key: cord-0706455-6eokug5t authors: Li, Shengjie; Li, Danhui; Fang, Jianchen; Liu, Qiang; Cao, Wenjun; Sun, Xinghuai; Xu, Gezhi title: SARS-CoV-2 receptor ACE2 is expressed in human conjunctival tissue, especially in diseased conjunctival tissue date: 2020-09-28 journal: Ocul Surf DOI: 10.1016/j.jtos.2020.09.010 sha: 13044a0a4e269f2e592bcf949ca477b582ecbd46 doc_id: 706455 cord_uid: 6eokug5t nan SARS-CoV-2 receptor ACE2 is expressed in human conjunctival tissue, especially in diseased conjunctival tissue To the Editor: In December 2019, coronavirus disease 2019 (COVID-19) was first reported in people in Wuhan, China, and has rapidly spread throughout the world [1] . As of August 11, 2020, 19 ,936,210 laboratory-confirmed cases were reported. Furthermore, the World Health Organization reported 732,499 fatalities [2] . The typical signs and symptoms of COVID-19 are fever, dry cough, and fatigue. The virus is mainly transmitted through respiratory droplets and by close contact with infected individuals. Furthermore, about 2.78% of COVID-19 patients showed symptoms of conjunctival congestion [3] . Thus, because the ocular surface is always exposed to the environment, it may serve as a possible site of virus entry and also as a source of contagious infection. Studies have provided powerful evidence that angiotensin-converting enzyme 2 (ACE2) is the major cellular-entry receptor for the SARS-CoV-2 virus, and higher ACE2 expression increases susceptibility to SARS-CoV-2 [4] . Numerous studies, along with the typical symptoms exhibited by COVID-19 patients [3, 5] , suggest that the expression of the ACE2 gene may be a factor involved in the mode of transmission of the virus. Recently, Collin et al. [6] and Zhou et al. [7] reported that ACE2 was expressed in normal human adult conjunctival, limbal and corneal epithelium, suggesting that ocular surface cells could be susceptible to infection by SARS-CoV-2. These initial reports led us to question whether and how the ACE2 gene is expressed in diseased conjunctival tissues. In the current study, we analysed both normal and diseased human conjunctival tissue using RT-qPCR, western blot, immunohistochemistry, and immunofluorescence in order to determine both expression and localization of this key viral susceptibility factor in the human conjunctival tissue. Sixty-eight subjects with a mean age of 47.42 ±16.19 years [(32 males (47.06%); 36 females (52.94%)] were recruited for the study, which included 10 subjects with conjunctival nevi, 20 subjects with conjunctivitis, 9 J o u r n a l P r e -p r o o f subjects with conjunctival papilloma, 16 subjects with conjunctival cyst, and 7 subjects with conjunctival polyps; 6 ocular traumas were used as normal control subjects. More information related to materials and methods can be found in online supplementary materials. All analyses were performed using the Statistical Package for the Social Sciences software, version 13.0 (SPSS Inc., Chicago, IL, USA). As presented in Figure 1A and 1B, ACE2 mRNA and protein levels were significantly overexpressed in conjunctival tissues of inflamed conjunctiva, conjunctival nevi, conjunctival cyst, conjunctival papilloma, and conjunctival polyps, as compared to normal conjunctival tissues (p<0.001). Furthermore, a significant difference in ACE2 mRNA and protein levels between normal tissue and diseased conjunctival tissues was observed (p<0.001; Figure 1A Figure 1E⑥ ). The levels of ACE2 staining were significantly different between diseased and normal conjunctival epithelial cells. A summary of the quantitative data is presented in Figure 1F -H. Significant differences in ACE2 expression between normal and diseased conjunctival epithelial cells were observed (p<0.05). Among diseased conjunctival tissues, the level of ACE2 was significantly (p<0.001) lower in inflamed conjunctiva as compared to other diseased tissue. The most significant finding was the surface expression of ACE2 in conjunctival epithelial cells. Recently, Collin et al. [6] reported that ACE2 was J o u r n a l P r e -p r o o f expressed in the human adult conjunctival, limbal and corneal epithelium. Meanwhile, Zhou et al. [7] also found ocular surface expression of ACE2 proteins across all human ocular specimens. Our results confirm these reports, but go further in showing that ACE2 was overexpressed in diseased tissue. Several respiratory viruses have been shown capable of using the eye as both a site for replication and a port of entry, which could result in a productive respiratory infection [8] . Peiris JS et al. [9] reported that SARS-CoV can be transmitted either through direct or indirect contact with ocular mucous membranes. Collectively, it is evident that the virus, via droplets, might be transmitted through the human conjunctival epithelium. Our study shows that ACE2 was significantly overexpressed in diseased epithelial cells when compared to normal conjunctival epithelial cells. Collin et al. [6] suggested that local inflammation can enhance ACE2 expression, which may explain why ACE2 is expressed at higher levels in diseased conjunctiva. Guangfa Wang, a member of the national expert panel on pneumonia, was infected by the COVID-19 virus during an inspection in Wuhan. Wang complained of redness of the eyes, which suggests that he was infected through the ocular surface [10] . Xia J et al. [11] reported that conjunctival swab samples collected from patients with conjunctivitis yielded positive SARS-CoV-2 RT-qPCR results, whereas negative RT-qPCR results were obtained in patients without conjunctivitis. One explanation for why patients with conjunctivitis are more likely to exhibit positive SARS-CoV-2 RNA levels is that conjunctivitis may have resulted from the active presence of SARS-CoV-2. On the other hand, the subjects with conjunctivitis may have had higher levels of ACE2, which may lead to a much stronger susceptibility to SARS-CoV-2 infection. Our results show that ACE2 is present in human conjunctival tissue, especially in diseased conjunctival tissue. Collectively, the clinical features of reported COVID-19 patients combined with our results indicate that COVID-19 is likely to be transmissible through the conjunctiva. It is easy to forget that conjunctivae are generally exposed to the environment. Thus, increasing the awareness of eye protection during COVID-19 is necessary, especially for subjects with diseased conjunctivae. The ethics committee of the Renji Hospital of Shanghai Jiao Tong University approved this study, and it adhered to the principles of the Declaration of Helsinki. Informed consent was obtained from all participating subjects. Clinical Characteristics of Coronavirus Disease 2019 in China The evidence of SARS-CoV-2 infection on ocular surface. The ocular surface A pneumonia outbreak associated with a new coronavirus of probable bat origin Ocular Findings and Proportion with Conjunctival SARS-COV-2 in COVID-19 Patients Co-expression of SARS-CoV-2 entry genes in the superficial adult human conjunctival, limbal and corneal epithelium suggests an additional route of entry via the ocular surface. The Ocular Surface ACE2 and TMPRSS2 are expressed on the human ocular surface, suggesting susceptibility to SARS-CoV-2 infection. The Ocular Surface Ocular tropism of respiratory viruses The severe acute respiratory syndrome 2019-nCoV transmission through the ocular surface must not be ignored Evaluation of coronavirus in tears and conjunctival secretions of patients with SARS-CoV-2 infection The authors declare that they have no competing interests.