key: cord-0946426-hsy9qts3
authors: Au, Chun Hang; Chan, Wai Sing; Lam, Ho Yin; Ho, Dona N.; Lam, Simon Y. M.; Zee, Jonpaul S. T.; Chan, Tsun Leung; Ma, Edmond S. K.
title: Genome Sequences of SARS-CoV-2 Strains Detected in Hong Kong
date: 2020-07-30
journal: Microbiol Resour Announc
DOI: 10.1128/mra.00697-20
sha: 2b005f429d646f6130ad607505dda333746ba1c9
doc_id: 946426
cord_uid: hsy9qts3

We sequenced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from deep throat saliva samples of three imported cases in Hong Kong by Nanopore sequencing. Epidemiological and clinical features of these coronavirus disease 2019 (COVID-19) cases were presented for genomic epidemiology studies.

S evere acute respiratory syndrome coronavirus 2 (SARS-CoV-2; family Coronaviridae and genus Betacoronavirus) is causing an ongoing coronavirus disease 2019 (COVID-19) pandemic. There were 1,109 COVID-19 cases in Hong Kong as of 12 June 2020 (https://www.chp.gov.hk/files/pdf/local_situation_covid19_en.pdf), with most cases reported in March to April 2020. We report here the genome sequences of SARS-CoV-2 strains detected from three imported cases in Hong Kong.

Case 1 (58-year-old man, HKSH0003) and case 2 (61-year-old female, HKSH0004) are a couple who had symptom onset on 10 March 2020 after returning from the United States to Hong Kong and tested positive on 14 March. Case 1 was relatively asymptomatic apart from fever, but case 2 showed symptoms and signs of pneumonia during hospitalization. They were enrolled in a clinical trial of interferon beta-1b, lopinavirritonavir, and ribavirin (1) and were discharged after two negative PCR tests. Case 3 (59-year-old man, HKSH0007) had symptom onset on 21 March after returning from Germany to Hong Kong and tested positive on 1 April. He showed very mild symptoms and denied treatment during isolation. The patient was discharged after two negative PCR tests. Informed consent was obtained from every case. This study was reviewed and approved by the Research Committee of Hong Kong Sanatorium & Hospital (reference number RC-2020-11).

Deep throat saliva samples treated with Sputasol (Thermo Scientific, USA) were subjected to nucleic acid extraction using eMAG (bioMérieux, France). The full genome was amplified following the ARTIC nCoV-2019 V1 protocol (https://www.protocols.io/ view/ncov-2019-sequencing-protocol-bbmuik6w). Briefly, cDNA was synthesized using a SuperScript III first-strand synthesis system (Invitrogen, USA) with random hexamers. Two multiplex PCRs of 49 amplicons (V1 panel) were performed using AmpliTaq Gold DNA polymerase (Applied Biosystems, USA), combined, purified using AMPure XP beads (Beckman Coulter, USA), and quantified using a Qubit double-stranded DNA (dsDNA) high-sensitivity assay (Invitrogen, USA). Each genome was sequenced separately. For each sample, a separate Nanopore sequencing library was prepared using a ligation sequencing kit (SQK-LSK109; Oxford Nanopore Technologies, UK), sequenced using a new MinION R9 Nanopore sequencing runs yielded 0.497-Gbp reads for HKSH0003 (853,213 reads; mean length, 582 bp), 0.534-Gbp reads for HKSH0004 (781,048 reads; mean length, 684 bp), and 7.740-Gbp reads for HKSH0007 (16,122,660 reads; mean length, 480 bp). All three SARS-CoV-2 genomes were sequenced at a mean depth of Ͼ3,500ϫ, reference coverage of 92% to 96%, length of 27,384 to 28,723 nucleotides, and GC content of 38%. Phylogenetic analysis showed that they descended from European samples (88%, 88%, and 100% confidence for cases 1, 2, and 3, respectively) and were distinct from most other Hong Kong sequences (Fig. 1A) . Although the disease severities of cases 1 and 2 were different, there was no detectable difference between their SARS-CoV-2 genomes (Fig. 1B) . This suggests that host factors played a more important role in clinical outcome than viral genetic variation, as shown by emerging clinical studies (6) . The phylogeny of genomes related to case 3 was also consistent with a pattern of international dissemination by air travel (Fig. 1C) .

Data availability. These sequences were deposited in the Global Initiative on Sharing All Influenza Data (GISAID) database (strain identifiers EPI_ISL_430018, EPI_ISL_430063, and EPI_ISL_451957) and in NCBI GenBank under the accession numbers MT510744, MT510745, and MT517419, BioProject number PRJNA634965, BioSample numbers SAMN15013847, SAMN15013848, and SAMN15028143, and SRA numbers SRR11844878, SRR11844877, and SRR11852109.

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We thank all the institutions that shared their SARS-CoV-2 sequences with the GISAID database (a full list of institutions is available from https://github.com/ tommyau/sars-cov-2). We thank our colleagues at the Molecular Pathology Division, Department of Pathology, Hong Kong Sanatorium & Hospital, for their dedicated and professional work on routine laboratory diagnostics.