key: cord-307213-i8yijbiu
authors: Ip, Jonathan Daniel; Kok, Kin-Hang; Chan, Wan-Mui; Wing-Ho Chu, Allen; Wu, Wai-Lan; Chik-Yan Yip, Cyril; To, Wing-Kin; Tak-Yin Tsang, Owen; Leung, Wai-Shing; Shiu-Hong Chik, Thomas; Chan, Kwok-Hung; Fan-Ngai Hung, Ivan; Yuen, Kwok-Yung; Kai-Wang To, Kelvin
title: Intrahost non-synonymous diversity at a neutralising antibody epitope of SARS-CoV-2 spike protein N-terminal domain
date: 2020-11-02
journal: Clin Microbiol Infect
DOI: 10.1016/j.cmi.2020.10.030
sha: 
doc_id: 307213
cord_uid: i8yijbiu

OBJECTIVES: SARS-CoV-2 has evolved rapidly into several genetic clusters. However, data on mutations during the course of infection are scarce. This study aims to determine viral genome diversity in serial samples of COVID-19 patients. METHODS: Targeted deep sequencing of spike gene was performed on serial respiratory specimens from COVID-19 patients using nanopore and Illumina sequencing. Sanger sequencing was then performed to confirm the single nucleotide polymorphisms. RESULTS: A total of 28 serial respiratory specimens from 12 patients were successfully sequenced using nanopore and Illumina sequencing. A 75-year-old patient with severe disease had a mutation, G22017T, identified in the second specimen. The frequency of G22017T increased from ≤5% (nanopore: 3.8%; Illumina: 5%) from first respiratory tract specimen (sputum) to ≥60% (nanopore: 67.7%; Illumina: 60.4%) in the second specimen (saliva; collected 2 days after the 1(st) specimen). The difference in G22017T frequency was also confirmed by Sanger sequencing. G22017T corresponds to W152L amino acid mutation in the spike protein which was only found in <0.03% of the sequences deposited into a public database. Spike amino acid residue 152 is located within the N-terminal domain, which mediates the binding of a neutralizing antibody. CONCLUSIONS: A spike protein amino acid mutation W152L located within a neutralizing epitope has appeared naturally in a patient. Our study demonstrated that monitoring of serial specimens is important in identifying hotspots of mutations, especially those occurring at neutralizing epitopes which may affect the therapeutic efficacy of monoclonal antibodies.

A total of 28 serial respiratory specimens from 12 patients were successfully sequenced 49 using nanopore and Illumina sequencing. A 75-year-old patient with severe disease had a 50 mutation, G22017T, identified in the second specimen. The frequency of G22017T increased 51 from ≤5% (nanopore: 3.8%; Illumina: 5%) from first respiratory tract specimen (sputum) to 52 ≥60% (nanopore: 67.7%; Illumina: 60.4%) in the second specimen (saliva; collected 2 days after 53 the 1 st specimen). The difference in G22017T frequency was also confirmed by Sanger 54 sequencing. G22017T corresponds to W152L amino acid mutation in the spike protein which 55 was only found in <0.03% of the sequences deposited into a public database. Spike amino acid 56 residue 152 is located within the N-terminal domain, which mediates the binding of a 57 neutralizing antibody. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly, 66 resulting in more than 28 million laboratory-confirmed COVID-19 cases globally as of 67 September 14, 2020. SARS-CoV-2 mainly causes respiratory tract infection, although 68 extrapulmonary manifestations have been reported [1] . The efficient person-to-person 69 transmission may be related to the high viral load shortly after symptom onset and the large 

As an RNA virus, the genome replication of SARS-CoV-2 is prone to error, and gene 73 mutations arise frequently. Whole genome sequencing showed that the viral genomes may differ 74 between family members [4] . Major genetic diversity has already been seen [5] . Phylogenetic 75 analysis has demonstrated that even patients within the same geographical region were infected 76 with genetically-diverse SARS-CoV-2 strains [6] . previously [4] . Full-length spike gene was amplified using SuperScript™ III one-step RT-PCR system with Platinum™ Taq High Fidelity DNA polymerase (Thermo Fisher Scientific, 111 Waltham, MA, USA) using primer set 1, or primer set 2, and 3 (Supplementary Table S1) Illumina adapters were removed from the reads. Any reads with length of at least 100 bp 158 and at least 90% of bases with quality score of ≥30 were retained during the quality filtering 159 process using FASTP [21] . Pair-end reads were aligned with the reference genome SARS-CoV-2 

We are grateful to the Centre for PanorOmic Sciences (CPOS) of the University of Hong 295 Kong for providing Illumina sequencing. We gratefully acknowledge the originating and 296 submitting laboratories who contributed sequences to GISAID (Supplementary Table S5 

Gastrointestinal 306 Manifestations of SARS-CoV-2 Infection and Virus Load in Fecal Samples from the Hong Kong 307 Cohort and Systematic Review and Meta-analysis

Temporal profiles of 310 viral load in posterior oropharyngeal saliva samples and serum antibody responses during 311 infection by SARS-CoV-2: an observational cohort study

SARS-CoV-2 shedding 314 and seroconversion among passengers quarantined after disembarking a cruise ship: a case 315 series

MinIONQC: fast and 352 simple quality control for MinION sequencing data

Fast and accurate long-read alignment with Burrows-Wheeler transform

BCFtools/RoH: a 356 hidden Markov model approach for detecting autozygosity from next-generation sequencing 357 data

Alignment/Map format and SAMtools

VarScan 2: 362 somatic mutation and copy number alteration discovery in cancer by exome sequencing

fastp: an ultra-fast all-in-one FASTQ preprocessor

High prevalence of four 367 novel astrovirus genotype species identified from rodents in China

A neutralizing human antibody 370 binds to the N-terminal domain of the Spike protein of SARS-CoV-2

Broad 373 neutralization of SARS-related viruses by human monoclonal antibodies

Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes 377 and Recurrent Features of Antibodies

Human 379 coronaviruses OC43 and HKU1 bind to 9-O-acetylated sialic acids via a conserved receptor-380 binding site in spike protein domain A

CoV spike glycoprotein in complex with sialoside attachment receptors

Structure of mouse coronavirus 385 spike protein complexed with receptor reveals mechanism for viral entry

Nextstrain: 388 real-time tracking of pathogen evolution

Triple combination of 390 interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to 391 hospital with COVID-19: an open-label, randomised, phase 2 trial

Saliva or Nasopharyngeal Swab Specimens for Detection of SARS-CoV-2

Viral load dynamics and disease 397 severity in patients infected with SARS-CoV-2 in Zhejiang province, China