by Steven Henck, PhD, Vice President, R&D, Integrated DNA Technologies
Globally, acute respiratory infections are responsible for about four million deaths every year. It is estimated that 80 percent of these infections are caused by viruses, especially influenza viruses, respiratory syncytial viruses (RSV), parainfluenza viruses, and human rhinoviruses.1 More recently, the world has been brought to a near-standstill with emerging respiratory viruses, including the most recent SARS-CoV-2 virus, with its devastating human and economic effects.
Respiratory virus season in the Northern Hemisphere typically occurs from October to March. However, these viruses aren’t strictly seasonal, as COVID-19 demonstrated, and can arise at any time of the year. But even with COVID, seasonality is beginning to set in, as the disease is expected to come back stronger in the fall and winter. The seasonal occurrence of respiratory viruses is typically a consequence of environmental factors and human behavior. Recent studies suggest a primary role for environmental factors, particularly temperature and humidity, on stabilizing respiratory viruses and modulating host immune response in the respiratory tract2. Some of these viruses, including the flu virus, can cause severe disease, particularly in infants, the elderly, and those with weakened immune systems.
So, what lies ahead? Much is known about existing viruses, but we need to be prepared for the next wave of viral pathogens—variants of the existing viruses that may escape current preventive and curative measures, as well as emerging viruses that could steer us into the next global pandemic. Equipping ourselves with the appropriate tools helps to ensure readiness to quickly and effectively combat novel variants of respiratory viruses. Next generation sequencing (NGS) is an extremely powerful tool for this, especially when performed using techniques that are unaffected by the myriad variants that may arise.
Challenges of preparing for respiratory virus season
A primary challenge for health authorities is predicting what to expect—which viruses and strains are likely to emerge, where they will cluster, how fast they will spread (how virulent), and how quickly they will mutate. In the United States, the Centers for Disease Control and Prevention (CDC) makes judgments based on what has occurred in other regions, particularly the Southern Hemisphere, and then predicts what to expect. Another challenge is that because viruses mutate, sometimes rapidly, it is always difficult to make predictions a priori.
The best preparation for respiratory virus season is to assess the prevailing conditions. Examining the local environment informs what is present, what is mutating, and where the mutations are occurring. Surveillance using wastewater has proven to be an effective method for assessing community spread.
Choosing the right technology for monitoring viruses
Nucleic acid amplification tests (NAATs) are PCR-based assays that are the go-to method for virus identification. Considered to be faster, cheaper, and easier to use than other methods, NAATs typically allow identification of existing viruses and surveillance of point mutations but are limited in the number of changes that can be identified and their ability to ascertain different variants at the same base. Using multiplexed assays, it is possible to analyze multiple loci simultaneously; however, the inherent design of PCR may result in variant strains and novel viruses evading detection.
NGS, on the other hand, allows the entire genome of the virus to be scrutinized in a single experiment to provide more comprehensive information. Various NGS techniques are available for virus detection and monitoring, including shotgun, hybridization capture, and amplicon sequencing. Shotgun sequencing, in which the viral nucleic acid is fragmented or reverse transcribed and then ligated with adapters for sequencing, requires no prior knowledge of the viral sequence and can detect novel, unknown pathogens, but host nucleic acid contamination can significantly increase sequencing costs.
For hybridization capture, sections along the length of a known virus are enriched from host nucleic acid using oligonucleotide probes, and then sequenced. This technique does not require prior knowledge of the viral strain as it is insensitive to mutations, so it is effective at identifying novel variants. However, the workflow can be long and onerous. It also typically requires reference sequences for the viruses (or virus families) of interest.
Amplicon sequencing is a powerful technique that is fast and simple to perform even with samples that have a low viral load. Highly multiplexed primer pairs designed to target regions along the length of the viral genome are used to produce short amplicons that are easily sequenced. An even more rigorous and reliable approach is to use overlapping amplicons in a single-tube multiplex PCR to enable contiguous genome coverage, even in instances where a mismatch between primer and target prevents binding. In specific single-tube chemistries, super amplicons are produced that cannot form when alternating primer pairs are placed into two separate reactions (Figure 1). These super amplicons enable full target coverage, so that a single panel design can be used to detect a variety of mutations and viral strains without experiencing coverage dropouts.
