Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease (COVID-19) pandemic. SARS-CoV-2 emerged late 2019 in China and had spread globally within a few months (Zhu et al., 2020). An unprecedented rapid vaccine development response has led to approval of the first COVID-19 vaccines at the end of 2020. Conversely, the quest for efficacious specific antiviral therapies against SARS-CoV-2 was not successful. The lack of antivirals, the high adaptive capacity of the virus, and the emergence of new strains, indicate that further research on SARS-CoV-2 biology is necessary.

The first step in most SARS-CoV-2 laboratory studies is in vitro virus propagation to obtain highly concentrated virus stocks. Despite recent advances in physiologically relevant in vitro cell culture systems, methods to propagate clinical isolates have not changed since the first cell lines were established. Traditionally, virus propagation relies on transformed cell lines to produce progeny viruses after inoculation of these cells with a clinical specimen containing the virus. The most widely used cell line in virology is the Vero cell line, which is derived from the kidney of an African green monkey. This cell line and its derivatives (e.g. VeroE6, Vero118, etc) contain genomic deletions of genes involved in the antiviral interferon response (Osada et al., 2014). Such mutations are common in transformed cell lines and allow unbridled virus replication, facilitating the production of high titer virus stocks and allowing research on a wide range of viruses. These isolated viruses are often adapted to their cell culture systems due to their high mutation rates. The development of first and next generation sequencing methods revealed that these adapted viruses were heavily mutated and had drifted significantly from their natural counterparts (Alfson et al., 2018; Lau et al., 2020; Sutter and Moss, 1992; Tamura et al., 2013; Wei et al., 2017). Cell culture adaptive mutations often affect viruses phenotypically, both in vitro and in vivo.

Coronavirus replication is initiated when the viral spike protein binds to the entry receptor on the cell and fuses viral and cellular membranes, allowing the viral RNA to enter the cytoplasm (Hulswit et al., 2016). The spike protein is composed of two domains, the S1 receptor binding domain and the S2 fusion domain, which are separated by the S1/S2 cleavage site. Proteolytic cleavage at the S1/S2 site and the more C-terminal S2’ site is required for coronavirus infectivity as this turns on the fusogenic activity of the S2 domain (Millet and Whittaker, 2015). A distinctive feature of SARS-CoV-2 is the presence of a specific S1/S2 cleavage site in the viral spike protein (Coutard et al., 2020). The SARS-CoV-2 S1/S2 cleavage site contains three basic arginines interrupted by a non-polar alanine (RRAR) and is therefore referred to as a multibasic cleavage site (MBCS). This feature is remarkable as all other viruses within the clade of SARS-related viruses, including SARS-CoV, lack a PRRA insertion that creates this multibasic cleavage site, leading to speculations on whether this site is essential for efficient replication in the human respiratory tract (Hoffmann et al., 2020). Importantly, SARS-CoV-2 isolates that are cultured in the lab rapidly obtain mutations or deletions in the MBCS (Davidson et al., 2020; Klimstra et al., 2020; Lau et al., 2020; Liu et al., 2020; Ogando et al., 2020). On VeroE6 cells, these mutated viruses have a large plaque phenotype, grow to higher titers and outcompete the wild-type virus within 1–4 passages. These mutations have rarely been observed in human clinical specimens (Liu et al., 2020; Wong et al., 2020) and purified MBCS mutants do not efficiently replicate in hamsters (Johnson et al., 2020; Lau et al., 2020). We have recently shown that the MBCS is not required for entry into VeroE6 cells, but is essential for entry into human airway organoids (Mykytyn et al., 2021). We also reported that the MBCS facilitated plasma membrane serine protease-mediated entry, whereas it decreased the dependency on endosomal cathepsins for entry. The serine protease inhibitor camostat mesylate, but not a cathepsin inhibitor, effectively inhibited SARS-CoV-2 entry in human airway organoids, whereas the opposite was observed in VeroE6 cells. These findings demonstrate that SARS-CoV-2 enters relevant airway cells using serine proteases but not cathepsins, and suggest that the multibasic cleavage site is an adaptation to this viral entry strategy. The loss of the MBCS may be an adaptation to the cathepsin-mediated entry pathway present in VeroE6 cells.

In this study, we investigated whether mutations in the SARS-CoV-2 spike MBCS could be prevented in a human airway cell line (Calu-3) and 2D air-liquid interface (ALI) airway organoids in which SARS-CoV-2 enters using serine proteases.

SARS-CoV-2 isolates that are cultured in the lab rapidly lose their spike MBCS (Davidson et al., 2020; Klimstra et al., 2020; Lau et al., 2020; Liu et al., 2020; Ogando et al., 2020). To investigate the extent of cell culture adaptation in our SARS-CoV-2 stocks, we deep-sequenced passage 2, 3, and 4 stocks (P2, P3, and P4) of the BavPat-1 or Munich-1 strain propagated on VeroE6 cells. These stocks were produced from a P1 virus stock grown on VeroE6 cells (Figure 1—figure supplement 1A). In the P2 stock, the majority of reads (65.3%) in the MBCS were identical to the WT sequence (Figure 1A). In the multibasic RxxR motif both the first (R682L) and the last (R685H) arginine were mutated in 3.5% and 6.1% of reads, respectively. An additional mutation (S686G) directly C-terminal to the MBCS was detected at 25.1%. As this variant increased during passaging and therefore could be an adaptation to cell culture, we included it in our analyses. The P3 stock contained 18.8% wild-type (WT) viruses and the S686G was the major MBCS variant at 45.4% while mutations R685H and R682L were present at 22.4% and 7.3%, respectively. A deletion (Del679-688) of the entire MBCS was found as well at 6.1% (Figure 1B). In the P4 virus, the dominant variant contained the R685H mutation at 33.4%, while the Del679-688 and R682L increased to 13.9% and 10.4%, respectively. In the P4 virus, only 9% of reads were WT. Despite the strikingly low level of WT viruses in the VeroE6 stocks, the predominant cleavage motif for the P2 and P3 was still RRARS since mutations never co-occurred (Figure 1A–C). Our results show that a thorough analysis of deep-sequencing data is required to critically assess culture adaptation. The observations from deep-sequencing data were consistent with Sanger sequencing analysis (Figure 1D–F). In agreement with the mixed population of wildtype (WT) and mutant viruses, we observed small (non-adapted) and large (cell culture-adapted) plaque phenotypes in a plaque assay for the P2 virus, but plaques increased in size during passaging (Figure 1G).

