key: cord-0958963-4w5o8jfu
authors: Caly, Leon; Druce, Julian; Roberts, Jason; Bond, Katherine; Tran, Thomas; Kostecki, Renata; Yoga, Yano; Naughton, William; Taiaroa, George; Seemann, Torsten; Schultz, Mark B; Howden, Benjamin P; Korman, Tony M; Lewin, Sharon R; Williamson, Deborah A; Catton, Mike G
title: Isolation and rapid sharing of the 2019 novel coronavirus (SARS‐CoV‐2) from the first patient diagnosed with COVID‐19 in Australia
date: 2020-04-01
journal: Med J Aust
DOI: 10.5694/mja2.50569
sha: 995283975d8501145551f88510d350ad296e58c0
doc_id: 958963
cord_uid: 4w5o8jfu

OBJECTIVES: To describe the first isolation and sequencing of SARS‐CoV‐2 in Australia and rapid sharing of the isolate. SETTING: SARS‐CoV‐2 was isolated from a 58‐year‐old man from Wuhan, China who arrived in Melbourne on 19 January 2020 and was admitted to the Monash Medical Centre, Melbourne from the emergency department on 24 January 2020 with fever, cough, and progressive dyspnoea. MAJOR OUTCOMES: Clinical course and laboratory features of the first reported case of COVID‐19 (the illness caused by SARS‐CoV‐2) in Australia; isolation, whole genome sequencing, imaging, and rapid sharing of virus from the patient. RESULTS: A nasopharyngeal swab and sputum collected when the patient presented to hospital were each positive for SARS‐CoV‐2 (reverse transcription polymerase chain reaction). Inoculation of Vero/hSLAM cells with material from the nasopharyngeal swab led to the isolation of SARS‐CoV‐2 virus in culture. Electron microscopy of the supernatant confirmed the presence of virus particles with morphology characteristic of viruses of the family Coronaviridae. Whole genome sequencing of the viral isolate and phylogenetic analysis indicated the isolate exhibited greater than 99.99% sequence identity with other publicly available SARS‐CoV‐2 genomes. Within 24 hours of isolation, the first Australian SARS‐CoV‐2 isolate was shared with local and overseas reference laboratories and major North American and European culture collections. CONCLUSIONS: The ability to rapidly identify, propagate, and internationally share our SARS‐CoV‐2 isolate is an important step in collaborative scientific efforts to deal effectively with this international public health emergency by developing better diagnostic procedures, vaccine candidates, and antiviral agents.

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200μL aliquots from swab (nasopharyngeal in VTM), sputum, urine, faeces and serum samples were subjected to RNA extraction using the QIAamp 96 Virus QIAcube HT Kit (Qiagen, Hilden, Germany) and eluted in 60μL. Reverse transcription was performed using the BioLine SensiFAST cDNA kit (Bioline, London, United Kingdom), total reaction mixture (20μL), containing 10μL RNA extract, 4μL 5x TransAmp buffer, 1μL reverse transcriptase and 5μL nuclease-free water. The reactions were incubated at 25°C for 10 min, 42°C for 15 min and 85°C for 5 min.

A PCR mixture containing 2μL cDNA, 1.6 μl 25mM MgCl2, 4μL 10x Qiagen Taq Buffer, 0.4μL 20mM dNTPs, 0.3μL Taq polymerase (Qiagen, Hilden, Germany) and 2μL of 10 μM primer pools as described 2 . Briefly, first round included the forward (5'-GGKTGGGAYTAYCCKAARTG-3') and reverse (5'-GGKTGGGAYTAYCCKAARTG-3') primers. Cycling conditions were 94°C for 10min, followed by 30 cycles of 94°C for 30s, 48°C for 30s and 72°C for 40s, with a final extension of 72°C for 10 min. 2μL of resultant product was used as template for the second round which included the forward (5'-GGTTGGGACTATCCTAAGTGTGA-3') and reverse (5'-CCATCATCAGATAGAATCATCAT-3') primers. Cycling conditions were 94°C for 10min, followed by 40 cycles of 94°C for 30s, 50°C for 30s and 72°C for 40s, with a final extension of 72°C for 10min. Resultant products had an expected size of approximately 440bp on a 2% agarose gel.

The PCR products were purified using ExoSAP-IT (Affymetrix, Santa Clara, CA, USA) and sequenced using an Applied Biosystems SeqStudio Genetic Analyzer (Life Technologies, Carlsbad, CA, USA) using Big Dye Terminator 3.1 (Life Technologies, Carlsbad, CA, USA) and Round 2 PCR primers above. SARS-CoV and 229e-CoV cDNA were used as positive control material.

