Clinical Microbiology
Announcement
15 April 2021

Genome Sequences of Three SARS-CoV-2 ORF7a Deletion Variants Obtained from Patients in Hong Kong

ABSTRACT

We report the genome sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains from the clinical samples of three coronavirus disease 2019 (COVID-19) patients in Hong Kong. All the genome sequences showed a 370-nucleotide deletion resulting in the complete loss of ORF7a.

ANNOUNCEMENT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel member of the genus Betacoronavirus in the family Coronaviridae and the causative agent of coronavirus disease 2019 (COVID-19). More than 100 million cases of infection had been reported globally by the end of January 2021 (1). In this report, we present the genome sequences of SARS-CoV-2 strains from 3 COVID-19 patients in Hong Kong obtained during investigation of a local cluster in mid-January 2021. The study protocol was approved by the institutional review board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster (UW 13-372).
Saliva specimens were collected by patients as described previously (2). Total nucleic acid extraction was performed using the MagNA Pure 96 DNA and viral NA small-volume kit (Roche, Switzerland). Tiling PCR for specific preamplification of the SARS-CoV-2 genome was performed using the ARTIC v3 primer set (https://github.com/artic-network/artic-ncov2019/tree/master/primer_schemes/nCoV-2019/V3) according to the specified protocol (https://www.protocols.io/view/ncov-2019-sequencing-protocol-v3-locost-bh42j8ye). Library preparation for multiplexed sequencing, with up to 32 samples per run, was performed using IDT for Illumina DNA/RNA unique dual (UD) indexes and the Illumina DNA prep kit (California, USA). Sequencing of the pooled library was performed on an iSeq 100 sequencer (Illumina) to obtain paired-end 150-bp reads.
Fastp version 0.21.0 (3) was used for quality control and preprocessing of demultiplexed read data. Subsequent processing was performed according to the Utah Department of Health ARTIC/Illumina Bioinformatic Workflow (https://github.com/CDCgov/SARS-CoV-2_Sequencing/tree/master/protocols/BFX-UT_ARTIC_Illumina), except for the use of bwa-mem2 instead of bwa for mapping reads. Briefly, reads were mapped to a reference SARS-CoV-2 genome sequence (GenBank accession number MN908947.3) with bwa-mem2 version 2.0 (4), and primer trimming was performed using iVar version 1.3 (5). Variant calling and consensus sequence generation were performed using SAMtools version 1.10 (6). The genome assembly was visualized and examined using Tablet version 1.20.12.24 (7).
The sequencing statistics and genomic features of the genome assemblies are summarized in Table 1. All 3 strains belonged to the PANGO lineage B.1.36, as classified by Pangolin version 2.3.0 (lineages version 2021-02-21) (https://github.com/cov-lineages/pangolin). In comparison, most local cases in Hong Kong since September 2020 have been caused by viruses from lineage B.1.36.27 (https://cov-lineages.org/lineages/lineage_B.1.36.27.html). Notably, the present sequences contained a 370-nucleotide deletion spanning positions 27387 to 27756 of the reference SARS-CoV-2 genome. This major deletion was confirmed independently by long-range PCR and Sanger sequencing (with primers 5′-AACTCGTAATCGGAGCTGTGA-3′ and 5′-TGTCATTCTCCTAAGAAGCTATTAAA-3′). As a result, the entire ORF7a and the original stop codon for ORF6 were lost. The latter resulted in an extension of the Orf6 protein by 2 amino acids. Furthermore, ORF7b was potentially extended in the 5′ direction by 27 nucleotides due to the occurrence of an in-frame start codon.
TABLE 1
TABLE 1 Sequencing statistics and features of the SARS-CoV-2 genome assembliesa
StrainSequence length (nucleotides)G+C content (%)Mean read depthb (×)Genomic coverageb (%)PANGO lineageSpecific nucleotide (and amino acid) substitution(s)b,cOther genomic featureb
HKCH_93229,42838.01,107.498.8B.1.36C11563T, C17590T370-nucleotide deletion from reference genome position 27387 to 27756
HKCH_93329,42838.01,046.698.8B.1.36 370-nucleotide deletion from reference genome position 27387 to 27756
HKCH_93429,42838.01,176.598.8B.1.36C11563T370-nucleotide deletion from reference genome position 27387 to 27756
a
Nucleotide (and amino acid) substitutions common to all three strains (mapped against the genome sequence of reference SARS-CoV-2 strain Wuhan-Hu-1; GenBank accession number MN908947.3) include C241T, C3037T, C4276T, C5339T (nsp3 P874S), C5812T, C6027T (nsp3 P1103L), C10039T, C10456T, C14408T (nsp12 P323L), C18877T, C19572T, C20844T, C22444T, T22882G (spike N440K), A23403G (spike D614G), G23587T (spike Q675H), G25563T (orf3a Q57H), G25785T (orf3a W131C), C26625T, C26735T, G28690T (nucleocapsid L139F), and C28854T (nucleocapsid S194L). The nucleotide substitution is synonymous or in a noncoding region if no accompanying amino acid substitution is shown.
b
When mapped against the genome sequence of reference SARS-CoV-2 strain Wuhan-Hu-1 (GenBank accession number MN908947.3).
c
The nucleotide substitution is synonymous or in a noncoding region if no accompanying amino acid substitution is shown.
Various major ORF7a deletions, sometimes alongside other major deletions, have been reported previously (810). The repeated reemergence of ORF7a deletion variants may suggest that the accessory protein could impose a fitness cost that exceeds the benefits of its canonical function in certain situations.

