SARS-CoV-2 infection in conjunctival tissue
Authors: Yu-Chi Liu, Marcus Ang, Hon Shing Ong, Tien Yin Wong, Jodhbir S Mehta
Journal: The Lancet Respiratory Medicine (2020)

In a recent study published in The Lancet Respiratory Medicine in May, 2020, Kenrie Hui and colleagues1Hui KPY Cheung M-C Perera RAPM et al.Tropism, replication competence, and innate immune responses of the coronavirus SARS-CoV-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures.Lancet Respir Med. 2020; 8: 687-695Summary Full Text Full Text PDF PubMed Scopus (384) Google Scholar described the tropism, replication competence, and innate immune responses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the human respiratory tract and conjunctiva. The authors concluded that SARS-CoV-2 replication in the conjunctiva was greater than SARS-CoV replication, and that the conjunctival epithelium was a potential portal of infection. However, this finding could be misleading. The main issue with the ex-vivo experimental model used in this study1Hui KPY Cheung M-C Perera RAPM et al.Tropism, replication competence, and innate immune responses of the coronavirus SARS-CoV-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures.Lancet Respir Med. 2020; 8: 687-695Summary Full Text Full Text PDF PubMed Scopus (384) Google Scholar is that it does not consider the fact that the ocular surface is covered by a tear film. The tear film forms a dynamic innate protection for the ocular surface that is regularly turned over at a rate of 5·4–20·6% of its volume per minute. Tears are secreted at a rate of 2 μL/min, which is even higher during reflex tearing in response to inflammation, such as in viral conjunctivitis. Hence, in a clinical setting, viral pathogens reaching the ocular surface are unlikely to inoculate the conjunctiva at a fixed viral load for 1 h, as in the described experimental model,1Hui KPY Cheung M-C Perera RAPM et al.Tropism, replication competence, and innate immune responses of the coronavirus SARS-CoV-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures.Lancet Respir Med. 2020; 8: 687-695Summary Full Text Full Text PDF PubMed Scopus (384) Google Scholar since the viral titre will be diluted by tear fluid. The authors should test the effect of different viral concentrations on infectivity to mimic the dilution effect of the tear film. Constant tear production will also wash the viral load away from the ocular surface and into the nasolacrimal duct. Most published clinical studies2Wu P Duan F Luo C et al.Characteristics of ocular findings of patients with coronavirus disease 2019 (COVID-19) in Hubei province, China.JAMA Ophthalmol. 2020; 138: 575-578Crossref PubMed Scopus (913) Google Scholar, 3Seah IYJ Anderson DE Kang AEZ et al.Assessing viral shedding and infectivity of tears in coronavirus disease 2019 (COVID-19) patients.Ophthalmology. 2020; (published online March 24.)DOI:10.1016/j.ophtha.2020.03.026Summary Full Text Full Text PDF PubMed Scopus (334) Google Scholar support this notion and have found that the proportion of PCR-positive patients with COVID-19 who test positive for SARS-CoV-2 RNA in conjunctival swabs or tears is very low (0–5·2%). In addition, the tear film contains several antimicrobial properties. Tear lactoferrin, mucins, lysozyme, lipocalin, IgA, and complement work synergistically to facilitate the clearance of viral pathogens. Lactoferrin, in particular, has been shown to have a protective function by inhibiting the early cellular attachment phase of SARS-CoV.4Lang J Yang N Deng J et al.Inhibition of SARS pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans.PLoS One. 2011; 6e23710Crossref PubMed Scopus (330) Google Scholar All of these factors will substantially affect viral kinetics on the conjunctiva, but these factors were not taken into account in the experimental model or discussed in the study by Hui and colleagues.1Hui KPY Cheung M-C Perera RAPM et al.Tropism, replication competence, and innate immune responses of the coronavirus SARS-CoV-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures.Lancet Respir Med. 2020; 8: 687-695Summary Full Text Full Text PDF PubMed Scopus (384) Google Scholar Conjunctivitis has been reported as an ocular manifestation of COVID-19, with a reported prevalence ranging from 0·9% to 31·6%.2Wu P Duan F Luo C et al.Characteristics of ocular findings of patients with coronavirus disease 2019 (COVID-19) in Hubei province, China.JAMA Ophthalmol. 2020; 138: 575-578Crossref PubMed Scopus (913) Google Scholar, 5Guan WJ Ni ZY Hu Y et al.Clinical characteristics of coronavirus disease 2019 in China.N Engl J Med. 2020; 382: 1708-1720Crossref PubMed Scopus (20495) Google Scholar However, the number of conjunctival samples positive for SARS-CoV-2 RNA has been low.2Wu P Duan F Luo C et al.Characteristics of ocular findings of patients with coronavirus disease 2019 (COVID-19) in Hubei province, China.JAMA Ophthalmol. 