key: cord-260315-uau554jj
authors: Ramirez, Santseharay; Fernandez-Antunez, Carlota; Pham, Long V.; Ryberg, Line A.; Feng, Shan; Pedersen, Martin S.; Mikkelsen, Lotte S.; Belouzard, Sandrine; Dubuisson, Jean; Gottwein, Judith M.; Fahnøe, Ulrik; Bukh, Jens
title: Efficient culture of SARS-CoV-2 in human hepatoma cells enhances viability of the virus in human lung cancer cell lines permitting the screening of antiviral compounds
date: 2020-10-04
journal: bioRxiv
DOI: 10.1101/2020.10.04.325316
sha: 
doc_id: 260315
cord_uid: uau554jj

Efforts to mitigate COVID-19 include screening of existing antiviral molecules that could be re-purposed to treat SARS-CoV-2 infections. Although SARS-CoV-2 propagates efficiently in African green monkey kidney (Vero) cells, antivirals such as nucleos(t)ide analogs (nucs) often exhibit decreased activity in these cells due to inefficient metabolization. Limited SARS-CoV-2 replication and propagation occurs in human cells, which are the most relevant testing platforms. By performing serial passages of a SARS-CoV-2 isolate in the human hepatoma cell line clone Huh7.5, we selected viral populations with improved viability in human cells. Culture adaptation led to the emergence of a significant number of high frequency changes (>90% of the viral population) in the region coding for the spike glycoprotein, including a deletion of nine amino acids in the N-terminal domain and 3 amino acid changes (E484D, P812R, and Q954H). We demonstrated that the Huh7.5-adapted virus exhibited a >3-Log10 increase in infectivity titers (TCID50) in Huh7.5 cells, with titers of ~8 Log10TCID50/mL, and >2-Log10 increase in the human lung cancer cell line Calu-1, with titers of ~6 Log10TCID50/mL. Culture adaptation in Huh7.5 cells further permitted efficient infection of the otherwise SARS-CoV-2 refractory human lung cancer cell line A549, with titers of ~6 Log10TCID50/mL. The enhanced ability of the virus to replicate and propagate in human cells permitted screening of a panel of nine nucs, including broad-spectrum compounds. Remdesivir, EIDD-2801 and to a limited extent galidesivir showed antiviral effect across these human cell lines, whereas sofosbuvir, uprifosbuvir, valopicitabine, mericitabine, ribavirin, and favipiravir had no apparent activity. Importance The cell culture adapted variant of the SARS-CoV-2 virus obtained in the present study, showed significantly enhanced replication and propagation in various human cell lines, including lung derived cells otherwise refractory for infection with the original virus. This SARS-CoV-2 variant will be a valuable tool permitting investigations across human cell types, and studies of identified mutations could contribute to our understanding of viral pathogenesis. In particular, the adapted virus can be a good model for investigations of viral entry and cell tropism for SARS-CoV-2, in which the spike glycoprotein plays a central role. Further, as shown here with the use of remdesivir and EIDD-2801, two nucs with significant inhibitory effect against SARS-CoV-2, large differences in the antiviral activity are observed depending on the cell line. Thus, it is essential to select the most relevant target cells for pre-clinical screenings of antiviral compounds, facilitated by using a virus with broader tropism.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), responsible for COVID-19, in order to generate a large volume of supernatant that could be used for characterization of the Huh7.5 136 adapted virus (this virus will be referred to as P5 Huh7.5 virus). 137 We performed a comparative titration in various cells of the P2 VeroE6 and the P5 Huh7.5 viruses ( Figure   138 1b) and found that the infectivity titers in Huh7.5 cells after culture adaptation had increased by more 139 than 3 logs (mean of 4.7 and 8.0 Log 10 TCID 50 /mL, respectively). The Huh7.5 adapted virus also 140 exhibited significantly increased titers in Vero E6 cells (mean of 7.0 Log 10 TCID 50 /mL for the P5 Huh7.5 141 virus versus 5.5 Log 10 TCID 50 /mL for the P2 VeroE6 virus). Interestingly, the original P2 VeroE6 virus was 142 less viable in the Huh7 parental cell line than in the Huh7.5 clone, however, adaptation to the Huh7.5 143 clone also led to a significant increase in infectivity titers in Huh7 cells (3.8 and 7.7 Log 10 TCID 50 /mL 144 for the P2 VeroE6 and P5 Huh7.5 viruses, respectively). 145 Visual observations of P5 Huh7.5 virus infected cultures in the light microscope indicated an increase in 146 CPE. To better quantify this, we performed viral cytopathic effect assays (CPE assays, Figure 1c ), in 147 which we detected an evident increase in CPE titers (Log 10 CPE 50 /mL) in all cells. CPE significantly 148 increased from 4.6 to 6.2 Log 10 CPE 50 /mL in Vero E6 cells. In Huh7.5 cells, the P2 VeroE6 virus was not 149 cytopathic (we obtained a value just above the assay threshold in one of the independent experiments), 150 whereas the P5 Huh7.5 virus led to high titers of 7.3 Log 10 CPE 50 /mL. For the Huh7 parental cells the 151 P2 VeroE6 virus was also non-cytopathic, but the adapted P5 Huh7.5 yielded 6.1 Log 10 CPE 50 /mL. 152 In addition to the increase in infectivity titers observed after infection with the adapted P5 Huh7.5 virus in 153 Huh7.5 cells, we also noticed an evident increase in the intensity of the antigen staining (α-spike 154 protein antibody), and in the number of infected cells at non-cytopathic virus dilutions of the P5 Huh7.5 155 virus compared to the P2 VeroE6 virus (Figure 1d ). This suggest that the P5 Huh7.5 virus might both replicate and propagate at higher levels in Huh7.5 cells, as also indicated by the significant increase in refractory to infection 12, 16 . To our knowledge, the ability of the Calu-1 cell line to support SARS-CoV-178 2 replication and propagation has not been previously reported. The Calu-1 and the A549 cell lines are 179 widely available and well-characterized standards among the human lung carcinoma/alveolar cell lines 180 used in cancer research 17, 18 . Further, the A549 cell line is also a model for the study of respiratory 181 viruses, such as respiratory syncytial virus and influenza 19, 20 .

