The coronavirus SARS-CoV-2 and resulting disease COVID-19 have reached almost every country on Earth… but compared with other infectious diseases such as the flu, there are still many unknowns about SARS-CoV-2. Scientists around the world are tirelessly researching the virus in order to understand it better and endeavor to find treatments. What is currently known about the genomics of SARS-Cov-2 and how is the information used in diagnostics?
What kind of virus is SARS-CoV-2?
SARS-CoV-2 is a RNA virus and belongs to the family of Coronaviridae and the genus Betacoronavirus. Its genome consists of a single-stranded positive-sense RNA. The peculiarity of single-stranded positive-sense RNA is that it can directly function as mRNA in the host cell and, therefore, is directly translated. In contrast, the RNA of a negative-sense single-stranded RNA virus first needs to be converted into positive-sense RNA in order to be translated (Figure 1).
Figure 1: Expression strategies of positive-sense and negative-sense single-stranded RNA viruses.
What are the genes of SARS-CoV-2?
The RNA genome of SARS-CoV-2 is approximately 30,000 nucleotides long and encodes 27 non-structural proteins and 4 structural proteins.
The 27 non-structural proteins include RNA-dependent RNA polymerase (RdRP) that was found to be highly similar to parts of the RdRP gene of coronavirus RaTG13 in bats. Other non-structural proteins are proteases and helicases for instance.
The 4 genes encoding structural proteins that essentially form the envelope of the virus are: 1. the spike surface glycoprotein of the virus (S), 2. the matrix protein (M), 3. the nucleocapsid protein (N), and 3. the envelope protein (E) (Figure 2).
Figure 2: Structure of SARS-CoV-2
The spike proteins on the surface of the virus seem to mediate the binding of the virus to the angiotensin converting enzyme 2 (ACE2) receptor of host cells and the entry into the host cells.
The M protein seems to be involved in formation of the spherical shape of the virus.
The N protein also called nucleoprotein is associated with the single-stranded RNA and together they form the nucleocapsid.
The E protein has been found to be the smallest of the 4 structural proteins.
The genomic composition of SARS-CoV-2 has been analyzed and the sequence is publicly available at the GenBank sequence repository. As of now, 104 strains of SARS-CoV-2 have been isolated and sequenced.
How is SARS-CoV-2 identified?
Molecular techniques have been shown to be highly suitable for the identification of SARS-CoV-2 infections. Specifically real-time RT-PCR (qRT-PCR) is utilized for the analysis of respiratory samples. Here, the RNA of SARS-CoV-2 is extracted from the respiratory samples and subsequently reverse transcribed into complementary DNA (cDNA). The cDNA is then used for real-time PCR amplification with specific primers and probes that target three regions of the viral genome:
- The RdRP gene that is situated in open reading frame ORF1ab.
- The E gene
- The N gene
It has been suggested that the RdRP gene and the E genes show slightly higher analytical sensitivity than the N gene.
For identification purposes, it has been suggested to use assays that target two regions. One primer pair for the identification of a broader range of coronaviruses including SARS-CoV-2, and one primer pair that is specific for SARS-CoV-2:
- The recommendation by a research group at Charité Universitätsmedizin Berlin, Germany, that is the basis of the WHO recommendation, is to first utilize a primer pair that targets different regions of the E gene to detect all SARS-related viruses. In case this leads to amplification, a second analysis with a primer pair that targets different regions of the RdRP gene is performed as confirmatory testing.
- The utilization of a primer pair that targets the N gene and one that targets the ORF1b/RNAse P gene to confirm the results have also been proposed and are recommended by Centers for Disease Control and Prevention (CDC) (Figure 3).
Figure 3: Genomic organization of SARS-CoV-2
qRT-PCR can be performed in a one-step or a two-step assay. The one-step assay with reverse transcription and PCR amplification in one reaction is fast and gives reproducible results for high-throughput analysis:
- The CDC utilizes a one-step qRT-PCR assay to detect a SARS-CoV-2 in samples. Here, primers and a fluorophore-quencher probe Black Hole Quencher-1 (BHQ1) and fluorescein amidite (FAM) are utilized. During amplification the fluorophore-quencher probe is cleaved, which results in the emission of a fluorescent signal.
- Charité Universitätsmedizin Berlin, Germany reported to also utilise a one-step qRT-PCR assay. Here, the probes for the assay contained Blackberry Quencher (BBQ) and 6-carboxyfluorescein (6-FAM).
The two-step assay, where reverse transcription and PCR amplification are performed in different tubes, is generally more sensitive but requires more time.
The utilization of qRT-PCR assays requires positive controls for valid and reliable analyses.
What are the positive controls for SARS-CoV-2 identification by qRT-PCR?
A range of validated positive controls as plasmids that include all genes of SARS-CoV-2 as recommended by the CDC and WHO for example is also provided: Control Plasmids for Coronavirus Research.
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