Figure 1 with 1 supplement see all

Download asset Open asset

Whereas the MBCS mutations directly removed arginines (R682L, R685H and the deletion) from the minimal RxxR furin motif, the most common stock mutation was the S686G. This site lies directly C-terminal from the MBCS at positions 682–685, indicating that it may also affect the MBCS functionally. To test this, we assessed the infectivity of the 686 mutation using vesicular stomatitis virus (VSV)-based pseudoviruses expressing a green fluorescent protein (GFP) as described before (Mykytyn et al., 2021). Western blot analysis of cleaved and uncleaved S1 revealed that proteolytic cleavage was observed for the WT SARS-CoV-2 pseudovirus and abrogated by all MBCS mutations tested (del-PRRA, del-RRAR, R682A, R685A, and R685H) (Figure 2A,B). For the S686G mutation ~10% cleaved S1 was observed, whereas this was ~80% for WT S (Figure 2A,B). The same difference in cleavage between WT and S686G pseudoviruses was observed for S2 (Figure 2C,D). As expected based on earlier work (Mykytyn et al., 2021), SARS-CoV-2 pseudoviruses with MBCS mutations were more infectious on VeroE6 cells and less infectious on Calu-3 cells (Figure 3A–C). A similar trend was observed for the S686G mutant spike. The infectivity on VeroE6-TMPRSS2 cells was similar for all spikes tested but the WT spike benefited more from TMPRSS2 expression (Figure 3D–E). Protease inhibitors camostat and E64D were then used to block serine proteases and cathepsins, respectively, to assess how spike mutations affect the route of entry. The stable expression of TMPRSS2 in VeroE6 cells leads to entry of WT pseudoviruses via this protease instead of cathepsin-mediated entry, but SARS-CoV-2 MBCS mutants and to a lesser extent the S686G mutant retained partial cathepsin mediated entry (Figure 3F–I). In addition, a GFP-complementation fusion assay, in which cell-cell fusion occurs at the plasma membrane, showed that MBCS mutations and to a lesser extent the S686G mutation abrogated fusion in VeroE6, VeroE6-TMPRSS2, and Calu-3 cells (Figure 4A–C). These data explain why VeroE6-propagated SARS-CoV-2 stocks rapidly accumulate mutations in the MBCS and at spike position 686. Despite being outside of the MBCS, the S686G mutation impairs spike cleavage, cell-cell fusion and serine protease usage, but not as dramatically as the MBCS mutations or deletions. The low infectivity of MBCS mutants and the S686G mutant on Calu-3 cells indicates that WT viruses could have a selective advantage in these cells.

Figure 2

Download asset Open asset

Figure 3

Download asset Open asset

Figure 4

Download asset Open asset

In order to establish culture conditions in which SARS-CoV-2 is genetically stable, we tested whether WT viruses would have a selective advantage on Calu-3 cells that possess serine protease mediated entry and little cathepsin-mediated entry (Mykytyn et al., 2021). For these experiments, we first produced a Calu-3 P2 virus from the VeroE6 P1 stock (Figure 5A). This stock was 100% WT in the MBCS and no major variants (>50%) were detected in the rest of the genome (Figure 5—figure supplement 1C). An additional round of passaging on Calu-3 cells did not lead to any MBCS mutations, or mutations elsewhere (Figure 5B, Figure 5—figure supplement 1C). A Calu-3 P3 from a VeroE6 P2 virus did still contain the S686G at low frequency (7.4%) (Figure 5C), but continued passaging to P5 completely removed the S686G (Figure 5D). Again, we did not observe any other major variant mutations in the rest of the genome (Figure 5—figure supplement 1C). We also produced Calu-3 P4 virus from a VeroE6 P3 stock and we show that while this Calu-3 P4 virus had lost all MBCS mutations, the S686G mutation remained at a frequency of 65.7% (Figure 5—figure supplement 1A). The addition of E64D to block any cathepsin-mediated entry decreased the frequency of S686G by ~11%, to 54.3%, but did not remove S686G entirely (Figure 5—figure supplement 1B). These results support our earlier findings (Figure 2; Figure 3; Figure 4) that the S686G is a less severe cell culture adaptation compared with MBCS mutations, and more importantly show that Calu-3 cells can be used to grow genetically stable stocks without MBCS mutations or S686G. Additionally, stocks grown on Calu-3 cells reached titers of 1.47 × 10⁶–2.1 × 10⁷ TCID50/ml, indicating that Calu-3 cells support the production of high titer stocks.

