Synthetic DNA’s Critical Role in Infectious Disease Research

On September 22, 2017, an 11-year-old boy was admitted to the Niger Delta University Teaching Hospital in Nigeria. For more than a week, the boy had been lethargic and feverish with large lesions dotting his body. Though it hadn’t been reported in Nigeria for nearly 40 years, the boy appeared to be infected with the monkeypox virus¹.

 

At the time, researchers knew of two dominant monkeypox viral clades in Africa: a west African clade known to be relatively mild, and the more severe central African (Congo Basin) clade. Previous outbreaks in Nigeria were small flare-ups brought on by west African strains. Patients were often children and there was little to no human-to-human transmission^2,3. This burgeoning outbreak, however, was different.

 

By December of 2017, approximately 200 suspected or confirmed cases had been reported, with many of the patients being young adult men². The rapidly growing case count paired with the abnormal patient demographics suggested that this strain of the monkeypox virus was more transmissible than what Nigeria had seen in the past³.

 

Through the use of next-generation sequencing, researchers from the Nigerian Centers for Disease Control, the U.S. Army Medical Research Institute of Infectious Diseases, and the Institut Pasteur in Dakar, Senegal were able to interrogate the virus’ genome and compile a phylogenetic map⁴. The data indicated this new strain had evolved from the west African clade and was likely the result of zoonotic spillover (animal to human transmission). Unlike previous strains, this virus had gained the ability to spread through bodily fluids, making it far more capable of spreading among humans.

 

 

News of the outbreak eventually faded as case counts dropped. Now, less than four years later, a monkeypox virus strain descended from the 2017-2018 outbreak is making headlines around the world, turning the monkeypox virus–like SARS-CoV-2 before it—into an infamous household name⁵.

 

As many learned during the COVID-19 pandemic, combating infectious diseases like monkeypox requires a myriad of molecular tools, many of which rely on synthetic DNA and RNA. The versatility of synthetic nucleotides allows them to be used in a broad spectrum of applications, from pathogen identification and characterization to therapeutic development. Twist Bioscience is a leader in this space, leveraging our industry-leading DNA synthesis platform to help researchers quickly build the tools they need to address infectious disease outbreaks.

 

Synthetic Nucleotides in Countering Infectious Disease

 

Quantitative real-time PCR (qPCR) has emerged as a powerful diagnostic tool, particularly when dealing with infectious diseases. Generally, these tests work by first collecting a tissue sample from the patient, specifically tissue where the virus is likely to be found (for SARS-CoV-2, mucus is sufficient; for the monkeypox virus, lesion tissue, dry skin, or blood is needed). DNA probes can then be used to target and amplify specific viral genes. As the genes are copied, a signal indicating their presence is also amplified, enabling minuscule amounts of viral DNA or RNA to be sensitively detected.

 

As with any experiment, qPCR assays require positive controls to prove that the test is capable of detecting genetic material from viruses when present. Traditionally, positive controls could be sourced from patient samples that are known to contain the target virus. However, working with a live virus is not only dangerous but is also quickly made obsolete. SARS-CoV-2 continues to remind us that viruses evolve rapidly and the mutations they accrue can cause PCR probes to no longer amplify the viruses’ RNA.

 

Synthetic nucleotides are a safer, more robust option. Twist Bioscience has the ability to precisely synthesize viral genes that can be used as positive controls during test development. These controls can be easily iterated, customized, and rapidly synthesized, enabling researchers to quickly update their tests as new viral variants arise.

 

 

Accuracy is a critical feature of synthetic controls. Small errors in DNA or RNA sequences can reduce the affinity between PCR probes and the synthetic control strand, resulting in a diminished signal. When this happens, test results may be rendered invalid. It is critical, then, that positive controls be precisely engineered.

 

Twist has leveraged its industry-leading DNA synthesis platform to produce synthetic RNA controls for most of the major SARS-CoV-2 viral strains. Similarly, Twist has recently announced synthetic DNA controls for the monkeypox virus. Rather than targeting an individual strain, Twist has produced controls that recognize viral strains from both the West African and Congo Basin clades.

 

Importantly, the dominant strain of monkeypox virus making its way around the world appears to have mutated much faster than expected, leaving open the possibility that the virus will continue to evolve at a rapid pace. Tests will need to keep up with this evolution by producing probes and controls that reflect new mutations as they arise, or by integrating new target genes for amplification. Twist’s monkeypox viral controls cover over 80% of the full monkeypox viral genome and are sequence-verified using both NGS and ddPCR. This means that, as tests evolve to include additional target genes over time, Twist monkeypox viral controls remain as valuable control samples.

 

Beyond Controls

 

In addition to synthetic controls, Twist also produces fixed and custom content target enrichment panels. Probes in these panels target and hybridize to viral DNA, enabling the isolation and sequencing of viral genomes from challenging tissue samples (such as blood and lesion tissue). Using this target enrichment approach, researchers can focus their sequencing resources on just the viral genes they’re interested in, enabling much more efficient sequencing. Additionally, enrichment panels can be designed to target specific genes from a large breadth of potential pathogens.

 

With this breadth, researchers can simultaneously test for multiple viruses (or viral strains) when they’re unsure which pathogen is affecting patients. Such a panel was used to shed light on the origin of the monkeypox viral strain that was responsible for the 2017-2018 outbreak⁴.

 

 

Currently, Twist offers both a comprehensive viral panel as well as a more focused respiratory virus panel that targets reference sequences for 29 common human respiratory viruses. However, custom content can also be added to these panels to ensure coverage over specific viruses or variants (Twist provides design support to build custom panels from scratch).

 

We’re hopeful that vaccines and improved public health measures will help to quell the SARS-CoV-2 pandemic and monkeypox virus outbreak. However, we also know that viral outbreaks are inevitable. Researchers will always need cutting-edge tools to help identify and mitigate these outbreaks when they happen, and Twist is committed to empowering their efforts with high-quality synthetic DNA and RNA.

 

References

1. Ogoina, Dimie, et al. “The 2017 Human Monkeypox Outbreak in Nigeria—Report of Outbreak Experience and Response in the Niger Delta University Teaching Hospital, Bayelsa State, Nigeria.” PLOS ONE, vol. 14, no. 4, 17 Apr. 2019, p. e0214229, 10.1371/journal.pone.0214229.
2. Yinka-Ogunleye, Adesola, et al. “Outbreak of Human Monkeypox in Nigeria in 2017–18: A Clinical and Epidemiological Report.” The Lancet Infectious Diseases, vol. 19, no. 8, 1 Aug. 2019, pp. 872–879, 10.1016/S1473-3099(19)30294-4.
3. “He Discovered the Origin of the Monkeypox Outbreak — and Tried to Warn the World.” NPR.org, www.npr.org/sections/goatsandsoda/2022/07/28/1114183886/a-doctor-in-nigeria-tried-to-warn-the-world-that-monkeypox-had-become-a-global-t.
4. Faye, Ousmane, et al. “Genomic Characterisation of Human Monkeypox Virus in Nigeria.” The Lancet Infectious Diseases, vol. 18, no. 3, 1 Mar. 2018, p. 246, 10.1016/S1473-3099(18)30043-4.
5. Isidro, Joana, et al. “Phylogenomic Characterization and Signs of Microevolution in the 2022 Multi-Country Outbreak of Monkeypox Virus.” Nature Medicine, 24 June 2022, 10.1038/s41591-022-01907-y.