Isolation and Genome Sequencing of SARS-CoV-2 from Brazil

Ethics declarations - All methods were performed in accordance with relevant guidelines and regulations. This work was approved by the Ethics Committee on Research with Humans from the Institute of Biomedical Sciences, University of São Paulo, Brazil (permission number 74683917.1.0000.5467). All specimens were handled under the Laboratory biosafety guidance required for the novel coronavirus (2019-nCoV) by the World Health Organization (WHO)¹³ at BLS3 facilities at the Institute of Biomedical Sciences, University of São Paulo.
Clinical specimen collection - Nasopharyngeal (NP) swab samples were collected from symptomatic patients who had acquired COVID-19 during travels to northwest of Italy (Lombardia region) and returned to the São Paulo city in late February. These patients were treated in the same hospital and were the two first confirmed cases of COVID-19 in the São Paulo city. The specimens were collected on day 2-4 post-symptom onset, placed in 1-2 mL of saline medium and used for molecular diagnosis and virus isolation.
Nucleic acid extraction and real-time RT-qPCR for virus detection - In order to perform the identification of SARS-CoV-2, the extraction of total nucleic acid (RNA and DNA) from the collected samples (200 µL of initial material) were carried out using the semi-automated NucliSENS^® easyMag^® platform (bioMérieux, Lyon, France), following the manufacturer’s’ instructions. All specimens were handled under the laboratory biosafety guidance required for the novel coronavirus (2019-nCoV) by WHO¹³ at BLS3 facilities at the Institute of Biomedical Sciences, University of São Paulo. The detection of viral RNA was carried out using the AgPath-ID One-Step RT-PCR Kit (Applied Biosystems Inc., Waltham, USA) on an ABI 7500 SDS real-time PCR machine (Applied Biosystems, Weiterstadt, Germany), using a published protocol and sequence of primers and probe for E gene.¹⁴ RNA copies/mL was quantified by real-time RT-qPCR using a specific in vitro-transcribed RNA quantification standard, kindly granted by Christian Drosten, Charité - Universitätsmedizin Berlin, Germany, as described previously.²Virus isolation - We used Vero E6 cells for isolation and initial passages. We cultured Vero E6 in Dulbecco minimal essential medium (DMEM) supplemented with 10% of heat-inactivated foetal bovine serum (FBS) (Vitrocell Embriolife, Campinas, Brazil).
We used NP swab specimen for virus isolation. For isolation and first passage, we sow cells in a 25 cm² cell culture flask in a concentration of 5 × 10⁵ cells/mL. After 24 h, we removed the culture medium, washed three times with FBS free-DMEM and inoculated aliquots (500 μL) of the clinical specimens into the flask. After 1 h of incubation (adsorption), we completed the volume for 5 mL with DMEM supplemented with 2.5% FBS and 1% of penicillin-streptomycin. We grew the inoculated cultures in a humidified 37°C incubator in an atmosphere of 5% CO₂ and observed for cytopathic effects (CPE) daily up to 72 h. Supernatant was collected daily, and virus replication was confirmed through CPE, gene detection and electron microscopy.
Virus titration - Median tissue culture infectious dose (TCID₅₀/mL) - Vero E6 and CCL-81 cells were seeded into 96-well plate (5 × 10⁴ cells/mL), 24 h before the experiment. Virus was 10-fold serially diluted in medium (10^-1 to 10^-12). Medium was removed from plates, virus dilutions applied in sextuplicate and incubated at 37°C. Visualisations were performed daily in an inverted light microscope (Axiovert 100, Carl Zeiss Oberkochen) to observe the CPE. After 72 h, the last reading was performed, and the monolayers were fixed and stained with Naphthol Blue Black (Sigma-Aldrich Co., Deisenhofen, Germany) dissolved in sodium acetate-acid acetic. The viral titre was expressed in TCID₅₀/mL and calculated using the Spearman & Kärber algorithm, as described by Hierholzer & Killington.¹⁵Plaque forming units (PFU/mL) - Virus titration was carried out in 24 wells plates seeded with Vero E6 and CCL-81 cells at a concentration of 1 × 10⁵ cells/well. After 24 h and a cell confluence of 80-90%, dilutions 10^-1 to 10^-10 in DMEM 2.5% FBS of the virus was transferred in duplicate (100 µL/well) to the seeded plates. After 1 h adsorption at 37^oC 5% CO₂, the wells were completed with an overlay of carboxymethyl cellulose (CMC) with DMEM, 2% FBS and 1% of penicillin-streptomycin, and plates incubated at 37^oC in 5% CO₂ and stained with Naphtol Blue Black dissolved in sodium acetate-acid acetic. Plates were observed and stained from 48 to 96 h post-inoculation (h.p.i.). Both virus titration (TCID₅₀/mL and PFU/mL) were made after the third passage of the isolated virus (T2).