Figure 5 with 1 supplement see all

Download asset Open asset

To confirm that serine proteases are responsible for the reversal of cell culture adaptation observed in Calu-3 cells, we passaged the adapted VeroE6 P3 stock (Figure 1B) on regular VeroE6 cells or VeroE6-TMPRSS2 cells. P4 viruses grown on VeroE6 cells were only 9% WT and R685H was the dominant variant at 33.4% (Figure 6A; redisplay of Figure 1C). R682L and Del679-688 were present at 10.4% and 13.9%, respectively. Propagation of SARS-CoV-2 in VeroE6-TMPRSS2 cells resulted in an increase in the frequency of WT viruses at 21.7% and a decrease in the frequency of MBCS mutations (7.9% R685H; 4.2% R682L; 1.5% Del679-688), but the S686G remained at 64.6% (Figure 6C). The addition of the serine protease inhibitor camostat (10 μM) to the VeroE6-TMPRSS2 culture, but not the VeroE6 culture (Figure 6B), increased the frequency of MBCS mutations (36.6% R685H; 13% R682L; 8.8% Del679-688), confirming that serine proteases prevent cell culture adaptation (Figure 6D). As TMPRSS2 expression prevented MBCS mutations, we tested whether the addition of trypsin (0.7 μg/ml TPCK-Trypsin) would have a similar effect. Surprisingly, the addition of trypsin to VeroE6 cells, but not VeroE6-TMPRSS2 cells, led to deletion of the entire MBCS (Figure 6—figure supplement 1A,C). This deletion may arise due to the complete cleavage (S1/S2 and S2’) of virus particles that are not bound to the cellular membranes, which would inactivate them. Cell surface expressed TMPRSS2 could accelerate TMPRSS2-mediated entry and cell-cell spread, reducing the chance of trypsin cleavage in the supernatant. Additionally, we tested whether the addition of fetal bovine serum (FBS, heat-inactivated, 10% final concentration) affected culture adaptation as this is commonly added when producing viral stocks. FBS had a similar effect to trypsin in the VeroE6, but not the VeroE6-TMPRSS2 culture, indicating that proteases capable of cleaving spike may be present in serum and that FBS should be avoided when propagating SARS-CoV-2 (Figure 6—figure supplement 1B,D).

Figure 6 with 1 supplement see all

Download asset Open asset

Next, we hypothesized that the best way to prevent cell culture adaptation would be to propagate the virus in non-transformed human airway cells. Recent advances in stem cell biology have enabled the establishment of human organoid culture systems (Katsura et al., 2020; Nikolić et al., 2017; Sachs et al., 2019; Salahudeen et al., 2020; Sato et al., 2009; Youk et al., 2020). These organoid cultures consist of stem cells that self-renew, allowing prolonged passaging and expansion, but can also differentiate to mature cell types, such as ciliated cells, goblet cells, and club cells. Organoids of the airways, the alveoli, and the intestine have recently been used by us to study SARS-CoV-2 entry and pathogenesis (Katsura et al., 2020; Lamers et al., 2020a; Lamers et al., 2020b; Mykytyn et al., 2021; Salahudeen et al., 2020; Youk et al., 2020; Zhou et al., 2020). Pseudoviruses containing MBCS mutations infected human airway organoids poorly (Mykytyn et al., 2021), indicating that these mutations could be prevented by virus propagation in these cells. To produce stocks in human airway organoids, we differentiated the organoids at 2D in transwell inserts at air-liquid interface for 12 weeks as described before (Figure 7A; Mykytyn et al., 2021). Apical cells, including ciliated cells, in these cultures expressed TMPRSS2 as shown by immunohistochemistry (Figure 7B). To produce viral stocks, 2D airway organoids were inoculated at the apical side at a MOI of 0.05 using the VeroE6 P2 stock (Figure 1A). After a 2-hr incubation, cells were washed three times to remove unbound particles. On day 2–5 post-infection, apical washes were collected and stored at 4°C. During virus collections, bound virus particles were released from cells by pipetting directly on the cell layer. Virus collections from day 2 and day 3 (d2+3), and day 4 and day 5 (d4+5) were pooled, centrifuged, and filtered to remove debris, dead cells and mucus. In these cultures, ciliated cells were infected, as shown by confocal imaging at day 3 post infection (Figure 7C). At day 5, cultures exhibited widespread infection (Figure 7D) and significant cytopathic effects including loss of ciliated cells (Figure 7D–E) and syncytium formation (Figure 7E). To remove cytokines that could interfere in downstream experiments (such as interferons), we exchanged the medium in the filtered virus collections three times using an Amicon Ultra-15 column (100 kDa cutoff). The resulting titers from the d2+3 and d4+5 stocks were 5.64 × 10⁵ and 1.00 × 10⁷ TCID₅₀/ml, respectively, indicating that high titer virus stocks can be made in human airway organoids. Sequencing demonstrated that the high titer organoid stock (d4+5) had a 98.9% WT spike sequence, without multibasic cleavage site mutations and the S686G mutation at only 1.1% (Figure 8A–B). In accordance, the Organoid P3 virus produced small plaques (Figure 8C). No major variants were detected in the rest of the genome (Figure 8D). Next, we investigated S1/S2 cleavage of the VeroE6 P2, P3, Calu-3 P3, and Organoid P3 virus stocks by immunoblot (Figure 8E). The non-adapted Calu-3 and organoid stocks were >85% cleaved, while the VeroE6 P2 and P3 stocks were 71.2% and 33% cleaved, respectively (Figure 8F). The findings support that the Calu-3 and organoid stocks are non-adapted and indicate that in vivo the S1/S2 cleavage takes place in the producing cell.

Figure 7

Download asset Open asset

Figure 8 with 1 supplement see all

Download asset Open asset

The rapid loss of the SARS-CoV-2 MBCS in cell culture has underlined that some in vitro propagation systems may fail to model key aspects of the viral life cycle. As these mutations directly affect the relevance and translatability of all laboratory SARS-CoV-2 experiments, it is pivotal to sort out exactly why these occur in order to prevent them. We and others have previously reported that the SARS-CoV-2 MBCS enhances serine protease-mediated entry, the dominant entry pathway in human airway cells (Hoffmann et al., 2020; Mykytyn et al., 2021). VeroE6 cells, commonly used in the lab to grow virus stocks, lack this entry pathway, forcing the virus to use endosomal cathepsins for entry. Cell culture adaptations promoting cathepsin-mediated entry were also observed for the human coronavirus 229E and adapted viruses showed a reduced ability to replicate in differentiated airway epithelial cells (Bertram et al., 2013; Shirato et al., 2017). These observations led us to hypothesize that mutations in the MBCS could be prevented in cells with an active serine protease-mediated entry pathway. In this study, we show that the ectopic expression of the serine protease TMPRSS2 in VeroE6 cells prevented MBCS mutations. Virus propagation in Calu-3 cells, which naturally express serine proteases, also prevented cell culture adaptation. Similar results were obtained using a human airway organoid-based culture system for SARS-CoV-2 propagation.

