key: cord-1053060-1cc9ig04 authors: Milewska, Aleksandra; Kula-Pacurar, Anna; Wadas, Jakub; Suder, Agnieszka; Szczepanski, Artur; Dabrowska, Agnieszka; Owczarek, Katarzyna; Ochman, Marek; Stacel, Tomasz; Rajfur, Zenon; Labaj, Pawel; Branicki, Wojciech; Pyrc, Krzysztof title: Replication of SARS-CoV-2 in human respiratory epithelium date: 2020-03-21 journal: bioRxiv DOI: 10.1101/2020.03.20.999029 sha: 4c0b20ced6658e131d7a180dae0a0b82e30cc00c doc_id: 1053060 cord_uid: 1cc9ig04 SARS-CoV-2 emerged by the end of 2019 to rapidly spread in 2020. At present, it is of utmost importance to understand the virus biology and to rapidly assess the potential of existing drugs and develop new active compounds. While some animal models for such studies are under development, most of the research is carried out in the Vero E6 cells. Here, we propose fully differentiated human airway epithelium cultures as a model for studies on the SARS-CoV-2. Further, we also provide basic characteristics of the system. Coronaviruses constitute a large family of RNA viruses that infect mainly mammals and 41 birds. In humans, there are four species associated with mild-to-moderate respiratory infections. 42 While these viruses are present in the human population for a long time, they are believed to 43 enter the human population in a zoonotic event, and one may speculate that they may have 44 caused epidemics similar to the one observed for the SARS-CoV-2. Time to the most recent 45 ancestor analysis suggests that human coronavirus HCoV-NL63 is the oldest species in humans, 46 followed by its cousin HCoV-229E and two betacoronaviruses, which emerged in humans in a 47 relatively near past 1, 2, 3, 4 . In the 21 st century, we already faced the emergence of the three novel 48 coronaviruses in humans, of which SARS-CoV disappeared after one season never to come 49 back, and MERS-CoV never fully crossed the species border, as its transmission between 50 humans is highly ineffective 5, 6, 7 . The 2019 zoonotic transmission, however, resulted in the 51 emergence of a novel human coronavirus, which seems to carry an optimal set of features 52 allowing for its rapid spread with considerable mortality. Whether the virus will become 53 endemic in humans is an open question 8, 9, 10 . At present, the studies on the virus are carried out using a surrogate system based on the 55 immortalized simian Vero E6 cell line 11 . While this model is convenient for diagnostics and 56 testing of some antiviral drugs, it has serious limitations and does not allow for the 57 understanding of virus biology and evolution. To make an example, the entry route of human 58 coronaviruses varies between the cell lines and differentiated tissue, not mentioning the immune 59 responses or virus-host interactions 12, 13, 14 . Here we used the fully differentiated epithelium cultures to study the infection with the science, but also the antiviral drug development 12, 13, 14, 20 . Here we verified whether HAE cultures may be used to study the SARS-CoV-2 infection The analysis clearly showed that the sg mRNA are abundant in the infected HAE cultures. As this is generally considered to be the hallmark of an active replication, we believe that it 107 provides sufficient proof that the virus is indeed actively replicating in the cultures. Origin and evolution of pathogenic coronaviruses Mosaic structure of human coronavirus NL63, one thousand years of evolution Identification of a new human coronavirus Molecular Evolution of Human Coronavirus Genomes Middle East respiratory syndrome coronavirus (MERS-CoV): A review. Germs Emergence of MERS-CoV in the Middle East: origins, transmission, treatment, and perspectives A novel coronavirus associated with severe acute respiratory syndrome LANCOVID-19). Clinical, laboratory and imaging features of COVID-19: A systematic review and meta-analysis Zhong NS; China Medical Treatment Expert Group for Covid-19. Clinical Characteristics of Coronavirus Disease 2019 in China Escaping Pandora's Box -Another Novel Coronavirus Severe Acute Respiratory Syndrome Coronavirus 2 from Patient with 2019 Novel Coronavirus Disease, United States. Emerg Infect Dis Wild-type human coronaviruses prefer cell-surface TMPRSS2 to endosomal cathepsins for cell entry Entry of Human Coronavirus NL63 into the Cell Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry Air-liquid interface cell culture: From airway epithelium to the female reproductive tract Coronaviruses and the human airway: a universal system for virus-host interaction studies Novel coronavirus-like particles targeting cells lining the respiratory tract APOBEC3-mediated restriction of RNA virus replication Culturing the unculturable: human coronavirus HKU1 infects, replicates, and produces progeny virions in human ciliated airway epithelial cell cultures