Abstract Title
Advanced Techniques in Calicivirus Detection and Genotyping Through DNA Hybridisation Enrichment Combined with Nanopore Sequencing
Presenter
Lucas Jollie, University of Amsterdam
Co-Author(s)
L. Jollie1,2; F. Harders2; S. Brul1; M.S.M. Brouwer2; L.P.B. Verhoef1,31 Molecular Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam2 Wageningen Bioveterinary Research, Wageningen University & Research3 Nederlandse Voedsel- en Warenauthoriteit
Abstract Category
Advances in diagnostics and detection
Abstract
Norovirus and other caliciviruses were the third most common cause of foodborne outbreaks and the second most common cause of human cases of foodborne illness in the European Union in 2023. Like other viruses, caliciviruses can be subdivided into genotypes; norovirus and sapovirus have 49 and 33 genotypes, respectively. Caliciviruses usually display high host-specificity, but fear exists that animal reservoirs containing multiple genetically diverse Caliciviridae may harbour zoonotic potential via recombination events. Accurate calicivirus subtype tracking and identification is therefore crucial from epidemiological and public health perspectives, especially in animals close to humans, such as livestock. We aim to develop a monitoring method to detect and genotype caliciviruses simultaneously at slaughterhouses.
Previously, sequencing libraries have been successfully enriched for viral sequences using a probe-based hybridisation and capture approach that allows for highly specific and sensitive detection of viral targets. As such, we hypothesise that following this approach the RdRP and VP1 regions of Caliciviruses can be captured and used for accurate genotyping. To this purpose, DNA probes corresponding to the RdRP (norovirus ) and VP1 regions (norovirus and sapovirus) were designed. Captured sequences will be sequenced using Oxford Nanopore Technologies and genotyped by comparing captured sequences to genotype-specific reference genomes.
Using ProbeTools DNA hybridisation-probe design software, probes were designed for the appropriate genomic targets for norovirus and sapovirus. These probes were capable of detecting 83/84 norovirus reference genotypes using 33 probes, as well as 30/33 sapovirus reference genotypes using 24 probes in silico, ahead of implementation in the lab.
Previously, sequencing libraries have been successfully enriched for viral sequences using a probe-based hybridisation and capture approach that allows for highly specific and sensitive detection of viral targets. As such, we hypothesise that following this approach the RdRP and VP1 regions of Caliciviruses can be captured and used for accurate genotyping. To this purpose, DNA probes corresponding to the RdRP (norovirus ) and VP1 regions (norovirus and sapovirus) were designed. Captured sequences will be sequenced using Oxford Nanopore Technologies and genotyped by comparing captured sequences to genotype-specific reference genomes.
Using ProbeTools DNA hybridisation-probe design software, probes were designed for the appropriate genomic targets for norovirus and sapovirus. These probes were capable of detecting 83/84 norovirus reference genotypes using 33 probes, as well as 30/33 sapovirus reference genotypes using 24 probes in silico, ahead of implementation in the lab.