Compared to the P2 VeroE6 virus, the P5 Huh7.5 virus exhibited a significant increase in the ability to infect 183 Calu-1 cells with >2-log increase in infectivity titers. For the original P2 VeroE6 virus, observed titers in 184 Calu-1 were 3.5 Log 10 TCID 50 /mL, which increased to 6.0 Log 10 TCID 50 /mL for the P5 Huh7.5 virus 185 ( Figure 2a) . Surprisingly, the P5 Huh7.5 virus was able to efficiently infect A549 cells, with titers of 6.0 

The adapted P5 Huh7.5 virus permitted drug testing in Huh7.5, Calu-1 and A549 cells. We focused our 208 screen on nucs previously shown to have antiviral effect against HCV, including sofosbuvir, a 209 pangenotypic HCV drug used in the clinic 24 , but we also included broader-spectrum molecules such as 210 galidesivir, favipiravir and ribavirin.

Among the nucs tested only remdesivir and EIDD-2801 displayed significant effect across the human 212 cells (Table 3) , as previously described 25 . Remdesivir was most active in Huh7.5 cells, with ~6-fold 213 lower EC 50 values when compared to Calu-1 and A549 cells (Figure 4a ). Despite being less active in 214 lung carcinoma than in hepatoma cells, remdesivir was still more active in lung carcinoma Calu-1 and 215 A549 than in Vero E6 cells (about 5-fold lower EC 50 compared to the P2 VeroE6 virus). The opposite was 216 observed for EIDD-2801 (Figure 4b) , which was more active in A549 and Calu-1 cells (6-and 3-fold 217 more active with EC 50 of 1.3 µM and 2.7 µM, respectively) than in Huh7.5 cells (8.5 µM). Finally, 218 galidesivir exhibited limited activity (Figure 4c ) with relatively high EC 50 (>20 µM); the best inhibitory effect was observed in A549 cells (EC 50 of 24 µM). Other nucs, including sofosbuvir, had no apparent 220 activity (EC 50 >50 µM) in these human cell lines with our experimental conditions (Table 3) .

In this study, we performed isolation of SARS-CoV-2 (isolate DK-AHH1) in Vero E6 cells and in the type-1 interferon response, one of the features that has been correlated to increased 238 permissiveness to HCV replication 26 . Interestingly, Huh7 cells are highly susceptible and permissive to CoV-2 is more sensitive to type-1 interferons than SARS-CoV 28 . Therefore decreased type-1 interferon 241 responses might be a key feature for enhancing the cell culture viability of SARS-CoV-2, correlating 242 with the high permissiveness of the Vero cell lines, which lack genes encoding type-1 interferons 29 . led to the acquisition of a mutation in the S-protein creating a novel furin site downstream of the S1/S2 site that was implicated in the entry and syncytium formation in Vero cells 36 . On the other hand, 263 deletions in the S1-S2 furin cleavage site have been found during culture adaptation in Vero E6 264 cells 23,37 . Finally, Q954H was present at low frequency in P1 but increased significantly in the last 265 passages, consistent with the maximum increase in viral infectivity. Residue Q954 locates within the 266 S2 subunit, which undergoes conformational rearrangements from prefusion to post fusion states.