Our study shows that SARS-CoV-2 rapidly adapts to VeroE6 cell culture. Therefore, deep-sequencing of viral stocks, which offers a thorough analysis beyond the consensus sequence, is essential. As none of the MBCS mutations co-occurred, consensus sequence logos of culture adapted stocks were often WT, while actually only 10–20% of viral reads contained the WT sequence. Therefore, besides reporting the consensus sequence SARS-CoV-2 studies should preferably also report the percentage of WT reads in the MBCS. The first adaptation to occur in our stocks was the S686G mutation, which lies directly adjacent to the MBCS and decreased Calu-3 infectivity, fusogenicity and S1/S2 cleavage, but not as severely as MBCS mutations. Interestingly, this mutation is rapidly positively selected in ferrets (Richard et al., 2020), and also transmitted, suggesting that there are key differences in transmission between humans and ferrets. Alternatively, it is possible that S686G optimizes cleavage by a specific ferret protease.

SARS-CoV-2 is generally grown on VeroE6 cells in the presence of 1–10% FBS, as this allows the production of highly concentrated virus stocks. Here, we show that SARS-CoV-2 rapidly acquired mutations in the MBCS upon passaging in VeroE6 cells and that the addition of FBS increases the frequency of MBCS mutations. Currently, we do not know which components of FBS lead to the increased rate of cell culture adaptation, but we hypothesize that (serine) proteases, naturally present in serum (Shimomura et al., 1992) may be responsible as the addition of trypsin dramatically increased the frequency of MBCS mutations and even led to the deletion of the entire MBCS. The observation that the MBCS is a disadvantage in the presence of trypsin indicates that S2’ proteolytic cleavage (performed by trypsin Hoffmann et al., 2020) should not occur in the supernatant where it would cause all spikes to shed their S1 and adopt their post fusion conformation prior to encountering the plasma membrane. Alternatively, protease inhibitors in serum (Gstraunthaler, 2003) may block transmembrane serine proteases. For these reasons, the use of serum should be avoided when producing virus stocks. The use of defined serum-free media avoids the uncertainty that factors in serum affect the genetic stability of a virus and increase experimental reproducibility due to variations in serum sources.

We show that the expression of the serine protease TMPRSS2 decreases the replicative fitness of MBCS mutant SARS-CoV-2 viruses, which can then be outcompeted by WT viruses. This indicates that the MBCS is an adaptation to serine proteases and that the serine protease-mediated entry pathway is used for entry in vivo. This is in agreement with our earlier observations that SARS-CoV-2 enters using serine proteases on airway organoids (Mykytyn et al., 2021) and that MBCS mutant pseudoviruses could not efficiently infect these cells. Low infectivity of MBCS mutants on the airway cell line Calu-3 was also noted by Hoffmann et al., 2020. In contrast, two CRISPR-based survival screens recently identified several endosomal proteins, including cathepsin L, as essential SARS-CoV-2 genes (Daniloski et al., 2021; Wei et al., 2020). As noted recently by Bailey and Diamond, 2021, the identification of endosomal host factors as proviral in cell-line-based CRISPR screens requires validation in primary cells. Our observation that WT viruses have a selective advantage in 2D airway organoids confirms that the endosomal entry pathway is of little significance in relevant cells.

As new SARS-CoV-2 strains are emerging now and will continue to emerge for as long as SARS-CoV-2 circulates in humans, there is a need to develop propagation systems that will preserve genetic stability for any given SARS-CoV-2 mutant originating from a human respiratory sample. The human airway organoid model for SARS-CoV-2 propagation established in this study (Figure 8) allows high titer SARS-CoV-2 production and was most successful in removing MBCS mutations. We have not observed any major variants associated with virus propagation in this system, but we cannot exclude that some minor variants could be culture adaptations. The self-renewing capacity of organoids allows labs to share organoid lines, allowing a level of reproducibility similar to that of transformed cell lines. In addition, organoids can be grown from a wide range of organs and species to best model the in vivo environment of a particular virus. As an accurate modeling of viral target cells is likely to prevent most forms of cell culture adaptation, we expect that in the future organoid-based systems are likely to replace transformed cell lines for viral stock production.

In conclusion, this study shows that SARS-CoV-2 rapidly adapts to VeroE6 cell culture propagation and that this can be prevented by using cell lines with an active serine protease-mediated entry pathway (VeroE6-TMPRSS2 or Calu-3). Alternatively, a 2D airway organoid-based cell culture model can be used for SARS-CoV-2 propagation if in the future new variants emerge that are not genetically stable on Calu-3 cells. Our study also shows that deep-sequencing rather than consensus sequencing of viral stocks is critical for obtaining relevant and reproducible results in SARS-CoV-2 studies.

All scripts used for data processing are deposited in GitHub: https://github.com/wchnicholas/SARS_CoV2_mutation (copy archived at https://archive.softwareheritage.org/swh:1:rev:0e62dbb51f3a470936981d205320ac4ac863e3c3). Raw sequencing data are available at the NIH Short Read Archive under accession number: BioProject PRJNA694097.

