key: cord-1018986-ez2u8gn7
authors: Saccon, Elisa; Krishnan, Shuba; Vinhas, Beatriz Sá; Byrareddy, Siddappa N.; Mirazimi, Ali; Neogi, Ujjwal; Gupta, Soham
title: Replication dynamics and cytotoxicity of SARS-CoV-2 Swedish isolate in commonly used laboratory cell lines
date: 2020-09-16
journal: bioRxiv
DOI: 10.1101/2020.08.28.271684
sha: c329ec39756b9a7a32d082ff52182b9c618d7d10
doc_id: 1018986
cord_uid: ez2u8gn7

We assessed the infectivity, replication dynamics and cytopathogenicity of the first Swedish isolate of SARS-CoV-2 in six different cell lines of human origin and compared their growth characteristics. High replication kinetics in absence of cytopathic-effect observed in many cell lines provided important clues on SARS-CoV-2 pathogenesis.

In the current pandemic of Coronavirus disease 2019 , caused by the novel Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), numerous research activities have been focusing on better understanding the mechanisms of viral pathogenesis as well as developing effective antivirals against the infection (1) (2) (3) (4) (5) . In this scenario, Vero-E6 cell line has been widely employed for viral isolation, propagation and antiviral testing, due to its high infectivity, virus production and a prominent cytopathic effect (CPE) upon infection. However, since Vero-E6 originates from monkey, a more physiologically relevant cell line of human origin is warranted, that is better able to recapitulate the complexity of host cellular response to the SARS-CoV-2.

In this study, we aimed at determining the SARS-CoV-2 tropism and replication capacity in six different human cell lines that are commonly used in laboratories. Our data provide a comprehensive view on how SARS-CoV-2 behaves differently in different human cell lines depending on the viral strain and, possibly, the isolation method. Overall, our work represents a useful tool for choosing a suitable cell system to investigate the viral pathogenicity and drug susceptibility.

We infected Vero-E6 (ATCC#CRL-1586), Calu-3 (ATCC#HTB-55), A549 (ATCC#CCL-185), Caco2 (CLS-300137), Huh7, 293T and 16HBE with the first Swedish isolate of SARS-CoV-2 virus (SWE/01/2020) at a multiplicity of infection (moi) of 1 and 0.1 as previously described(1).

Virus-induced cytotoxicity was evaluated by measuring the cellular ATP using Viral-ToxGlo™ 3 assay (Promega) and virus replication kinetics were determined in the cell culture supernatant by qPCR targeting the N-gene(6) using PrimeDirect™ Probe RT-qPCR mix (Takara) starting at 3h post-infection (hpi) and followed up to 120hpi (1) . As shown in Figure 1A , infection with moi 0.1 showed better kinetics than moi 1 and attained similar viral-copies at 120hpi. At moi 0.1 Vero-E6 showed a complete CPE (viability<3%) by 48hpi and a significant increase of 3.8 log10 viralcopies at 24hpi. Of the six human cell lines that were tested, Caco2 (intestinal) showed highest infection with a 4.3 log10 copies rise in viral load at 120hpi, with moi 0.1 (p<0.001). 293T (kidney;p<0.01), Huh7 (liver;p<0.02) and Calu-3 (lung;p<0.05) showed moderate infection with a change of >1 log10 viral-copies at 120hpi, while 16HBE (lung) and A549 (lung) showed very poor infectivity with a change of <0.6 log10 viral-copies. Interestingly, viral-induced cytotoxicity was only observed in Calu-3 cells (viability at moi 1= 50% and moi 0.1=77%) while none of the other cell lines showed any apparent cytotoxicity (viability>85%) ( Figure 1B ). ACE2 receptor and TMPRSS2 activity has been shown to be critical for SARS-CoV-2 entry into the cell (5, 7) . To correlate the ACE2 and TMPRSS2 expression with the infectivity, we determined the protein expression in the cell lysates by western blot (Figure 2A ). Among the infected cell lines, co-expression of ACE2 and TMPRSS2 was most prominent in Caco2, followed by Vero-E6 and Calu-3 that showed poor TMPRSS2 expression. Contrarily, Huh7 and 293T strongly expressed TMPRSS2 but lacked ACE2 expression. Our results showed no strong correlation between co-expression of the receptors and infectivity, indicating that each receptor has an individual role in aiding the infection.

