Nanopore Sequencing as a Rapid Tool for Discrimination between Epidemic and Vaccine Strains of Classical Swine Fever Viruses in Japan

Abstract

In 2018, an outbreak of classical swine fever (CSF) was identified in Japan for the first time in 26 years. To prevent the spread of CSF to wild boars, bait vaccination with a commercial vaccine derived from the live attenuated C-strain for wild boars has been introduced in Japan since March 2019. Although conventional and real-time RT-PCR methods are suitable for screening and confirmation of suspected cases of the disease, neither of the methods used in Japan can discriminate between CSF virus field strains and live attenuated vaccines. Currently, Sanger sequencing of PCR products is used to distinguish them. In recent years, nanopore sequencing, which is based on a novel sequencing technology, has been adopted for laboratory diagnosis. To save the time and effort required for Sanger sequencing of PCR products, we aimed to determine whether nanopore sequencing would be applicable to CSF virus strain genotyping. PCR products from twenty-nine wild boar samples were evaluated and nanopore sequencing succeeded in discriminating between vaccine and epidemic strains in 28 of 29 samples in less than 90 minutes of total examination time per test. The comparative studies of the sequencing accuracy between Sanger and nanopore sequencing indicated a concordance of ＞96% suggesting that nanopore sequencing could be a promising method for CSF virus strain genotyping despite the short examination time.