1. 1. Lamers MM
   2. Mykytyn AZ
   3. Breugem TI
   4. Wang Y
   5. Wu DC
   6. Riesebosch S
   7. van den Doel PB
   8. Schipper D
   9. Bestebroer T
   10. Wu NC
   11. Haagmans BL

   (2021) NCBI BioProject

   ID PRJNA694097. Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation.

   https://www.ncbi.nlm.nih.gov/bioproject/PRJNA694097

1. 1. Alfson KJ
   2. Avena LE
   3. Delgado J
   4. Beadles MW
   5. Patterson JL
   6. Carrion R
   7. Griffiths A

   (2018) A single amino acid change in the marburg virus glycoprotein arises during serial cell culture passages and attenuates the virus in a macaque model of disease

   mSphere 3:e00401-17.

   https://doi.org/10.1128/mSphere.00401-17

   • PubMed
   • Google Scholar
2. 1. Bailey AL
   2. Diamond MS

   (2021) A crisp(r) New perspective on SARS-CoV-2 biology

   Cell 184:15–17.

   https://doi.org/10.1016/j.cell.2020.12.003

   • PubMed
   • Google Scholar
3. 1. Bertram S
   2. Dijkman R
   3. Habjan M
   4. Heurich A
   5. Gierer S
   6. Glowacka I
   7. Welsch K
   8. Winkler M
   9. Schneider H
   10. Hofmann-Winkler H
   11. Thiel V
   12. Pöhlmann S

   (2013) TMPRSS2 activates the human coronavirus 229E for cathepsin-independent host cell entry and is expressed in viral target cells in the respiratory epithelium

   Journal of Virology 87:6150–6160.

   https://doi.org/10.1128/JVI.03372-12

   • PubMed
   • Google Scholar
4. 1. Coutard B
   2. Valle C
   3. de Lamballerie X
   4. Canard B
   5. Seidah NG
   6. Decroly E

   (2020) The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade

   Antiviral Research 176:104742.

   https://doi.org/10.1016/j.antiviral.2020.104742

   • PubMed
   • Google Scholar
5. 1. Daniloski Z
   2. Jordan TX
   3. Wessels HH
   4. Hoagland DA
   5. Kasela S
   6. Legut M
   7. Maniatis S
   8. Mimitou EP
   9. Lu L
   10. Geller E
   11. Danziger O
   12. Rosenberg BR
   13. Phatnani H
   14. Smibert P
   15. Lappalainen T
   16. tenOever BR
   17. Sanjana NE

   (2021) Identification of required host factors for SARS-CoV-2 infection in human cells

   Cell 184:92–105.

   https://doi.org/10.1016/j.cell.2020.10.030

   • PubMed
   • Google Scholar
6. 1. Davidson AD
   2. Williamson MK
   3. Lewis S
   4. Shoemark D
   5. Carroll MW
   6. Heesom KJ
   7. Zambon M
   8. Ellis J
   9. Lewis PA
   10. Hiscox JA
   11. Matthews DA

   (2020) Characterisation of the transcriptome and proteome of SARS-CoV-2 reveals a cell passage induced in-frame deletion of the furin-like cleavage site from the spike glycoprotein

   Genome Medicine 12:68.

   https://doi.org/10.1186/s13073-020-00763-0

   • PubMed
   • Google Scholar
7. 1. Gstraunthaler G

   (2003)

   Alternatives to the use of fetal bovine serum: serum-free cell culture

   Altex-Alternativen Zu Tierexperimenten 20:275–281.

   • Google Scholar
8. Book

   1. Hadley W

   (2016) Ggplot2

   Springer.

   https://doi.org/10.1007/978-0-387-98141-3

   • Google Scholar
9. 1. Hoffmann M
   2. Kleine-Weber H
   3. Pöhlmann S

   (2020) A multibasic cleavage site in the spike protein of SARS-CoV-2 is essential for infection of human lung cells

   Molecular Cell 78:779–784.

   https://doi.org/10.1016/j.molcel.2020.04.022

   • PubMed
   • Google Scholar
10. 1. Hulswit RJ
    2. de Haan CA
    3. Bosch BJ

    (2016) Coronavirus spike protein and tropism changes

    Advances in Virus Research 96:29–57.

    https://doi.org/10.1016/bs.aivir.2016.08.004

    • PubMed
    • Google Scholar
11. Preprint

    1. Johnson BA
    2. Xie X
    3. Kalveram B
    4. Lokugamage KG
    5. Muruato A
    6. Zou J
    7. Menachery VD

    (2020) Furin cleavage site is key to SARS-CoV-2 pathogenesis

    bioRxiv.

    https://doi.org/10.1101/2020.08.26.268854

    • Google Scholar
12. 1. Jun G
    2. Wing MK
    3. Abecasis GR
    4. Kang HM

    (2015) An efficient and scalable analysis framework for variant extraction and refinement from population-scale DNA sequence data

    Genome Research 25:918–925.

    https://doi.org/10.1101/gr.176552.114

    • PubMed
    • Google Scholar
13. 1. Katsura H
    2. Sontake V
    3. Tata A
    4. Kobayashi Y
    5. Edwards CE
    6. Heaton BE
    7. Konkimalla A
    8. Asakura T
    9. Mikami Y
    10. Fritch EJ
    11. Lee PJ
    12. Heaton NS
    13. Boucher RC
    14. Randell SH
    15. Baric RS
    16. Tata PR

    (2020) Human lung stem Cell-Based alveolospheres provide insights into SARS-CoV-2-Mediated interferon responses and pneumocyte dysfunction

    Cell Stem Cell 27:890–904.

    https://doi.org/10.1016/j.stem.2020.10.005

    • PubMed
    • Google Scholar
14. 1. Klimstra WB
    2. Tilston-Lunel NL
    3. Nambulli S
    4. Boslett J
    5. McMillen CM
    6. Gilliland T
    7. Dunn MD
    8. Sun C
    9. Wheeler SE
    10. Wells A
    11. Hartman AL
    12. McElroy AK
    13. Reed DS
    14. Rennick LJ
    15. Duprex WP

    (2020) SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients

    Journal of General Virology 101:1156–1169.

    https://doi.org/10.1099/jgv.0.001481

    • Google Scholar
15. 1. Koboldt DC
    2. Zhang Q
    3. Larson DE
    4. Shen D
    5. McLellan MD
    6. Lin L
    7. Miller CA
    8. Mardis ER
    9. Ding L
    10. Wilson RK

    (2012) VarScan 2: somatic mutation and copy number alteration discovery in Cancer by exome sequencing

    Genome Research 22:568–576.

