key: cord-0842997-1lms9n4f authors: Mathez, Gregory; Cagno, Valeria title: Clinical severe acute respiratory syndrome coronavirus 2 isolation and antiviral testing date: 2021-11-20 journal: Antivir Chem Chemother DOI: 10.1177/20402066211061063 sha: 577d9273ae111ee4494abf11d180cf72e7b9ece6 doc_id: 842997 cord_uid: 1lms9n4f Severe acute respiratory syndrome coronavirus 2 is an RNA virus currently causing a pandemic. Due to errors during replication, mutations can occur and result in cell adaptation by the virus or in the rise of new variants. This can change the attachment receptors' usage, result in different morphology of plaques, and can affect as well antiviral development. Indeed, a molecule can be active on laboratory strains but not necessarily on circulating strains or be effective only against some viral variants. Experiments with clinical samples with limited cell adaptation should be performed to confirm the efficiency of drugs of interest. In this protocol, we present a method to culture severe acute respiratory syndrome coronavirus 2 from nasopharyngeal swabs, obtain a high viral titer while limiting cell adaptation, and assess antiviral efficiency. At the end of 2019, a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified in Wuhan. 1 Since then, it has caused millions of deaths. 2 Different strategies of treatment are available or under investigation such as protease inhibitor, nucleoside analogs, monoclonal antibodies, or compounds targeting the secondary structure of viral RNA. [3] [4] [5] [6] [7] Several cell lines can be infected by this virus, which has as primary entry receptor angiotensin-converting enzyme 2 (ACE-2). 1, [8] [9] [10] Vero E6, a monkey epithelial kidney cell line, is the most used for viral culture due to the extensive cytopathic effect after infection and fast viral growth, 8, 9, 11 also if it is not representative of the natural tropism of the virus. When viruses replicate inside the host cell, mutations can occur and may be selected due to an advantage in replication in the cell line used in the laboratory. The same mutations will not occur in vivo and might cause an evolutionary divergence. Viruses can acquire the ability to bind different receptors and increase their affinity for cells that do not represent the natural tropism. For instance, these changes might affect viral entry. Cell adaptation is well known for several viruses: the use of heparan sulfate as an attachment receptor is increased after cell passaging [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] ; human immunodeficiency virus 1 (HIV-1) can adapt to have a higher fitness for infection of MT4 cells 29 ; and SARS-CoV-2 was reported as well to adapt to cell culture: increasing plaques size was observed due to a mutation in the furin-like S1/S2 cleavage site. 11 Adaptations can be an issue during antiviral design since the antiviral activity of the drug of interest can rely on the cause or the consequences of this effect. For example, antivirals acting on the viral entry of yellow fever virus might be effective against the Asibi derived strain (17D), but not on the original strain, because they have a distinct entry route. 21 Experiments with circulating strains with low cell passaging are therefore essential to validate antivirals' activity. At the time of writing the emergence of new variants is the major concern of vaccine escape and increased transmissibility of the virus. [30] [31] [32] Moreover, this phenomenon is likely to increase in the future, due to the selective pressure caused by the increase of vaccine coverage and exposure of the population to natural infection. 33 Hence, it is important to assess the antiviral efficacy of a molecule against multiple variants of interest circulating in the population, rapidly isolated from patient samples. In this context, we established a simple pipeline to culture a clinical sample of SARS-CoV-2 for antiviral assays. With our protocol, it is possible to isolate SARS-CoV-2 with a minimal number of passages from a nasopharyngeal sample. We present as well methods to characterize the stock by titration, reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) and sequencing ( Figure 1 ). In the end, we show how to test antiviral activity in Vero E6 cells with isolated clinical strains. Other protocols are available for the isolation of SARS-CoV-2 from different clinical samples, but the goal was to assess the reliability of qPCR and the correlation among RNA copies and infectious viruses. [34] [35] [36] [37] In opposition, our protocol achieves a better success rate from nasopharyngeal RT-qPCR positive samples and can produce viral stocks with high titer and minimal adaptations. This protocol must be performed by personnel who received specific training for a biosafety level (BSL) 3 laboratory authorized to work with SARS-CoV-2. In the following section, we describe the different steps to culture SARS-CoV-2 from clinical samples, characterize the newly produced virus stock (titration, RNA quantification, sequencing) (Figure 1 ), and test the antiviral activity of the drug of interest. Viral yield reduction can be used to evaluate the release of viral particles by RT-qPCR, or titration of supernatants. [45] [46] [47] The percentage of infected cells or levels of viral protein can be assessed by immunofluorescence or flow cytometry with a primary antibody targeting viral proteins or with reporter genes. 46, 48 After isolation of different clinical isolates as described above, examples of titers obtained for antiviral assays and mutation that occurred during the isolation are shown in Table 1 . Sixteen clinical samples out of 18 were isolated with this protocol. The two non-isolated samples had high initial CT and low starting material. The percentage of samples without any mutation after the isolation represents 54%. Samples with at least two mutations represent 15%. An example of dose-response antiviral activity against SARS-CoV-2 is shown in Figure 4 . The results were assessed by plaque assay, as described in this protocol, using merafloxacin that was previously reported to inhibit SARS-CoV-2 by interacting with its viral RNA. 7 The data are presented as a percentage of infection calculated in comparison to untreated cells. The dose axis is represented in a logarithmic scale. IC 50 can be calculated with GraphPad Prism software (nonlinear regression -> log(inhibitor) vs. response (four parameters) -> bottom and top constraints are 0, 100, respectively). R 2 values and confidence interval have to be critically observed. IC 50s with R 2 values < 0.5 and a large confidence interval represent high variability between the replicates and require additional experiments. IC 50s greater than the higher dose tested should be discarded. Drugs with low IC 50s and high selectivity index (ratio between the half-maximal cytotoxic concentration (CC 50 ) and the IC 50 ) (e.g. > 10) warrant further investigation. SARS-CoV-2 caused an important pandemic and treatments are still under investigation. 3, 4 Since the adaptation of viruses to cell culture can bias the results, and viral variants are emerging with high frequency, drug efficiency should be confirmed with circulating strains. However, material from a nasopharyngeal sample is insufficient for characterization and antiviral testing. Here, we presented a simple way to culture clinical SARS-CoV-2 without extensive passaging. Our method allowed us to isolate clinical strains from nasopharyngeal samples and limit cell adaptation. Our strategy can be applied as well to other viruses. The limitation of our protocol is that cell adaptation cannot be fully omitted since a balance between fast viral growth and cytopathic effect evaluation obliged us to use Vero E6 cells. These cells are not the natural host cell for SARS-CoV-2. Therefore, confirmation of the antiviral activity should be performed in a representative model such as human lung adenocarcinoma cell line (Calu-3) 42,49 or respiratory tissues. 50 This protocol was used to isolate different SARS-CoV-2 variants from patients for quality control of sequencing and antiviral assays for manuscripts in preparation. • 500 mL DMEM. • 50 mL FBS (It has been previously decomplemented for 30 min at 56°C and filtered with a 0.22 µm filter). • 5 mL P/S. • 500 mL DMEM. • 12.5 mL FBS decomplemented and filtered. • 5 mL P/S. • 7.2 g Avicel. • 300 mL water. Dissolve with a magnetic bar for 10 min and autoclave. Crystal violet solution • 0.5 g crystal violet. • 100 mL ethanol absolute. • 400 mL water. • 108.1 mL formaldehyde 37%. • 891.9 mL PBS. 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