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Matthew Keller: Deployable NGS for Influenza virus field surveillance and outbreak response

Matthew Keller, PhD, of the CDC Influenza Genomics Team (IGT), presented his team’s development of a rapid, portable, end-to-end influenza A virus sequencing pipeline using Oxford Nanopore’s MinION device.

Matthew began by describing the scale of seasonal influenza, which causes ~290-650,000 deaths a year; in 2017-8, it was responsible for ~80,000 deaths in the United States alone, with influenza A virus (IAV) the primary cause. The RNA virus is highly variable “to the point that virions are essentially unique”; it is able to rapidly mutate, via antigenic drift and reassortment of its segmented genome with that of other coinfecting viruses. He stressed the need for a concerted surveillance effort of this process: this is the main goal of the IGT. The team, together with the NIRC, have sequenced over 25,000 influenza samples from across the world using their high-throughput Global Surveillance pipeline: RNA is extracted, reverse transcribed and amplified via M-RTPCR, barcoded and prepared for sequencing in a total of ~11.5 hours, then sequenced for 24 hours using a short-read technology; sequencing data is then analysed and curated in ~2 hours. Matthew explains that, whilst most of the samples sequenced represent seasonal influenza, the team also look for novel viruses which could cause future pandemics.

Matthew described the potential for zoonoses, viruses which can transmit from animal to human hosts, and reassortment of viruses, to cause pandemics. He noted that the Spanish flu virus, which caused more deaths than there were casualties in World War One, transferred to humans from wild water fowl, whilst the virus behind the 2009 H1N1 outbreak had complicated origins, from four viruses in three species, with pigs acting as the “mixing vessel”. With transfer of zoonoses from swine to humans posing an important source of potential pandemics, Matthew asked: where could swine-human contact occur? One answer is exhibition swine shows: with pigs and humans travelling from far and wide, and with lots of contact between the two, these could pose a zoonosis risk: “sick pigs and transmission everywhere.”

Matthew then introduced MIA: IGT’s Mobile Influenza Analysis pipeline for rapid, portable IAV sequencing and analysis using nanopore sequencing. To enable faster, on-site sequencing, the team redesigned the Global Surveillance pipeline from start to finish, cutting RNA extraction, M-RTPCR and barcoded sequencing library prep down to ~3.5 hours and sequencing samples in multiplex on the MinION device for 1-6 hours; analysis was run in real-time alongside sequencing for 1-12 hours.

Matthew notes that the high-powered laptop used for analysis was “actually the least portable part of our pipeline.” The team then took the MIA pipeline on the road, in two small suitcases and a cool box, to a swine show, for on-site testing. They initially screened ~100 pigs using rapid tests, noting that these are known to frequently produce false-negatives; that night, they returned to the 7 pigs that had tested positive for influenza, taking nasal wipes from these and their neighbours. They then set up in the barn and, overnight, extracted RNA from the samples, sequenced the 24 samples in multiplex on the MinION and ran analysis. Data was basecalled, demultiplexed then mapped via IRMA.

By the afternoon of the next day, they were able to report virus coverage for every barcoded sample and construct a phylogenetic tree of the 13 influenza A viruses identified in the samples. 11 of these clustered as an outbreak of H1N2, which Matthew noted had originated from a human H1N1 virus (for which a vaccine was made), passed into swine and subsequently evolved to the H1 delta 2 lineage; this data indicated that the strain was circulating in the swine population. He described how children <10, of which there were many at the swine show, could be particularly vulnerable to the strain. Could the H1N2 identified pose a risk of a future zoonosis event? The data was then used to search for candidate vaccines; in the absence of a good match with any current candidates, the data was emailed to the CDC, from which a synthetic vaccine could be synthesised – “18 hours after unpacking MIA.”

The samples were subsequently run through the IGT Global Analysis pipeline and produced the same 13 genomes, with 99.3% consensus accuracy between MinION-generated data and that of the short-read technology. A follow-up phylogenetic tree constructed with collaborators at USDA allowed for tracking of the evolution of H1 delta 2 through swine until detection. Since the study, two cases of H1N2 have been identified in humans, whose ZIP codes matched the locations of the pigs; the strain matched that identified by MIA.

Matthew concluded that MIA allows for the successful on-site sequencing of swine influenza virus isolates. The pipeline demonstrates the ability of rapid sequencing to improve surveillance of influenza and potential zoonisis risks. The team plan to deploy the pipeline again in Thailand and Puerto Rico, and hope to further improve the screening process for even faster sample-to-answer time.

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