Specifically, this position is located within the heptad repeat 1 (HR1), which is part of the fusion-active 268 core structure 38 . We demonstrated that the activity of remdesivir in Huh7.5 cells was very similar between the P2 VeroE6 316 and the adapted P5 Huh7.5 virus, despite the later exhibiting multiple changes in the genome. Since these 317 changes concentrated in the S-protein and not in the nsp12 protein, which is the main target of nucs, 318 this virus represents an excellent tool to study this drug class in human cells. However, the mutations 319 present in the adapted virus could potentially interfere with entry processes and therefore this 320 experimental system might not be an optimal tool for the screening of entry/fusion inhibitors.

It is important to continue evaluating antiviral strategies against SARS-CoV-2, that could also be Table 3 . Anti-SARS-CoV-2 activity of a panel of nucleos(t)ide analogs in different human cells. 530 For each compound, the antiviral activity in Huh7.5, Calu-1 or A549 cells is indicated by EC 50 values 531 (µM). These values were inferred from concentration-response curves as shown in Figure 4 . All 532 compounds were tested at non-cytotoxic concentrations as described in Materials and Methods. For 533 remdesivir the maximum concentration tested was 10 µM. For EID-2801 the maximum concentration 534 tested was 50 µM. ">50" indicates that the maximum concentration tested was 50 µM and that no viral 535 inhibition tending towards or reaching 50% was observed at this concentration. The maximum 536 concentration tested for galidesivir was 100 µM (since clear inhibitory effects were observed at 50 µM 537 and 100 µM was non-cytotoxic). 

Genomic characterisation and epidemiology of 2019 novel coronavirus: Infectivity of the COVID-19 Virus

Comparative tropism, replication kinetics, and cell damage profiling of SARS-578

CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory 579 studies of COVID-19: an observational study

Effect of inducible FHIT and p53 expression in the Calu-1 lung cancer cell 29

Regulation of the interferon system: evidence that Vero cells 606 have a genetic defect in interferon production

Deducing the N-and O-glycosylation 608 profile of the spike protein of novel coronavirus SARS-CoV-2

Mutations Strengthened SARS-CoV-2 Infectivity

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked 613 by a Clinically Proven Protease Inhibitor

Cleavage Site in the Spike 615 Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells

Activation of the SARS coronavirus spike protein 618 via sequential proteolytic cleavage at two distinct sites

Entry from the cell surface of severe acute respiratory syndrome 621 coronavirus with cleaved S protein as revealed by pseudotype virus bearing cleaved S protein

Expression of SARS-CoV-2 receptor ACE2 and TMPRSS2 in human primary 637 conjunctival and pterygium cell lines and in mouse cornea

Activates Respiratory Viruses and Is Expressed in Viral Target Cells

Transmembrane serine protease TMPRSS2 activates hepatitis C virus 641 infection

Advantages of the Parent Nucleoside GS-441524 over Remdesivir 643 for Covid-19 Treatment

Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious 645 culture system

Cell Culture System Permits Identification of Escape Variants With Resistance to Sofosbuvir

Highly efficient infectious cell culture of three hepatitis C virus genotype 2b 650 strains and sensitivity to lead protease

Cell Culture Studies of the Efficacy and Barrier to Resistance of Sofosbuvir-653

Velpatasvir and Glecaprevir-Pibrentasvir against Hepatitis C Virus Genotypes 2a, 2b, and 2c

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

Full-Length Open Reading Frame Amplification of Hepatitis C Virus

MicroRNA-122 antagonism 674 against hepatitis C virus genotypes 1-6 and reduced efficacy by host RNA insertion or mutations in the 675 HCV 5' UTR

Evolutionary Pathways to Persistence of Highly Fit and Resistant Hepatitis 677 C Virus Protease Inhibitor Escape Variants

Cutadapt removes adapter sequences from high-throughput sequencing reads

Novel infectious cDNA clones of hepatitis C virus genotype

S52) and 4a (strain ED43): genetic analyses and in vivo pathogenesis studies

Differential efficacy of 685 protease inhibitors against HCV genotypes 2a, 3a, 5a, and 6a NS3/4A protease recombinant viruses

MUSCLE: multiple sequence alignment with high accuracy and high throughput

Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal 699 a prerequisite conformational state for receptor binding

Potential to Target New Outbreaks

 423 We thank Bjarne Ørskov Lindhardt (Hvidovre Hospital) and Carsten Geisler (University of 424 Copenhagen) for their support of these studies, and Anna-Louise Sørensen, Susanne Ruszczycka and 425 Louise Barny Christensen, Hvidovre Hospital, for technical support.