2020; 138: 575-578Crossref PubMed Scopus (913) Google Scholar SARS-CoV-2 gains cellular entry through the angiotensin-converting enzyme 2 (ACE2) receptor after cellular protease priming by transmembrane protease serine 2 (TMPRSS2).6Lange C Wolf J Auw-Haedrich C et al.Expression of the COVID-19 receptor ACE2 in the human conjunctiva.J Med Virol. 2020; (published online May 6.)DOI:10.1002/jmv.25981.sCrossref PubMed Scopus (102) Google Scholar By use of single-cell sequencing and high-throughput RNA sequencing, two independent studies6Lange C Wolf J Auw-Haedrich C et al.Expression of the COVID-19 receptor ACE2 in the human conjunctiva.J Med Virol. 2020; (published online May 6.)DOI:10.1002/jmv.25981.sCrossref PubMed Scopus (102) Google Scholar, 7Sungnak W Huang N Becavin C et al.SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes.Nat Med. 2020; (published online April 23.)DOI:10.1038/s41591-020-0868-6Crossref PubMed Scopus (1803) Google Scholar have shown that the expression of ACE2 and TMPRSS2 on the ocular surface is significantly lower than that in the respiratory tract. Immunohistochemical staining studies6Lange C Wolf J Auw-Haedrich C et al.Expression of the COVID-19 receptor ACE2 in the human conjunctiva.J Med Virol. 2020; (published online May 6.)DOI:10.1002/jmv.25981.sCrossref PubMed Scopus (102) Google Scholar have shown no ACE2 expression in conjunctival tissue using two different monoclonal antibodies. However, the nasal cavity, which is anatomically contiguous with the conjunctival mucosa, has one of the highest levels of both ACE2 and TMPRSS2 expression in the human body.7Sungnak W Huang N Becavin C et al.SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes.Nat Med. 2020; (published online April 23.)DOI:10.1038/s41591-020-0868-6Crossref PubMed Scopus (1803) Google Scholar Comparing entry of SARS-CoV-2 in the conjunctiva with the nasal mucosa would have been key to understanding the potential portal of entry of this virus in the study by Hui and colleagues.1Hui KPY Cheung M-C Perera RAPM et al.Tropism, replication competence, and innate immune responses of the coronavirus SARS-CoV-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures.Lancet Respir Med. 2020; 8: 687-695Summary Full Text Full Text PDF PubMed Scopus (384) Google Scholar Finally, the appearance of the immunostaining results for SARS-CoV-2 in the conjunctiva was unusual in this study,1Hui KPY Cheung M-C Perera RAPM et al.Tropism, replication competence, and innate immune responses of the coronavirus SARS-CoV-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures.Lancet Respir Med. 2020; 8: 687-695Summary Full Text Full Text PDF PubMed Scopus (384) Google Scholar as positive nucleoprotein staining was found in the conjunctival stroma, but not in the epithelium. One would have expected SARS-CoV-2 staining on the conjunctival epithelial surface to be consistent with the authors' theory. Performing the analysis at an earlier timepoint (eg, at 24 h) could help to explain this finding. Another possible explanation could be that the mechanism of SARS-CoV-2 entry into the conjunctiva is different from other types of SARS-CoV. All of these issues should have been addressed, and the need for further studies should have been suggested, before concluding that the conjunctival epithelium was a potential portal of infection for SARS-CoV-2. We declare no competing interests. Tropism, replication competence, and innate immune responses of the coronavirus SARS-CoV-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro culturesThe conjunctival epithelium and conducting airways appear to be potential portals of infection for SARS-CoV-2. Both SARS-CoV and SARS-CoV-2 replicated similarly in the alveolar epithelium; SARS-CoV-2 replicated more extensively in the bronchus than SARS-CoV. These findings provide important insights into the transmissibility and pathogenesis of SARS-CoV-2 infection and differences with other respiratory pathogens. Full-Text PDF SARS-CoV-2 infection in conjunctival tissue – Authors' replyWe thank Yu-Chi Liu and colleagues for their correspondence on our study1 on the tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in ex-vivo cultures of human ocular and respiratory tissues, in which they highlight three key points. The first point involves the dynamic nature and antimicrobial properties of the tear film, and the challenge of mimicking these properties in model systems. With regards to the antiviral effects of tear fluid, it is relevant to note that other enveloped viruses (eg, herpes simplex virus type 1 [HSV-1]2 and the H7N7 avian influenza A virus3) and non-enveloped viruses (eg, enteroviruses and adenoviruses)4 can effectively infect the conjunctiva (leading to conjunctivitis), and that the comparative replication of HSV-15 and pandemic H1N1 influenza viruses6 has also been shown in ex-vivo explant cultures of human ocular tissue, even in the presence of the same antiviral and viral clearance mechanisms. Full-Text PDF