During the beginning of this year, a novel coronavirus disease — termed Covid-19 by the WHO — was identified as being responsible for a sudden increase in cases of pneumonia in hospitalized patients. We have seen this rapidly escalate into a global crisis with this new coronavirus named severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2. As of February 25, 2020, a total of 81,109 laboratory-confirmed cases had been documented globally (1). A recent paper in the New England Medical Journal describes how SARS-CoV-2 was initially identified and characterized (2).
Coronaviruses are positive-sense single-stranded RNA viruses ((+)ssRNA virus), meaning the viral RNA genome can serve as messenger RNA (mRNA) and can be directly translated into protein in the host cell. Other examples of (+)ssRNA viruses include hepatitis C virus, West Nile virus, dengue virus, rhinoviruses that cause the common cold as well as SARS (Severe acute respiratory syndrome) and MERS (Middle East respiratory syndrome) (3-5). While SARS-CoV and MERS-CoV can cause fatality, the other 4 known coronaviruses — 229E, OC43, NL64 and HKU1 — typically manifest as a common cold (6). However, given the prevalence of coronaviruses and their ability to undergo genetic recombination combined with ever increasing human-animal interaction, zoonosis is likely to occur from time to time as we have witnessed and are witnessing now.
A link between seafood and animal wholesale markets in Wuhan, China, alerted the Chinese Center for Disease Control and Prevention (China CDC) to a potential outbreak, and they rapidly implemented an investigation to identify the source. Following isolation of virus from supernatants of lung samples and infection of cultured human airway epithelial cells as well as permissive Vero E6 and Huh-7 cell lines, cytopathic effects were observed. RT-PCR confirmed the presence of viral nucleic acid in the supernatant. Electron micrography (EM) of negative-stained virus revealed spherical particles varying in diameter (60 to 140 nm) with distinctive spikes. This and the presence of virus particles engulfed in cytoplasmic membranes, is consistent with the morphology of the Coronaviridae family, which includes coronavirus.
Respiratory tract samples from patients, analysed by PCR, tested negative for 22 known pathogens including members of the coronavirus family. In order to identify the pathogen, a combination of Illumina and nanopore sequencing together with 5’ and 3’ RACE (rapid amplification of cDNA ends) was employed using RNA from lung samples as a template for cloning and contig assembly. Multiple-sequence alignment and phylogenetic analysis revealed an 85% identity to the genome from lineage B of the genus betacoronavirus (β-CoV); namely a bat SARS-like CoV (bat-SL-CoVZC45, MG772933.1). Additional confirmation that the pathogen was an RNA-virus was obtained by RT-PCR targeting a the RdRp sequence common to β-CoV. However, while the genome contained typical β-CoV sequences including 5’UTR, replicase complex, (orf1ab), S gene, E gene, M gene, N gene and 3′ UTR, several unidentified open reading frames were evident. The virus was named SARS-CoV-2 and confirmed as a novel β-CoV belonging to the sarbecovirus subgenus of Coronaviridae family.
In summary, through the use of several molecular biology techniques and unbiased DNA sequencing, the above describes the initial sampling and subsequent identification of the novel betacoronavirus — SARS-CoV-2. While the outcome of Covid-19 is currently unclear, a rapid approach as taken here to identify the pathogen is essential when responding to new infectious disease outbreaks and ultimately improves our chances of preventing a pandemic and developing an effective vaccine.
VectorBuilder is carefully following the Covid-19 situation. To date, we are pleased to say that we have not experiecned any major delays in production and hope for a speedy end to this unfortunate crisis.
1. Guan, W. et al. Clinical Characteristics of Coronavirus Disease 2019 in China , N Engl J Med. 2020. February 28; DOI: 10.1056/NEJMoa2002032.
2. Zhu, N. et al. A Novel Coronavirus from Patients with Pneumonia in China, 2019 , N Engl J Med. 2020. February 20; 382:727-733.
3. Nagy, P et al. The dependence of viral RNA replication on co-opted host factors , Nature Reviews Microbiology. 2011; 10 (2): 137–149.
4. Ahlquist, P. et al. Host Factors in Positive-Strand RNA Virus Genome Replication , Science. 2014;346(6213):1258096.
5. Modrow, S. et al. Viruses with Single-Stranded, Positive-Sense RNA Genomes , Molecular virology. 2013; pp. 185–349.
6. Su, S. et al. Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses , Trends Microbiol. 2016; 24:490-502.