key: cord-343827-jo61t3m0 authors: Qian, Qun; Fan, Lifang; Liu, Weicheng; Li, Jin; Yue, Junqiu; Wang, Mingwei; Ke, Xianliang; Yin, Yan; Chen, Quanjiao; Jiang, Congqing title: Direct evidence of active SARS-CoV-2 replication in the intestine date: 2020-07-08 journal: Clin Infect Dis DOI: 10.1093/cid/ciaa925 sha: doc_id: 343827 cord_uid: jo61t3m0 BACKGROUND: Currently, there is no direct evidence to prove the active SARS-CoV-2 replication in the intestinal tract and relevant pathological changes in the colon and rectum. We investigated the presence of virions and pathological changes in surgical rectal tissues of a clinically confirmed COVID-19 patient with rectal adenocarcinoma. METHODS: Here, the clinical data were collected during hospitalization and follow-up of this patient. Quantitative RT-PCR was performed on the rectal tissue specimens obtained from surgical resection, succus entericus and intestinal mucosa of ileostomy, and rectal mucosa during follow-up after recovery. Ultrathin sections of surgical samples were observed for SARS-CoV-2 virions using electron microscopy. Histopathological examination was performed using hematoxylin-eosin stain. Immunohistochemical analysis and immunofluorescence were carried out on rectal tissues to evaluate the distribution of SARS-CoV-2 antigen, and immune cell infiltrations. RESULTS: The patient had fever and cough on day 3 postoperatively, was diagnosed with COVID-19 on day 7, and was discharged from the hospital on day 41. RNA of SARS-CoV-2 was detected in surgically resected rectal specimens, but not in samples collected on 37 day after discharge. Notably, coincidence with rectal tissues of surgical specimens tested nucleic acid positive for SARS-CoV-2, typical coronavirus virions in rectal tissue were observed under electron microscopy. Moreover, abundant lymphocytes and macrophages (some are SARS-CoV-2 positive) infiltrating the lamina propria were found with no significant mucosal damage. CONCLUSIONS: We firstly reported that direct evidence of the active SARS-CoV-2 replication in the patient's rectum during the incubation period, which might explain SARS-CoV-2 fecal-oral transmission. The current coronavirus disease identified in 2019 , caused by a novel coronavirus, has become a global public health problem [1, 2] . As of May 27, 2020, a total of 5,451,532 cases of COVID-19 have been confirmed globally, including 345,752 deaths [3] . There are many reports suggesting that SARS-CoV-2 RNA can be detected and identified in anal/rectal swabs [4, 5] and stool specimens [6, 7] . In fact, one recent small sample study found that RNA was consistently detected in rectal swabs, even after viral clearance from the upper respiratory tract, indicating extended duration of viral shedding in fecal samples and raising the possibility of fecal-oral transmission of SARS-CoV-2 [5] . Similar results were reported in another study with more cases involved, raising the possibility of prolonged presence of SARS-CoV-2 in stools. Notably, fecal samples remained positive for SARS-CoV-2 RNA nearly 5 weeks after the viral clearance from the upper respiratory tract in COVID-19 patients [8] . Considering a high degree of sequence homology between the SARS-CoV-2 and SARS-CoV, angiotensin-converting enzyme II (ACE2) has been identified as the entry receptor of SARS-CoV-2. Since this receptor is highly expressed on the epithelial cells from the ileum and colon [9] , the intestinal tract may be a potential route for SARA- Patients with cancer are considered to be more susceptible to SARS-CoV-2 [10, 11] . One patient with rectal cancer was admitted to Zhongnan Hospital of Wuhan University for radical operation. On postoperative day 3, the patient began to develop cough and A c c e p t e d M a n u s c r i p t fever; chest CT revealed radiologic characteristics of viral pneumonia. On postoperative day 7, the patient was confirmed to be infected with SARS-CoV-2. Although live SARS-CoV-2 had been successfully isolated from the fecal sample of a laboratory confirmed SARS-CoV-2 patient [12] , until now, there has been no direct evidence to prove active SARS-CoV-2 virus replication in the intestinal tract. It remains unknown whether there are pathological changes