Outbreak surveillance of viral pathogens

For outbreak surveillance, rapid time to result is essential to enable the early implementation of effective containment and treatment strategies. However, as explained by Rambo-Martin et al., ‘[the traditional approach of] collecting samples in the field, shipping them to centralised laboratories, sequencing their genomes, and analysing them often takes several weeks or months’1.

To overcome these challenges, researchers are now utilising the advantages of portable, realtime nanopore sequencing technology to perform outbreak surveillance at sample source. For example, nanopore technology has been successfully used to characterise and inform public health responses for a number of emerging pathogens, including coronavirus 2,3, Ebola virus 4, Zika virus 5, and Lassa virus 6.

Rambo-Martin et al.1 described the development and deployment of Mia (Mobile influenza analysis), a rapid and portable influenza A virus (IAV) sequencing pipeline, which incorporates the MinION device.

Influenza A viruses (IAVs) have multiple enzootic reservoirs, including swine, and have known potential to infect humans, as evidenced by the 2009 H1N1 virus pandemic. Since that time, there have been 465 human cases of swine-origin influenza in the United States, most of which arise from exposure to the pathogen at agricultural fairs.

The team set up their mobile sequencing station at a large US swine exhibition and sequenced 24 swabs taken from animals that had tested positive for IAV (using commercially available rapid testing kit) and their immediate neighbours (Figure 1). In brief, RNA was extracted from each swab prior to whole genome amplification, barcoding, and library preparation using the Ligation Sequencing Kit. All samples were run on a single MinION Flow Cell.

A novel automated, real-time data analysis pipeline performed basecalling, quality control, genome assembly, phylogenetic analysis, BLAST searches, and amino acid comparisons to current candidate vaccine viruses (CVVs; viruses that have been prepared for vaccine production) — all without the need for internet connection.

In total, 13 whole genome assemblies were obtained. Phylogenetic analysis identified three genetically distinct swine IAV lineages comprising the numerically dominant H1N2 subtype, plus H1N1 and H3N2 subtypes. Analysis of the haemagglutinin (HA) sequences of the H1N2 viruses identified >30 amino acid differences to the most closely related CVVs.

As an exercise in pandemic preparedness, all sequences were emailed to the US Centers for Disease Control and Prevention, who initiated the development of a synthetically derived CVV. Importantly, all data were obtained within 18 hours of set up — from sample to actionable results. According to the researchers, ‘Had this virus caused a severe outbreak or pandemic, our proactive surveillance efforts and vaccine derivation would have provided an approximate 8-week time advantage for vaccine manufacturing’ 1.

In a separate study, the researchers applied the direct RNA sequencing capabilities of nanopore technology to sequence a complete IAV genome, representing the first time that an RNA genome has been sequenced in its native form. 7

Figure 1: Real-time detection of influenza A virus at a swine exhibition using Mia. Images courtesy of Benjamin L. RamboMartin, Battelle Memorial Institute, and Matthew W. Keller, Oak Ridge Institute of Science and Education.

1. Rambo-Martin, B.L. et al. Influenza A virus field surveillance at a swine-human interface. mSphere 5(1):e00822-19 (2020).

2. Zhu, N. et al. A Novel coronavirus from patients with pneumonia in China, 2019. N. Engl. J. Med. 382(8):727–733 (2020).

3. Taiaroa, G. et al. Direct RNA sequencing and early evolution of SARS-CoV-2. bioRxiv 976167 (2020).

4. Quick J, et al. Real-time, portable genome sequencing for Ebola surveillance. Nature 530(7589):228- 32 (2016).

5. Quick, J. et al. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nat. Protoc. 12(6):1261–1276 (2017).

6. Kafetzopoulou, L. E. et al. Metagenomic sequencing at the epicenter of the Nigeria 2018 Lassa fever outbreak. Science 363(6422):74-77 (2019).

7. Keller, M.W. et al. Direct RNA sequencing of the coding complete influenza A virus genome. Sci. Rep. 8(1):14408 (2018).