Figure 1: Overlapping amplicons create “super amplicons” that provide coverage in situations of primer dropout. In a two-tube amplicon approach, Variant 1 is detected, but because of failure of primer F2 to bind, Amplicon 2 is not generated and so Variant 2 is not detected. Using a one-tube amplicon method, the formation of the super amplicon from primers F1 and R2 provides coverage across the entire region.
Considerations for expedient virus identification using NGS
For more comprehensive and insightful actionable information, NGS methods should be more widely adopted in public health settings. A key first step for using NGS for virus identification and monitoring is to start with a robust, reliable design. From library preparation through adapter choices to library normalization, there are various options available, many of which are agnostic to the sequencing platform.
Another important consideration is being open to trying new tools. PCR/NAAT is tried and tested but does not provide the breadth and depth of information that can be delivered by NGS. Recent NGS technologies provide a much more comprehensive picture. For example, multiplexed amplicon sequencing allows virus identification and lineage detection (full genome sequencing) in over 1,500 samples using a single sequencing run, far beyond what could possibly be achieved with PCR/NAAT.
Concerns about the cost of NGS are common and may be considered well-founded; however, viral genomes are small, and the cost of sequencing has decreased significantly, with a wider choice of available sequencing platforms, enabling much broader use of NGS, which is the ideal technology for quick identification of variants. Given the amount of information obtained using amplicon sequencing, this is a cost-efficient approach because it requires less sequencing coverage than shotgun sequencing and has a simpler workflow that makes it more cost-effective, from a labor perspective, than hybridization capture. Also, since the sequencing cost can be amortized over more than 1,500 samples, NGS is a more favorable option.
The choice of NGS panel is also an important consideration. Some panels detect a broad range of viral genomes, while others are more specific; for example, targeting only respiratory viruses. Using panels that are able to identify known and novel variants helps to keep costs down when targeting known respiratory viruses and their variants. Comprehensive panels that target a wider range of viruses could be useful for exploratory studies to identify unknown viruses. Focused and broad panels can be used for monitoring complex samples such as wastewater. However, the amount of sequencing required to generate useful data from comprehensive panels would make routine monitoring of known respiratory viruses by sequencing cost prohibitive. When multiplexing samples to help drive down per-sample sequencing cost, it is also important to normalize the amount of each sample to ensure sufficient and uniform coverage of each. This normalization can be performed by quantifying each sample library prior to pooling, or by employing enzymatic normalization approaches which have the advantage of being easier to automate and generate less read depth variation.
The future of respiratory virus season preparation
We have just emerged from the worst global pandemic in decades—for many, the only pandemic in living memory. COVID demonstrated the power of NGS for monitoring virus clustering, evolution, and spread, with significant impacts to public health approaches. Within a month of the first patient being identified, the SARS-CoV-2 genome had been sequenced and the sequence information made publicly available, allowing authorities to identify the threat and begin vaccine development.
NGS was also key to the unprecedented quick deployment of vaccines due to the ability to identify novel variants. With the ongoing war between viruses and their hosts, it’s a constant battle to stay ahead of the viruses to ensure timely development of effective vaccines and treatments. So, NGS is proving to be a powerful tool when preparing for respiratory virus season and future outbreaks of novel viral disease.
To promote increased adoption of NGS in public health, there is a need to continue to drive down the cost of sequencing to ensure the technology remains cost-effective for use in comprehensive surveillance of multiple seasonal viruses. Using highly multiplexed amplicon sequencing panels targeting respiratory viruses or other specific viral pathogens helps to minimize sequencing costs. Additionally, the industry needs robust, standardized NGS analysis pipelines to help ensure data integrity and to facilitate global data sharing and harmonization.
References
1. Wang X, Stelzer-Braid S, et al. Detection of respiratory viruses directly from clinical samples using next-generation sequencing: A literature review of recent advances and potential for routine clinical use. Rev Med Virol. 2022. 32:e2375. https://doi.org/10.1002/rmv.2375
2. Moriyama M, Hugentobler WJ, Iwasaki A. Seasonality of respiratory virus infections. Annu Rev Virol. 2020. 7:83–101. https://doi/10.1146/annurev-virology-012420-02244
About the author
Steven Henck, PhD, is vice president of R&D at Integrated DNA Technologies, a global genomics solutions provider whose mission is to accelerate the pace of genomics. IDT has developed proprietary technologies for genomics applications such as next generation sequencing, CRISPR genome editing, synthetic biology, digital PCR, and RNA interference.