TaqMan RT-PCR assay comprised of 2.5 μl cDNA, 10 μl Primer Design PrecisonPLUS qPCR Master 

Vero/hSLAM cells (African green monkey kidney cells transfected to express the human signaling lymphocytic activation molecule (SLAM; also known as CDw150) 1 were grown at 37C, 5% CO2 in maintenance media consisting of 10mL Earle's minimum essential medium (EMEM), 7% fetal bovine serum (FBS) (Bovogen Biologicals, Keilor East, AUS) 2mM L-glutamine, 1 mM sodium pyruvate, 1500mg/L sodium bicarbonate, 15 mM HEPES and 0.4mg/ml geneticin to 95% confluency in 25cm 2 flasks. Prior to use for isolation, maintenance media was removed from the flask and 500μL of respiratory swab inoculum was overlaid on the cell monolayer. The flask was returned to the 37C incubator to allow the virus to adsorb for 1 hour before addition of 10 mL viral culture media (EMEM as above but FBS reduced to 2%). Flasks were monitored for viral cytopathic effect and 140μL

aliquots of supernatant removed every 48 hours to assess virus burden by TaqMan real-time RT-PCR.

First passage culture grown virus isolate was subsequently shipped nationally and internationally in packaging compliant with UN 2814 Category A shipping requirements using credentialed, specialised courier services under the appropriate Australian government export approvals processes and receiving country import permissions.

Negative staining and thin sectioning of cell culture-derived supernatant was performed after low speed centrifugation at 1000g for 3 minutes. Copper electron microscopy grids coated with formvar and a continuous carbon layer were glow-discharged for 30 seconds using a Pelco EasiGlow system. 8μL of supernatant was applied directly to a glow-discharged grid and allowed to adsorb for 20 seconds, then negative stained with 8μL of 3% phosphotungstic acid (pH 7.0). The negative-stained grid was air-dried at room temperature and examined using an FEI T12 Spirit electron microscope operating at an acceleration voltage of 80keV. Electron micrographs were collected using an FEI Eagle 16MP CCD camera. Virion diameter and glycoprotein features were measured using the FEI TIA software package. After low speed centrifugation, the supernatant was removed and the remaining pellet was enrobed in 3% agarose, after fixation in 2% osmium tetroxide the sections were passed through a gradient series of dehydrating ethanol solutions and then transitioned to 100%

propylene oxide for embedding in Spurr's resin. Embedded samples were polymerised at 60ºC for 72 hours then thin-sectioned using a Leica UC7 ultramicrotome, 70nm sections were transferred to copper 100-mesh electron microscopy grids coated with formvar and a continuous carbon layer and examined using an FEI F30 Twin electron microscope operating at an acceleration voltage of 200keV.

Electron micrographs were collected using an FEI Ceta 16MP CCD camera. reaction volume) and eluted in 48μL nuclease-free water. cDNA was quantified using a Qubit highsensitivity double-stranded DNA (dsDNA) kit (Thermo Fisher Scientific, Waltham, MA, USA), according to manufacturers' instructions.

RNA

Sequencing libraries were prepared using 300ng cDNA as input for the SQK-LSK109 kit (Oxford Nanopore Technologies, Oxford, UK) as per manufacturer's instructions. Libraries were sequenced on FLO-MIN106 flow cells on the MinION Mk1b device (Oxford Nanopore Technologies, Oxford,

SISPA barcode sequences were removed using the "Trim Ends

Illumina library preparation and sequencing

USA) library was prepared using a Nextera XT kit, using 75b paired-end chemistry, and were sequenced on an Illumina MiSeq platform as per manufacturer's instructions. Approximately 30,000,000 reads were generated from the SISPA input material

Phylogenetic analysis and genome assembly The assembly of the SARS-CoV-2/VIC01/Australia genome (GenBank Accession MT007544.1, GISAID EPI_ISL_406844) was confirmed using both de novo assembly and alignment to a reference

De novo assembly of Illumina reads used Shovill

Reference guided assembly was performed by alignment of Illumina reads to the Wuhan

Geneious Prime 2020.0.0, and alignment of Nanopore reads using Minimap2

BetaCoV/Australia/VIC01/2020 genome demonstrated 3 single nucleotide polymorphisms (SNPs) compared to the Wuhan-Hu-1 reference genome (MN908947.3), with 2 SNPs having a 10% minor wildtype allele frequency. Further, a 10 bp deletion was detected in the 3' UTR

Measles viruses on throat swabs from measles patients use signaling lymphocytic activation molecule (CDw150) but not CD46 as a cellular receptor

Avian coronavirus in wild aquatic birds

Assessment of metagenomic Nanopore and Illumina sequencing for recovering whole genome sequences of chikungunya and dengue viruses directly from clinical samples

Metagenomic nanopore sequencing of influenza virus direct from clinical respiratory samples

Minimap2: pairwise alignment for nucleotide sequences