Combating viral contamination and disease outbreaks at source with rapid, low-cost nanopore sequencing

Infectious disease outbreaks and viral contamination naturally found during preharvest of food are a constant threat, requiring rapid delivery of genomic data to inform farmers, food processors, and public health responses by identifying effective control measures and treatments. Nanopore technology has been widely used for pathogen surveillance, from investigating clinical research samples to detecting foodborne pathogens, enabling comprehensive monitoring and rapid responses to infectious disease threats. Below are examples of how nanopore sequencing has been utilised to combat viral contamination and disease outbreaks at source.

The global burden of hepatitis B

Hepatitis B virus (HBV) causes liver infections, with chronic cases often leading to hepatocellular carcinoma and liver cirrhosis. This virus is a significant global health concern due to its high prevalence and the severe long-term consequences of chronic infections, which can result in prolonged illness and premature death ¹. In 2022 alone, there were approximately 257.5 million affected individuals, with only 6.8 million undergoing treatment².

HBV has a compact circular genome of approximately 3.2 kb, which comprises four genes. There are ten HBV genotypes (A–J), with C the most prevalent globally, and sub-genotypes A1 and D3 the most predominant in southern Africa³. Specifically, sub-genotype A1 is linked to severe liver disease and rapid progression to hepatocellular carcinoma.

Therapies and prophylactic measures are available to reduce prevalence of the infection, including HBV vaccinations in children to eliminate risk of infection in later life^1,2. However, an increase in accessible identification and effective treatment for chronic infection is required to lower disease burden and prevalence of the virus. Currently, serological tests are used to identify HBV but there are concerns that these tests may lack sufficient sensitivity or specificity and may lead to inaccurate results³.

Sanger sequencing is also used to characterise infected samples; however, only partial sequences of the virus are typically generated, meaning it is difficult to determine genotype and little information is provided on intra-sample HBV genetic diversity³. Since ‘coinfection and viral recombination can trigger greater virulence’ and therefore more severe illness, the ability to accurately genotype and identify recombinant strains holds significant potential to improve the treatment of HBV.

Challenging the gold-standard method with nanopore sequencing

Tshiabuila and colleagues investigated the use of nanopore technology to perform whole-genome sequencing of HBV viral amplicons to rapidly characterise complete HBV genomes. Whole-genome sequencing is ‘becoming a key tool for understanding the distribution, infection prevalence, and genetic diversity of HBV and other viral pathogens’. The team noted that nanopore sequencing is more efficient to run compared to legacy technologies due to fewer required reagents, and sequencing run time is shorter due to real-time data analysis. Furthermore, long nanopore reads are generated, ‘a factor that vastly simplifies the assembly of whole-genome sequences’.

The research team analysed 148 HBV-positive samples using multiplexed tiled PCR to sequence the entire HBV genome on a GridION device. By utilising nanopore sequencing for real-time analysis, sequencing data for 96 samples was delivered within 14 hours, nearly twice as fast as legacy short-read sequencing technologies. For 123 samples, uniform coverage of over 80% with a sequencing depth of over 2,300x was generated, enabling genotyping, recombination identification, and mutation detection, demonstrating the rich data this rapid approach generates.

Tshiabuila et al. highlighted that genome characterisation is critical ‘beyond identifying the infecting agent [because] it can reveal clinically relevant genetic variations’. With fully characterised HBV genomes, the team was able to identify the most prevalent genotype in the study — HBV-A — and revealed complex viral replication/recombination dynamics between genotypes A and D, likely due to the high prevalence of these genotypes in sub-Saharan Africa. The research team was also able to profile mutations associated with drug and vaccine resistance, critical to determine whether first-line treatments will be effective in a region.

‘While short-read sequencing is considered the gold standard for sequencing of viral genomes … [Oxford Nanopore Technologies] appears to generate high-quality data at a very affordable cost.’

Not only is nanopore sequencing rapid and efficient, Tshiabuila et al. noted that the analysis was also more user-friendly, ‘ensuring that valuable insights can be obtained without the dependency on specialised expertise’. Without the need for expert bioinformaticians, more researchers in decentralised labs can adopt this technology. Tshiabuila et al. concluded that ‘*[Oxford Nanopore Technologies]-based sequencing is presently the most cost-effective, high-throughput sequencing technology’ and is ‘especially well-suited for countries with limited resources for monitoring shifting viral demographics and tracking the prevalence and spread of drug resistance and vaccine evasion mutations*’.

Pre-empting infectious disease outbreaks

Nanopore sequencing of viral amplicons has also been used in food and environmental research to investigate factors that could lead to infectious disease outbreaks. Bioindicator animals, such as molluscs, can be used to assess qualitative changes in an ecosystem because these filter-feeding animals concentrate viruses in their tissues when living in contaminated water. Since they are consumed raw, molluscs can be a significant source of human exposure to viruses, including astroviruses (AstVs) and could be an investigative target to potentially pre-empt infectious disease outbreaks and protect the food supply chain⁴.

AstVs are known to infect mammals and birds, and have been reported in reptiles, amphibians, fish, and invertebrates. Human AstVs cause acute gastroenteritis and have been linked to large foodborne outbreaks⁴. Over the last decade, genetically divergent human AstV species, referred to as ‘animal-like’ or ‘atypical’ strains, have been identified. However, there is currently little information available on these pathogens⁵.

Beikpour et al. employed targeted nested RT-PCR with nanopore sequencing to assess AstVs present in 134 molluscs, allowing the identification of viral contamination in food sources and the environment. Legacy technologies, such as direct Sanger sequencing, are not suitable for this application because the viral amplicons are likely to consist of a mixed population, limiting sequencing sensitivity and potentially missing low abundance populations⁶. Whereas nanopore sequencing generates reads of any length, making it ideal for metagenomic analysis because long nanopore reads can span entire RT-PCR-enriched viral genomes. Beikpour et al. generated 37 AstV sequence contigs, predominantly avian, along with bat and reptilian AstVs. The diversity of viral species in the samples likely reflects faecal contamination in the water from the birds living in coastal areas where shellfish are commonly farmed.

Despite not detecting human AstVs, this study highlights the potential for nanopore sequencing to be used to explore the diversity of viruses in molluscs. Beikpour et al. concluded that because AstVs are common in several animals, this approach of using ‘astroviromic data as a proxy for the microbiological quality of water and food should be further explored’.