Why S-Gene Sequencing is Key for SARS-CoV-2 Surveillance

This blog represents the most up-to-date information about SARS-CoV-2 surveillance as of February 05 2021.

The Infectious Disease Research blog series provides perspectives on the value of genomics across the spectrum of pathogen research, host response, epidemiological studies and vaccine/therapeutic discovery and production to advance research in this area. To talk with our team on implementing your genetic-based initiatives, please contact us or browse our infectious disease research solutions.

Global epidemiological surveillance is vital for understanding the evolution of viral pathogens and monitoring for changes in transmissibility, virulence, and disease pathology. Molecular methods such as RT-PCR and Next Generation Sequencing (NGS) allow rapid and extensive laboratory testing to determine SARS-CoV-2 prevalence in the population. However, the emergence of new strain lineages arising from genomic mutation means that sequencing is key for SARS-CoV-2 surveillance. As scientists track virus evolution, they need to understand that not only can mutations affect virus behavior and infectivity, but they may also impact testing.

SARS-CoV-2 S-Gene and Virus Behavior

The S-gene encodes a surface protein¹, the spike protein, which is a homotrimeric glycoprotein complex essential for infectivity. The complex is made up of two subdomains; S1 contains a receptor binding domain (RBD) with high affinity for mammalian ACE2 or angiotensin converting enzyme 2. This cell surface protein modulates the activity of angiotensin II, a vasoactive polypeptide hormone active throughout the body.

When the S1 subdomain binds to ACE2, cleavage at the S1-S2 site facilitates SARS-CoV-2 entry to infect the host cell. Since the spike protein is on the virus surface, it plays a role in host immune response and also makes an excellent target for novel therapeutic strategies and vaccine development.

S-Gene Mutation, Virus Behavior and Public Health

Part of the current challenge is that viruses all show genomic mutation over time. RNA viruses, including coronaviruses, are no exception, though the rate of mutation may be slower for SARS-CoV-2². Although this may seem reassuring, the number of infections worldwide raises the chances that novel strains will appear in circulation.

Regardless of the rate of change, surveillance shows that mutations in the S-gene are more common³, which affects not only virus infectivity but also detection. Analysis of recent outbreaks has borne this out.

Several new strain lineages with documented increases in transmissibility and infectivity were identified in late 2020. This recent CDC review⁴ from mid-January lists three strains of concern to the United States:

• 1.1.7 lineage (a.k.a. 20I/501Y.V1 Variant of Concern (VOC) 202012/01) emerged in the United Kingdom in September 2020 and has been recorded in the US⁵.
• 1.351 lineage (a.k.a. 20H/501Y.V2), identified in October 2020, has spread out of South Africa recently⁶.
• 1 lineage (a.k.a. 20J/501Y.V3) was detected in Japan in late 2020 in travelers from Brazil.

In addition, there are recent reports that a new strain is emerging in southern California⁷.

Mutations in the S-gene are common to all these new strains. Research is ongoing to elucidate the mechanisms involved but the mutations may boost infectivity and transmissibility through increased spike density, enhanced cleavage and host cell uptake, increased viral load and ability to evade host immune responses⁸.

Download our white paper: “Overview of the pathology, pathogenicity, and epidemiology of SARS-CoV-2”

Impact on Laboratory Detection and Surveillance

Mutations may also interfere with laboratory testing and complicate epidemiological monitoring by interfering with detection in sequence-based tests.

Gene dropout is a situation wherein if a mutation occurs in the part of the virus’s genome assessed by a PCR test, the sample may result in gene “dropout”, as seen in the B.1.1.7 variant.  It is important for scientists to fully understand that not only must laboratory testing pick up all variants, but analysis also needs to characterize strain mutations.

Since mutations may affect treatment and prevention, it is important to identify strains correctly to direct public health containment strategies.

Sequencing Surveillance Solutions

While an NGS approach is useful for fast and widespread surveillance, Sanger sequencing provides a rapid method for tracking changes in S-gene sequences that fully characterizes new strains. Using primers with overlapping amplicons to give complete sequence coverage of the S-gene, the protocol developed for the Applied Biosystems BigDye Direct Cycle Sequencing Kit and BigDye XTerminator Purification Kit streamlines the sequencing process, providing data in about four hours.

Since each amplicon is several hundred bases long, any novel mutations outside the canonical sequence can be quickly identified. The panel of amplicons is also designed to be flexible; researchers can pick and choose primer pairs listed in the protocol that best suit their research needs, instead of having to use the entire primer set.

Because the importance of SARS-CoV-2 mutation in global spread is not yet well understood⁹, monitoring genomic changes in the S-gene is even more essential. This will not only track the spread of new variants for clues to the evolution of the virus but also play a central role in proactively managing pathogens..

For Research Use Only. Not for use in diagnostic procedures.

Find out more about our Sanger sequencing solutions for SARS-CoV-2 research

Find out more about our NGS solutions for SARS-CoV-2 research

If you have additional questions or would like to discuss your specific situation, please contact our technical support team at www.thermofisher.com/contactus.

Resources

• Quickly confirm S-gene dropouts seen with RT-PCR assays by identifying if the S gene 69/70 mutation is present.

Download Protocol I Order Primers

• Flexible protocol to confirm the presence of the highly transmissible SARS-CoV-2 B.1.1.7 and B.1.351 or B.1.1.28 strain lineages in your sample. Refer to the protocol to select primers pairs that best suit your needs.

Download Protocol I Download primer sequences for B.1.1.7 I Download primer sequences for B.1.351 | Download primer sequences for B.1.1.28

• Protocol to identify new variants in the Spike gene by sequencing the entire S-gene for vaccine development and epidemiological research.

Download Protocol I Download primer sequences

References

1. (Lan J. et al.  Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature May;581(7807):215-220. https://www.nature.com/articles/s41586-020-2180-5  doi: 10.1038/s41586-020-2180-5. Epub 2020 Mar 30. PMID: 32225176. (2020)
2. https://www.frontiersin.org/articles/10.3389/fimmu.2020.576622/full
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324922/
4. https://www.cdc.gov/coronavirus/2019-ncov/more/science-and-research/scientific-brief-emerging-variants.html
5. https://www.medrxiv.org/content/10.1101/2021.01.18.21249786v1
6. Wise J., Covid-19: New coronavirus variant is identified in UK. BMJ Dec 16;371:m4857. doi: 10.1136/bmj.m4857. (2020)
7. Tegally H., et al. Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa. medRxiv Dec 2020. doi: https://doi.org/10.1101/2020.12.21.20248640 (2020)
8. https://www.nature.com/articles/s41467-020-19808-4
9. https://www.nature.com/articles/d41586-020-02544-6