    https://doi.org/10.1101/gr.129684.111

    • PubMed
    • Google Scholar
16. 1. Lamers MM
    2. Beumer J
    3. van der Vaart J
    4. Knoops K
    5. Puschhof J
    6. Breugem TI
    7. Ravelli RBG
    8. Paul van Schayck J
    9. Mykytyn AZ
    10. Duimel HQ
    11. van Donselaar E
    12. Riesebosch S
    13. Kuijpers HJH
    14. Schipper D
    15. van de Wetering WJ
    16. de Graaf M
    17. Koopmans M
    18. Cuppen E
    19. Peters PJ
    20. Haagmans BL
    21. Clevers H

    (2020a) SARS-CoV-2 productively infects human gut enterocytes

    Science 369:50–54.

    https://doi.org/10.1126/science.abc1669

    • PubMed
    • Google Scholar
17. 1. Lamers MM
    2. van der Vaart J
    3. Knoops K
    4. Riesebosch S
    5. Breugem T
    6. Mykytyn AZ
    7. Haagmans BL

    (2020b) An organoid-derived bronchioalveolar model for SARS-CoV-2 infection of human alveolar type II-like cells

    The EMBO Journal 40:e105912.

    https://doi.org/10.15252/embj.2020105912

    • Google Scholar
18. 1. Langmead B
    2. Salzberg SL

    (2012) Fast gapped-read alignment with bowtie 2

    Nature Methods 9:357–359.

    https://doi.org/10.1038/nmeth.1923

    • PubMed
    • Google Scholar
19. 1. Lau SY
    2. Wang P
    3. Mok BW
    4. Zhang AJ
    5. Chu H
    6. Lee AC
    7. Deng S
    8. Chen P
    9. Chan KH
    10. Song W
    11. Chen Z
    12. To KK
    13. Chan JF
    14. Yuen KY
    15. Chen H

    (2020) Attenuated SARS-CoV-2 variants with deletions at the S1/S2 junction

    Emerging Microbes & Infections 9:837–842.

    https://doi.org/10.1080/22221751.2020.1756700

    • PubMed
    • Google Scholar
20. 1. Li H
    2. Handsaker B
    3. Wysoker A
    4. Fennell T
    5. Ruan J
    6. Homer N
    7. Marth G
    8. Abecasis G
    9. Durbin R
    10. 1000 Genome Project Data Processing Subgroup

    (2009) The sequence alignment/Map format and SAMtools

    Bioinformatics 25:2078–2079.

    https://doi.org/10.1093/bioinformatics/btp352

    • PubMed
    • Google Scholar
21. 1. Liu Z
    2. Zheng H
    3. Lin H
    4. Li M
    5. Yuan R
    6. Peng J
    7. Xiong Q
    8. Sun J
    9. Li B
    10. Wu J
    11. Yi L
    12. Peng X
    13. Zhang H
    14. Zhang W
    15. Hulswit RJG
    16. Loman N
    17. Rambaut A
    18. Ke C
    19. Bowden TA
    20. Pybus OG
    21. Lu J

    (2020) Identification of common deletions in the spike protein of severe acute respiratory syndrome coronavirus 2

    Journal of Virology 94:20.

    https://doi.org/10.1128/JVI.00790-20

    • Google Scholar
22. 1. Millet JK
    2. Whittaker GR

    (2015) Host cell proteases: critical determinants of coronavirus tropism and pathogenesis

    Virus Research 202:120–134.

    https://doi.org/10.1016/j.virusres.2014.11.021

    • PubMed
    • Google Scholar
23. 1. Mykytyn AZ
    2. Breugem TI
    3. Riesebosch S
    4. Schipper D
    5. van den Doel PB
    6. Rottier RJ
    7. Lamers MM
    8. Haagmans BL

    (2021) SARS-CoV-2 entry into human airway organoids is serine protease-mediated and facilitated by the multibasic cleavage site

    eLife 10:e64508.

    https://doi.org/10.7554/eLife.64508

    • PubMed
    • Google Scholar
24. 1. Nikolić MZ
    2. Caritg O
    3. Jeng Q
    4. Johnson JA
    5. Sun D
    6. Howell KJ
    7. Brady JL
    8. Laresgoiti U
    9. Allen G
    10. Butler R
    11. Zilbauer M
    12. Giangreco A
    13. Rawlins EL

    (2017) Human embryonic lung epithelial tips are multipotent progenitors that can be expanded in vitro as long-term self-renewing organoids

    eLife 6:e26575.

    https://doi.org/10.7554/eLife.26575

    • PubMed
    • Google Scholar
25. 1. Ogando NS
    2. Dalebout TJ
    3. Zevenhoven-Dobbe JC
    4. Limpens R
    5. van der Meer Y
    6. Caly L
    7. Druce J
    8. de Vries JJC
    9. Kikkert M
    10. Bárcena M
    11. Sidorov I
    12. Snijder EJ

    (2020) SARS-coronavirus-2 replication in vero E6 cells: replication kinetics, rapid adaptation and cytopathology

    Journal of General Virology 101:925–940.

    https://doi.org/10.1099/jgv.0.001453

    • PubMed
    • Google Scholar
26. 1. Osada N
    2. Kohara A
    3. Yamaji T
    4. Hirayama N
    5. Kasai F
    6. Sekizuka T
    7. Kuroda M
    8. Hanada K

    (2014) The genome landscape of the african green monkey kidney-derived vero cell line

    DNA Research 21:673–683.

    https://doi.org/10.1093/dnares/dsu029

    • PubMed
    • Google Scholar
27. 1. Oude Munnink BB
    2. Nieuwenhuijse DF
    3. Stein M
    4. O'Toole Á
    5. Haverkate M
    6. Mollers M
    7. Kamga SK
    8. Schapendonk C
    9. Pronk M
    10. Lexmond P
    11. van der Linden A
    12. Bestebroer T
    13. Chestakova I
    14. Overmars RJ
    15. van Nieuwkoop S
    16. Molenkamp R
    17. van der Eijk AA
    18. GeurtsvanKessel C
    19. Vennema H
    20. Meijer A
    21. Rambaut A
    22. van Dissel J
    23. Sikkema RS
    24. Timen A
    25. Koopmans M
    26. Dutch-Covid-19 response team

