Journal:Laboratory testing methods for novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

Full article title	Laboratory testing methods for novel Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2)
Journal	Frontiers in Cell and Developmental Biology
Author(s)	D'Cruz, Roshan J.; Currier, Arthur W.; Sampson, Valerie B.
Author affiliation(s)	Nemours/Alfred I. duPont Hospital for Children
Primary contact	Email: valerie dot sampson at nemours dot org
Year published	2020
Volume and issue	8
Article #	468
DOI	10.3389/fcell.2020.00468
ISSN	2296-634X
Distribution license	Creative Commons Attribution 4.0 International
Website	https://www.frontiersin.org/articles/10.3389/fcell.2020.00468/full
Download	https://www.frontiersin.org/articles/10.3389/fcell.2020.00468/pdf (PDF)

Abstract

Following the first reports of coronavirus disease 2019 (COVID-19) by China to the World Health Organization (WHO) on December 31, 2019, more than 4,302,774 cases of infection by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus have been reported by authorities in 212 countries and territories as of May 12, 2020. The outbreak and spread of COVID-19 worldwide highlights the critical need for developing rapid and accurate diagnostic testing methods for emerging human coronavirus (CoV) infections. Testing is crucial to tracking the spread of disease during a pandemic, and to swiftly permitting public health interventions, including isolation, quarantine, and appropriate clinical management of afflicted individuals. The key components of viral diagnostic tests are (1) collection of the appropriate sample (blood, nasal swab, and throat swab); (2) availability of the genetic and proteomic sequences of the novel virus for analysis; and (3) rapid and accurate laboratory testing methods. The current gold standard for the molecular diagnosis of SARS-CoV-2 infection is the real-time reverse transcription polymerase chain reaction (qRT-PCR) for the qualitative and quantitative detection of viral nucleic acids. Other relevant laboratory methods include enzyme-linked immunosorbent assays (ELISA) for viral antibody and antigen detection, and serum virus neutralization (SVN) assays for antibody neutralization determination. The challenges faced in developing a diagnostic test for a novel pathogen are the ability to measure low viral loads for early detection, to provide low or no cross-reactivity with other viral strains, and to deliver results rapidly. Several point-of-care molecular devices are currently being integrated for fast and accurate diagnosis of SARS-CoV-2 infections. This review discusses the current laboratory methods available to test for coronaviruses by focusing on the present COVID-19 outbreak.

Keywords: coronavirus, RT-PCR, ELISA, lateral flow diagnostics, convalescent plasma

Introduction

Coronavirus disease 2019 (COVID-19) is caused by a novel coronavirus (CoV) that was originally reported in Wuhan, Hubei province, China in December 2019. The International Committee on Taxonomy of Viruses named the virus "severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infection by SARS-CoV-2 causes a respiratory illness that varies in severity from mild upper respiratory symptoms akin to the seasonal flu, to severe progressive respiratory failure that requires intensive care and can lead to death. Asymptomatic carriers of the virus have also been reported and pose a significant public health threat due to their ability to unknowingly spread the virus. SARS-CoV-2 represents the third coronavirus in this millennium to cross species from animals to humans and cause a severe respiratory disease, after Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 and severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003. SARS-CoV-2 has now been identified as the seventh coronavirus that is transmissible between humans (a group which also includes HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1). On January 30, 2020, the World Health Organization (WHO) declared the SARS-CoV-2 epidemic a public health emergency of international concern, and the public health emergency was upgraded to a pandemic on March 11. At least 4,302,774 confirmed cases and 289,561 deaths worldwide were reported as of May 12. Diagnostic testing is critical during a pandemic as the ability to track the spread of SARS-CoV-2 is essential for effective disease management and control.

SARS-CoV-2 is a positive-sense, single-stranded RNA (ssRNA), group IV virus. The genome was sequenced from the bronchoalveolar lavage fluid of a patient (Genbank: MN908947) and shared through the Global Initiative on Sharing All Influenza Data (GISAID) platform on January 12, 2020. The ~30 k base pair genome is highly similar to the human SARS-CoV and bat CoV-SARS-like genomes, with 14 open reading frames (ORFs) that encode structural, replication, and non-structural accessory proteins, as depicted in Figure 1. Molecular modeling studies demonstrate that like SARS-CoV, SARS-CoV-2 is surrounded by a lipid bilayer membrane, containing structural membrane (M) and envelope (E) proteins that interact to form the viral envelope. This layer also contains spike glycoproteins (S) that give the characteristic “corona” appearance of this family of viruses. The spike proteins bind specific host cell receptors to facilitate host cell attachment and entry. The nucleic acid-associated protein binds the RNA genome and forms the nucleocapsid (N). Other proteins include replication and non-structural accessory proteins, listed in Table 1. Reports of different strains of SARS-CoV-2 suggest an early split from the SARS-CoV-2 lineage and/or that the virus is mutating. Ongoing research provides insight into the unique and conserved features of the genome and proteome of SARS-CoV-2 for tracking mutations and generates evidence about the evolution of the virus. This is important as these changes may affect key structural and non-structural components of SARS-CoV-2 that can render some diagnostic tests ineffective or less sensitive and can also impact the selection of epitopes for the development of new tests.

Figure 1. Schematic representation of SARS-CoV-2 genome. SARS-CoV-2 contains a positive-sense, positive-stranded mRNA genome with a 5′ capped mRNA sequence (C) and a 3′ poly-A tail. The coding genes are: ORF1a, ORF1b, Spike (S), ORF3a, ORF3b, Envelope (E), Membrane (M), ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF14, Nucleocapsid (N), and ORF10.

Table 1. Proteins associated with the 14 ORFs of SARS-CoV.

Notes

This presentation is faithful to the original, with only a few minor changes to presentation. In some cases important information was missing from the references, and that information was added. References in this version are listed in order of appearance—by design—rather than alphabetical order as the original was.