Negative stain transmission electron microscopy - Samples were adsorbed to glow-discharged carbon film-coated copper grids (400 Mesh, CF400-Cu, Electron Microscopy Sciences). The grids were washed with ultrapure water treated with DEPC and negatively stained with uranyl acetate 2% (w/v) with blotting on filter paper after each step. A FEI Tecnai G20 200 kV transmission electron microscope (Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo) was used for image acquisition.
Virus growth kinetics in different cell lines - Three Vero cell lines (E6, CCL-81 and hSLAM) plus a human epithelial type 2 (HEp-2) cells, at concentration of 5 × 10⁴ cells/mL, were tested for the propagation of the SARS-CoV-2 by inoculation at a multiplicity of infection (MOI) of 0.02. The culture medium consisted of DMEM supplemented with 2.5% of FBS. Aliquots of cell-associated and supernatants compartments were collected every 12 h up to 96 h.p.i. for virus quantification via TCID₅₀/mL and RNA copy number quantification by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The assay was conducted in triplicate, reproduced in two independent experiments and expressed by standard error of the mean (SEM). Graphics and SEM were done using GraphPad Prism software version 8.1 (GraphPad Software, San Diego, USA).
Next generation sequencing of viral full-length genome - We extracted total nucleic acid from the NP and oropharyngeal (OP) swab samples and cell supernatants isolates with the QIAamp Viral RNA Mini kit (QIAGEN, Hilden, Germany). The purification and concentration steps were carried out with RNA Clean & Concentrator kit (Zymo Research, Irvine, USA) with DNAse I treatment during the concentration process. Depletion of human ribosomal RNA was performed with the concentrated RNA product using the QIAseq Fast Select RNA Removal kit (QIAGEN). Finally, the RNA samples were submitted to random amplification following the methodology described in Greninger et al.¹⁶ with few modifications.
The preparation of sequencing libraries for the Illumina platform was carried out with the Nextera XT Kit (Illumina, San Diego, USA) and multiplex testing, using the random two-step PCR amplification product as input, followed the kit’s standard instructions. The libraries were quantified after fluorescence measuring with the Qubit instrument (Thermo Fisher Scientific, Waltham, USA) and loaded on the NextSeq 550 equipment (Illumina) for sequencing with MID 300 paired-end reads (Illumina).
Sequencing analysis - The sequencing data was analysed by a flow of bioinformatics analysis (pipeline) developed at Albert Einstein Hospital. In summary, raw sequencing data was subjected to sequence quality controls, removal of human contaminants by aligning against the HG19 reference genome, taxonomic identification of other pathogens and genome recovery through manual curing. Quality control was performed using cutadapt¹⁷ to filter sequences by length (< 50 bp), average quality (Q_p < 20) and trim options to remove low quality ends (9 bp to 5’ end and 5 bp to 3’ end). Passed QC reads were mapped to HG19 human reference genome using bwa¹⁸ mem with default parameters. Not mapped reads were submitted to assembly using SPADES 1.13.¹⁹ Contigs were inspected and manually curated using Geneious 2020.1 to generate a final assembly. Complete genome was compared to SARS-CoV-2 reference and close isolates by multiple sequence alignment. Final genome was deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/).
Production of virus isolates (VIS) and lysate (VLS) stocks and national distribution network - The preparation of VIS and VLS stocks was performed as described above for virus isolation. Related to VIS, one millilitre of VIS (passage 3, T2) was aliquoted on cryogenic vials (Corning Incorporated, Kennebunk, USA) and stored at -196°C. For VLS, 200 µL of T2 of SARS-CoV-2 isolate was added to 800 µL of NucliSENS^® Lysis Buffer (bioMérieux) for virus inactivation and conservation of genetic material in cryogenic vials (Corning Incorporated) and stored at -20°C. The delivery of VIS and VLS was made by the Brazilian Mail Company in partnership with Ministry of Science, Technology and Innovations (MCTIC) of Brazil (http://www.mctic.gov.br) in accordance with the Brazilian Health Regulatory Agency (ANVISA) biosafety rules. Upon formal request to acquire VIS and/or VLS, all recipients signed a term of commitment and responsibility for the use of such reagents within the norms stipulated by WHO.¹³