    (2020) Rapid SARS-CoV-2 whole-genome sequencing and analysis for informed public health decision-making in the netherlands

    Nature Medicine 26:1405–1410.

    https://doi.org/10.1038/s41591-020-0997-y

    • PubMed
    • Google Scholar
28. 1. Richard M
    2. Kok A
    3. de Meulder D
    4. Bestebroer TM
    5. Lamers MM
    6. Okba NMA
    7. Fentener van Vlissingen M
    8. Rockx B
    9. Haagmans BL
    10. Koopmans MPG
    11. Fouchier RAM
    12. Herfst S

    (2020) SARS-CoV-2 is transmitted via contact and via the air between ferrets

    Nature Communications 11:3496.

    https://doi.org/10.1038/s41467-020-17367-2

    • PubMed
    • Google Scholar
29. 1. Rockx B
    2. Kuiken T
    3. Herfst S
    4. Bestebroer T
    5. Lamers MM
    6. Oude Munnink BB
    7. de Meulder D
    8. van Amerongen G
    9. van den Brand J
    10. Okba NMA
    11. Schipper D
    12. van Run P
    13. Leijten L
    14. Sikkema R
    15. Verschoor E
    16. Verstrepen B
    17. Bogers W
    18. Langermans J
    19. Drosten C
    20. Fentener van Vlissingen M
    21. Fouchier R
    22. de Swart R
    23. Koopmans M
    24. Haagmans BL

    (2020) Comparative pathogenesis of COVID-19, MERS, and SARS in a nonhuman primate model

    Science 368:1012–1015.

    https://doi.org/10.1126/science.abb7314

    • PubMed
    • Google Scholar
30. 1. Sachs N
    2. Papaspyropoulos A
    3. Zomer‐van Ommen DD
    4. Heo I
    5. Böttinger L
    6. Klay D
    7. Weeber F
    8. Huelsz‐Prince G
    9. Iakobachvili N
    10. Amatngalim GD
    11. Ligt J
    12. Hoeck A
    13. Proost N
    14. Viveen MC
    15. Lyubimova A
    16. Teeven L
    17. Derakhshan S
    18. Korving J
    19. Begthel H
    20. Dekkers JF
    21. Kumawat K
    22. Ramos E
    23. Oosterhout MFM
    24. Offerhaus GJ
    25. Wiener DJ
    26. Olimpio EP
    27. Dijkstra KK
    28. Smit EF
    29. Linden M
    30. Jaksani S
    31. Ven M
    32. Jonkers J
    33. Rios AC
    34. Voest EE
    35. Moorsel CHM
    36. Ent CK
    37. Cuppen E
    38. Oudenaarden A
    39. Coenjaerts FE
    40. Meyaard L
    41. Bont LJ
    42. Peters PJ
    43. Tans SJ
    44. Zon JS
    45. Boj SF
    46. Vries RG
    47. Beekman JM
    48. Clevers H

    (2019) Long‐term expanding human airway organoids for disease modeling

    The EMBO Journal 38:e00300.

    https://doi.org/10.15252/embj.2018100300

    • Google Scholar
31. 1. Salahudeen AA
    2. Choi SS
    3. Rustagi A
    4. Zhu J
    5. van Unen V
    6. de la O SM
    7. Flynn RA
    8. Margalef-Català M
    9. Santos AJM
    10. Ju J
    11. Batish A
    12. Usui T
    13. Zheng GXY
    14. Edwards CE
    15. Wagar LE
    16. Luca V
    17. Anchang B
    18. Nagendran M
    19. Nguyen K
    20. Hart DJ
    21. Terry JM
    22. Belgrader P
    23. Ziraldo SB
    24. Mikkelsen TS
    25. Harbury PB
    26. Glenn JS
    27. Garcia KC
    28. Davis MM
    29. Baric RS
    30. Sabatti C
    31. Amieva MR
    32. Blish CA
    33. Desai TJ
    34. Kuo CJ

    (2020) Progenitor identification and SARS-CoV-2 infection in human distal lung organoids

    Nature 588:670–675.

    https://doi.org/10.1038/s41586-020-3014-1

    • PubMed
    • Google Scholar
32. 1. Sato T
    2. Vries RG
    3. Snippert HJ
    4. van de Wetering M
    5. Barker N
    6. Stange DE
    7. van Es JH
    8. Abo A
    9. Kujala P
    10. Peters PJ
    11. Clevers H

    (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche

    Nature 459:262–265.

    https://doi.org/10.1038/nature07935

    • PubMed
    • Google Scholar
33. 1. Shimomura T
    2. Ochiai M
    3. Kondo J
    4. Morimoto Y

    (1992) A novel protease obtained from FBS-containing culture supernatant, that processes single chain form hepatocyte growth factor to two chain form in serum-free culture

    Cytotechnology 8:219–229.

    https://doi.org/10.1007/BF02522039

    • PubMed
    • Google Scholar
34. 1. Shirato K
    2. Kanou K
    3. Kawase M
    4. Matsuyama S

    (2017) Clinical isolates of human coronavirus 229E bypass the endosome for cell entry

    Journal of Virology 91:e01387-16.

    https://doi.org/10.1128/JVI.01387-16

    • PubMed
    • Google Scholar
35. 1. Sutter G
    2. Moss B

    (1992) Nonreplicating vaccinia vector efficiently expresses recombinant genes

    PNAS 89:10847–10851.

    https://doi.org/10.1073/pnas.89.22.10847

    • PubMed
    • Google Scholar
36. 1. Tamura D
    2. Nguyen HT
    3. Sleeman K
    4. Levine M
    5. Mishin VP
    6. Yang H
    7. Guo Z
    8. Okomo-Adhiambo M
    9. Xu X
    10. Stevens J
    11. Gubareva LV

    (2013) Cell Culture-Selected substitutions in influenza A(H3N2) Neuraminidase affect drug susceptibility assessment

    Antimicrobial Agents and Chemotherapy 57:6141–6146.

