SARS-CoV-2 in 222 five of the six evaluated donors (5/6, 83.3%) (Figure 2A ). Using the area under the curve 223 analysis, we quantified the total virus growth from baseline between 2 hpi and 24 hpi. Our 224 results indicated that SARS-CoV-2 produced significantly less (40.9%, P < 0.03) virus 225 genome copies than that of SARS-CoV over the 22 hours incubation period ( Figure 2B ). We 226 further assessed the infectious virus titer from supernatants of the infected intestinal tissues 227 harvested at 24 hpi with plaque assays. Our results demonstrated that the SARS-CoV-2 228 generated significantly less (76.2%, P < 0.04) infectious particles than that of SARS-CoV at 229 24 hpi ( Figure 2C ). Overall, our findings indicated that SARS-CoV-2 could infect and 230 replicate in the human intestinal tissues. Together with the immunohistochemistry and virus 231 replication assays, we demonstrated that SARS-CoV-2 replicated less robustly than that of 232 SARS-CoV and induced less cytopathology than that of SARS-CoV in the human intestine 233 epithelium. 234

We recently demonstrated that SARS-CoV-2 triggered an attenuated interferon response in ex 238 Caco2. 19, 31 Interestingly, the expression of known SARS-CoV-2 and SARS-CoV entry 305 factors including ACE2 and TMPRSS2 were more robust in the human intestine than in the 306 human lung. 32, 33 In this regard, since SARS-CoV spike lacks the additional furin-like 307 cleavage site at the S 1 /S 2 junction comparing to the SARS-CoV-2 spike, 34, 35 the higher ACE2 308 and TMPRSS2 expression in the intestine may have a larger impact on facilitating SARS-309

CoV replication than that of SARS-CoV-2. In addition to virus replication, SARS-CoV-2 310 also triggered less severe cytopathology than SARS-CoV in the infected intestine epithelium. supplemented with 2mM HEPES, 1x GlutaMAX, 100 U/ml penicillin, and 100 μg/ml 365 streptomycin (Thermo Fisher Scientific). The human ex vivo intestinal tissue cubes were 366 incubated with SARS-CoV-2 or SARS-CoV inoculum of 1×10 6 PFU/ml for 2 hours. After 2 367 hours, the viruses were removed and tissue cubes were washed three times with PBS. The 368 infected human intestinal tissues were incubated in 1.2ml advanced DMEM/F12 medium 369 supplemented with 2mM HEPES, 1×GlutaMAX, 100U/ml penicillin, 100μg/ml 370 streptomycin, 20μg/ml vancomycin, 20μg/ml ciprofloxacin, 50μg/ml amikacin, and 50μg/ml 371 nystatin per well. Supernatants were harvested at 2, 12, 24 hours post infection (hpi) for real-372 time qPCR analysis. Intestinal tissues were collected at 2 and 24hpi in RL buffer (Qiagen, 373 Hilden, Germany) with 50mM DTT (Qiagen) for real-time qPCR analysis or were fixed 374 overnight in 4% paraformaldehyde (PFA) for immunohistochemistry staining. Plaque assays were performed as we previously described with slight modifications 21 . In 409 brief, VeroE6 cells were seeded in 12-well plates one day before the experiment. The 410 harvested supernatant samples were serially diluted and inoculated to the cells for 1h at 37°C. 411

After inoculation, the cells were washed with PBS 3 times, and covered with 3% 412 agarose/PBS mixed with DMEM / 1.5%FBS at 1:2 ratio. The cells were fixed in 4%PFA 413 after incubating at 37°C for 96h. Fixed samples were stained with 1% crystal violet in 20% 414 ethanol/distilled water for 15min for plaque visualization. 

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