related to SARS-CoV-2 infection existing in colorectal mucosa in COVID-19 patients. To clarify the above questions, we performed a retrospective study to detect the presence of SARS-CoV-2 virions and determine the pathological changes in rectal tissues of this patient. The patient's clinical information is described in Supplementary Samples of rectal tissues, succus entericus and intestinal mucosa of ileostomy, and rectal mucosa were tested for SARS-CoV-2 nucleic acid using qRT-PCR. The qRT-PCR analyses were performed following a previously described method [13] . The qRT-PCR test kits (BioGerm) were recommended by the Chinese Center for Disease Control A c c e p t e d M a n u s c r i p t and Prevention. The rectal tissue obtained by resection was soaked in RNAlater solution overnight, the solution was discarded and the tissue was frozen at -80°C. The rectal tissue was cut into 1 mm thick sections and fixed in 2.5% glutaraldehyde and 1% osmium tetroxide in a biosafety cabinet with level 2 protection, and subsequently dehydrated using different ascending concentrations of alcohol (30% to 100%), and immersed and embedded in epoxy resin. Ultrathin sections (80-100 nm) were prepared on formvar-coated copper grids (200 mesh). The virions were observed with a Tecnai G2 20 TWIN electron microscope (FEI company) under 200 kV. Immunohistochemical staining was performed on formalin-fixed and paraffinembedded tissue sections (4-μm). Sections of rectal tissues were immunostained to evaluate the expression and distribution of the SARS-CoV-2 antigen. Briefly, sections were deparaffinized with xylene and alcohol and subsequently heated in citrate buffer (pH 6.0) for antigen retrieval. After blockage with 3% BSA in PBS for 30 minutes, a rabbit antibody against Rp3 NP protein (kindly provided by Dr. Zhengli Shi, Wuhan Institute of Virology, Chinese Academy of Science [14] ) was incubated with the sections overnight at 4°C. For immunochemistry study, the slides were subsequently Figure 1 . The patient's clinical information is shown in supplementary Table 1 Clinical Characteristics of Coronavirus Disease 2019 in China The origin, transmission and clinical therapies on coronavirus disease 2019 (COVID-19) outbreak -an update on the status World Health Organization. Coronavirus disease (COVID-2019) situation reports Diarrhea may be underestimated: a missing link in 2019 novel coronavirus Characteristics of pediatric SARS-CoV-2 infection and potential evidence for persistent fecal viral shedding The presence of SARS-CoV-2 RNA in the feces of COVID-19 patients Epidemiologic Features and Clinical Course of Patients Infected With SARS-CoV-2 in Singapore Prolonged presence of SARS-CoV-2 viral RNA in faecal samples The digestive system is a potential route of 2019-nCov infection: a bioinformatics analysis based on single-cell transcriptomes Clinical features of patients infected with 2019 novel coronavirus in Wuhan SARS-CoV-2 Transmission in Patients With Cancer at a Tertiary Care Hospital in Wuhan, China Isolation of 2019-nCoV from a Stool Specimen of a Laboratory confirmed Case of the Coronavirus Disease 2019 (COVID-19) Positive RT-PCR Test Results in Patients Recovered From COVID-19 A pneumonia outbreak associated with a new coronavirus of probable bat origin molecular biology, and pathogenesis of avian influenza A (H5N1) infection in humans Influenza viremia and the potential for blood-borne transmission Influenza viral RNA detection in blood as a marker to predict disease severity in hematopoietic cell transplant recipients The fate of influenza A virus after infection of human macrophages and dendritic cells Viral infection triggers rapid differentiation of monocytes into dendritic cells Influenza virus A infection of human monocyte and macrophage subpopulations reveals increased susceptibility associated with cell differentiation From Influenza-Induced Acute Lung Injury to Annual Update in Intensive Care and Emergency Medicine Evidence for Gastrointestinal Infection of SARS-CoV-2 We are grateful to Center for Instrumental Analysis and Metrology, The CoreFacility and Technical Support, Wuhan Institute of Virology, CAS. We declare that we have no conflicts of interest.A c c e p t e d M a n u s c r i p t