    https://doi.org/10.1128/AAC.01364-13

    • Google Scholar
37. 1. Tareen A
    2. Kinney JB

    (2020) Logomaker: beautiful sequence logos in Python

    Bioinformatics 36:2272–2274.

    https://doi.org/10.1093/bioinformatics/btz921

    • PubMed
    • Google Scholar
38. 1. Wei H
    2. Audet J
    3. Wong G
    4. He S
    5. Huang X
    6. Cutts T
    7. Theriault S
    8. Xu B
    9. Kobinger G
    10. Qiu X

    (2017) Deep-sequencing of marburg virus genome during sequential mouse passaging and cell-culture adaptation reveals extensive changes over time

    Scientific Reports 7:3390.

    https://doi.org/10.1038/s41598-017-03318-3

    • PubMed
    • Google Scholar
39. Preprint

    1. Wei J
    2. Alfajaro MM
    3. Hanna RE
    4. DeWeirdt PC
    5. Strine MS
    6. Lu-Culligan WJ
    7. Wilen CB

    (2020) Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection

    bioRxiv.

    https://doi.org/10.1101/2020.06.16.155101

    • Google Scholar
40. 1. Wong YC
    2. Lau SY
    3. Wang To KK
    4. Mok BWY
    5. Li X
    6. Wang P
    7. Chen Z

    (2020) Natural transmission of bat-like SARS-CoV-2PRRA variants in COVID-19 patients

    Clinical Infectious Diseases : An Official Publication of the Infectious Diseases Society of America 10:ciaa953.

    https://doi.org/10.1093/cid/ciaa953

    • Google Scholar
41. Software

    1. Wu D
    2. Wang Y
    3. Wu N

    (2021) SARS-CoV-2_in_vitro_adaptation

    Zenodo.

    https://doi.org/10.5281/zenodo.4706095
42. 1. Youk J
    2. Kim T
    3. Evans KV
    4. Jeong YI
    5. Hur Y
    6. Hong SP
    7. Kim JH
    8. Yi K
    9. Kim SY
    10. Na KJ
    11. Bleazard T
    12. Kim HM
    13. Fellows M
    14. Mahbubani KT
    15. Saeb-Parsy K
    16. Kim SY
    17. Kim YT
    18. Koh GY
    19. Choi BS
    20. Ju YS
    21. Lee JH

    (2020) Three-Dimensional human alveolar stem cell culture models reveal infection response to SARS-CoV-2

    Cell Stem Cell 27:905–919.

    https://doi.org/10.1016/j.stem.2020.10.004

    • PubMed
    • Google Scholar
43. 1. Zhou J
    2. Li C
    3. Liu X
    4. Chiu MC
    5. Zhao X
    6. Wang D
    7. Wei Y
    8. Lee A
    9. Zhang AJ
    10. Chu H
    11. Cai JP
    12. Yip CC
    13. Chan IH
    14. Wong KK
    15. Tsang OT
    16. Chan KH
    17. Chan JF
    18. To KK
    19. Chen H
    20. Yuen KY

    (2020) Infection of bat and human intestinal organoids by SARS-CoV-2

    Nature Medicine 26:1077–1083.

    https://doi.org/10.1038/s41591-020-0912-6

    • PubMed
    • Google Scholar
44. 1. Zhu N
    2. Zhang D
    3. Wang W
    4. Li X
    5. Yang B
    6. Song J
    7. Zhao X
    8. Huang B
    9. Shi W
    10. Lu R
    11. Niu P
    12. Zhan F
    13. Ma X
    14. Wang D
    15. Xu W
    16. Wu G
    17. Gao GF
    18. Tan W

    (2020) A novel coronavirus from patients with pneumonia in China, 2019

    New England Journal of Medicine 382:727–733.

    https://doi.org/10.1056/NEJMoa2001017

    • Google Scholar

Author details

1. Mart M Lamers

   Viroscience Department, Erasmus Medical Center, Rotterdam, Netherlands

   Contribution

   Conceptualization, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1431-4022

2. Anna Z Mykytyn

   Viroscience Department, Erasmus Medical Center, Rotterdam, Netherlands

   Contribution

   Conceptualization, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7188-6871

3. Tim I Breugem

   Viroscience Department, Erasmus Medical Center, Rotterdam, Netherlands

   Contribution

   Conceptualization, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5558-7043

4. Yiquan Wang

   Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, United States

   Contribution

   Data curation, Software, Formal analysis, Visualization, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1954-9808

5. Douglas C Wu

   Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, United States

   Present address

   Invitae Corporation, San Francisco, United States

   Contribution

   Data curation, Software, Formal analysis, Visualization, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6179-3110

6. Samra Riesebosch

   Viroscience Department, Erasmus Medical Center, Rotterdam, Netherlands

   Contribution

   Validation, Investigation, Visualization, Methodology

   Competing interests

   No competing interests declared

7. Petra B van den Doel

   Viroscience Department, Erasmus Medical Center, Rotterdam, Netherlands

   Contribution

   Investigation, Writing - original draft

   Competing interests

   No competing interests declared

8. Debby Schipper

   Viroscience Department, Erasmus Medical Center, Rotterdam, Netherlands

   Contribution

   Investigation

   Competing interests

   No competing interests declared

9. Theo Bestebroer

   Viroscience Department, Erasmus Medical Center, Rotterdam, Netherlands

   Contribution

   Investigation, Methodology, Writing - original draft

   Competing interests

   No competing interests declared

10. Nicholas C Wu

    1. Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, United States
    2. Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, United States
    3. Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, United States

    Contribution

    Data curation, Software, Formal analysis, Supervision, Visualization, Writing - review and editing

    Competing interests

    No competing interests declared

11. Bart L Haagmans

    Viroscience Department, Erasmus Medical Center, Rotterdam, Netherlands

    Contribution

    Conceptualization, Supervision, Funding acquisition, Writing - review and editing

    For correspondence

    b.haagmans@erasmusmc.nl

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-6221-2015

• 3,972

  views

• 544

  downloads

• 78

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.