Coronavirus Detection in the Clinical Microbiology Laboratory

Abstract

Endemic species of coronavirus (HCoV-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1) are frequent causes of upper respiratory tract infections. Three highly pathogenic coronaviruses have been associated with outbreaks and epidemics and have challenged clinical microbiology laboratories to quickly develop assays for diagnosis. Their initial characterization was achieved by molecular methods. With the great advance in metagenomic whole-genome sequencing directly from clinical specimens, diagnosis of novel coronaviruses could be quickly implemented into the workflow of managing cases of pneumonia of unknown cause, which will markedly affect the time of the initial characterization and accelerate the initiation of outbreak control measures.

A Case of Whole Genome Analysis of SARS-CoV-2 Using Oxford Nanopore MinION System

“…Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first reported in December 2019 in Wuhan, China, is an RNA virus with a sequence of approximately 30,000 bases, consisting of open reading frames (ORFs) 1-10 that make 16 non-structural proteins and spike (S), envelope (E), membrane (M), and nucleocapsid (N) that make structural proteins [1] . Genomic analysis of SARS-CoV-2 plays an important role in epidemiological analysis and identification of variants [2] , and a range of nextgeneration sequencing (NGS) instruments are available from Illumina (San Diego, CA, USA) and Oxford Nanopore Technologies (ONT, Oxford, UK) [3][4][5][6][7] .…”

“…A Case of Whole Genome Analysis of SARS-CoV-2 Using Oxford Nanopore MinION System -10-Among the NGS instruments, the ONT MinION is small enough to fit in the palm of the human hand and can be analyzed in real-time using the company's cloud-based program, allowing it to be used in laboratory settings where NGS is not routinely performed. In addition, the ARTIC network in the United Kingdom provides various bioinformatic tools, and there is a system that can be used for genomic analysis of the Ebola virus in Africa, and it is also used for genomic analysis of the Lassa virus, yellow fever virus, influenza virus, and SARS-CoV-2 [1,8] .…”

“…Eleven sequence differences were observed against the reference sequence (Table 1), including T265I, F924, Y3884L, P4715L, L5462, and Q6804L in the ORF1ab region, D614G in the S gene, Q57H in the ORF3a region, and P302 in the N gene, and one gene sequence difference was observed in the 5'-UTR and 3'-UTR, respectively. sequencing (WGS) analysis is useful [1,2,9] . In the case of SARS-CoV-2, it was difficult to characterize the virus in the early stages of the outbreak, but several key variants may have different transmission, prognosis, and vaccine effectiveness, so it is important to understand these variants [2] .…”