Finally, to compare the tropism of Swedish SARS-CoV-2 virus with the other globally isolated strains, we performed a literature survey to determine the infectivity and cytopathogenicity in all the six cell lines as noted above. We have included virus isolates from Germany 4 (FFM1/2020(2) and Mun_IMB1/2020(4)), France (IHUMI2) (8) , USA (WA1/2020)(5), Canada (SB3-TYAGNC)(9), China (KMS1/2020)(10), Hong Kong (HKU-001a/2020(3) and VM20001061/2020 (11) , Taiwan (NTU01/2020 and NTU02/2020) (12) and Japan (TY-WK-521/2020)(7) and found major differences in cellular tropism and cytopathogenicity ( Figure 2B ).

Specifically, we observed that the majority of the strains were able to infect both Caco2 and Calu-3, except for the Muc_IMB1/2020 (no infectivity in both the cell lines)(4) and the Japanese strain 

Establishing SARS-CoV-2 infectivity and cytopathogenicity in a human cell line is key to testing new antivirals as well as understanding viral-host interplay in a more physiologically relevant setting. Several recent studies testing different local strains, show conflicting results concerning infectivity and cytopathogenicity in human cell lines ( Figure 2B ), especially when Caco2 and Calu-3 cells are employed. Particularly, the Swedish isolate tested here showed high infectivity in Caco2 and moderate infectivity for Calu-3, not aligning with any particular strain. A stark difference was also noted in cytopathogenicity, wherever reported. In our study, upon 5 infection, Calu-3 stopped growing, rounded up and mottled, as compared to the uninfected control.

The morphological change observed in Calu-3 might represent a cellular mechanism to control viral infection and therefore needs more investigation. In Caco2 we did not observe any appreciable cytopathogenicity as was reported for the French(8) and the Honk Kong strain(3).

However, the Frankfurt strain caused rapid CPE in Caco2 within 24hpi. Unlike others, the Frankfurt strain was the only strain that was isolated and adapted in Caco2 that could have possibly lead to higher infectivity and CPE of this strain in the cell line (2) . In addition, different infective doses, structural changes in the virus, culture conditions and clonal differences in the cell lines, could also govern strain specific differences in infectivity.

A major role in viral infectivity is played by envelope glycoproteins and cellular receptors, which initiate the early phases of the viral life cycle. Therefore, we first investigated whether mutations in the spike glycoproteins of the different strains presented above could affect their infectivity and cytopathogenicity. However, no major amino acid changes was observed in the spike protein, except for the Munich strain harboring D614G mutation that had become the dominant genotype in Europe and considered to be more infectious in humans (13) . Interestingly, this strain shows no infectivity in both Caco2 and Calu-3(4).We then correlated the viral infectivity with the expression of ACE2 and TMPRSS2 proteins that are considered essential for viral entry.

Our results showed no strong correlation between co-expression of the receptors and infectivity and suggest that while ACE2 expression is important for the virus infectivity, increased expression of TMPRSS2 alone can enhance virus uptake into the cell (14) . However, we cannot exclude the possibility that other molecules or endocytosis mechanisms are involved in viral recognition and entry. In addition, in the present study, we have investigated the replicative capacity of the virus by measuring the newly produced virions in the culture supernatant. In different cell lines there 6 might be other mechanisms that interfere with viral replication following entry, that require further validation.

In conclusion, our study warrants the need for physiologically relevant models to study the biological properties of SARS-CoV-2 which requires understanding of the viral and host factors that govern infectivity of SARS-CoV-2 in different cell lines. 

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We would like to thank Prof. Lena Palmberg, and Asst. Prof