Data availability.

These genome sequences were deposited in GenBank under accession numbers MW691225, MW691226, and MW691227. The raw reads were deposited in SRA under accession numbers SRR13775065, SRR13775066, and SRR13775067.

ACKNOWLEDGMENTS

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. We thank the Hospital Authority and the Food and Health Bureau for their administrative and policy support.

REFERENCES

1.
World Health Organization. 2021. Weekly epidemiological update—2 February 2021. World Health Organization, Geneva, Switerland. https://www.who.int/publications/m/item/weekly-epidemiological-update---2-february-2021.
2.
Wong SCY, Tse H, Siu HK, Kwong TS, Chu MY, Yau FYS, Cheung IYY, Tse CWS, Poon KC, Cheung KC, Wu TC, Chan JWM, Cheuk W, Lung DC. 2020. Posterior oropharyngeal saliva for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clin Infect Dis 71:2939–2946.
3.
Chen S, Zhou Y, Chen Y, Gu J. 2018. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34:i884–i890.
4.
Vasimuddin M, Misra S, Li H, Aluru S. 2019. Efficient architecture-aware acceleration of BWA-MEM for multicore systems, p 314–324. In 2019 IEEE International Parallel and Distributed Processing Symposium (IPDPS). IEEE, Rio de Janeiro, Brazil.
5.
Grubaugh ND, Gangavarapu K, Quick J, Matteson NL, De Jesus JG, Main BJ, Tan AL, Paul LM, Brackney DE, Grewal S, Gurfield N, Van Rompay KKA, Isern S, Michael SF, Coffey LL, Loman NJ, Andersen KG. 2019. An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar. Genome Biol 20:8.
6.
Li H. 2011. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics 27:2987–2993.
7.
Milne I, Stephen G, Bayer M, Cock PJA, Pritchard L, Cardle L, Shaw PD, Marshall D. 2013. Using Tablet for visual exploration of second-generation sequencing data. Brief Bioinform 14:193–202.
8.
Addetia A, Xie H, Roychoudhury P, Shrestha L, Loprieno M, Huang M-L, Jerome KR, Greninger AL. 2020. Identification of multiple large deletions in ORF7a resulting in in-frame gene fusions in clinical SARS-CoV-2 isolates. J Clin Virol 129:104523.
9.
Holland LA, Kaelin EA, Maqsood R, Estifanos B, Wu LI, Varsani A, Halden RU, Hogue BG, Scotch M, Lim ES. 2020. An 81-nucleotide deletion in SARS-CoV-2 ORF7a identified from sentinel surveillance in Arizona (January to March 2020.). J Virol 94:e00711-20.
10.
Tse H, Lung DC, Wong SC-Y, Ip K-F, Wu T-C, To KK-W, Kok K-H, Yuen K-Y, Choi GK-Y. 2021. Emergence of a severe acute respiratory syndrome coronavirus 2 virus variant with novel genomic architecture in Hong Kong. Clin Infect Dis:ciab198.

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cover image Microbiology Resource Announcements
Microbiology Resource Announcements
Volume 10Number 1515 April 2021
eLocator: e00251-21
Editor: John J. Dennehy, Queens College CUNY
PubMed: 33858933

History

Received: 4 March 2021
Accepted: 23 March 2021
Published online: 15 April 2021

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Contributors

Authors

Herman Tse
Department of Pathology, Hong Kong Children’s Hospital, Hong Kong Special Administrative Region, China
Sally Cheuk-Ying Wong
Department of Pathology, Hong Kong Children’s Hospital, Hong Kong Special Administrative Region, China
Ka-Fai Ip
Department of Pathology, Hong Kong Children’s Hospital, Hong Kong Special Administrative Region, China
Vincent Chi-Chung Cheng
Department of Microbiology, Queen Mary Hospital, Hong Kong Special Administrative Region, China
State Key Laboratory of Emerging Infectious Diseases, Carol Yu Centre for Infection, Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, China
Department of Microbiology, The University of Hong Kong, Hong Kong Special Administrative Region, China
David Christopher Lung [email protected]
Department of Pathology, Hong Kong Children’s Hospital, Hong Kong Special Administrative Region, China
Department of Pathology, Queen Elizabeth Hospital, Hong Kong Special Administrative Region, China
Garnet Kwan-Yue Choi [email protected]
Department of Pathology, Hong Kong Children’s Hospital, Hong Kong Special Administrative Region, China

Editor

John J. Dennehy
Editor
Queens College CUNY

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