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bioRxiv · 10.1101/2025.07.16.665162

Targeted Whole Genome Sequencing of African Swine Fever Virus and Classical Swine Fever Virus on the MinION Portable Sequencing Platform

McDowell, C. D.·Kwon, T.·Assato, P.·Mantlo, E.·Trujillo, J. D.·Gaudreault, N. N.·Caserta, L. C.·Morozov, I.·Souza-Neto, J. A.·Pogranichniy, R. M.·Diel, D.·Richt, J. A.
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Abstract

African swine fever virus (ASFV) and classical swine fever virus (CSFV) are important transboundary animal diseases (TADs) affecting swine. ASFV is a large DNA virus with a genome size of 170-190 kilobases (kB) belonging to the family Asfarviridae, genus Asfivirus. CSFV is a single-stranded RNA virus with genome size of approximately 12 kB belonging to the family Flaviviridae, genus Pestivirus. Outbreaks involving either one of these viruses result in similar disease syndromes and significant economic impacts from: (i) high morbidity and mortality events; (ii) control measures which include culling and quarantine; and (iii) export restrictions of swine and pork products. Current detection methods during an outbreak provide minimal genetic information on the circulating virus strains/genotypes that are important for tracing and vaccine considerations. The increasing availability and reduced cost of next-generation sequencing (NGS), allows for the establishment of vital NGS protocols for the rapid identification and complete genetic characterization of outbreak strains during an investigation. NGS data provides a better understanding of viral spread and evolution facilitating the development of novel and effective control measures. In this study, panels of primers spanning the genomes of ASFV and CSFV were independently developed to generate approximately 10kB and 6kB amplicons, respectively. The primer panels consisted of 19 primer pairs for ASFV and 2 primer pairs for CSFV providing whole genome amplification of each pathogen. These primer pools were further optimized for batch pooling and thermocycling conditions, resulting in a total of 5 primer pools/reactions used for ASFV and 2 primer pairs/reactions for CSFV. The ASFV primer panel was tested on viral DNA extracted from blood collected from pigs experimentally infected with ASFV genotype I and genotype II viruses. The CSFV primer panel was tested on 11 different strains of CSFV representing the 3 known CSFV genotypes, and 21 clinical samples collected from pigs experimentally infected with 2 different genotype 1 viruses. ASFV and CSFV amplicons from optimized PCR reactions were subsequently sequenced on the Oxford Nanopore MinION platform. The targeted protocols for these viruses resulted in an average coverage greater than 1000X for ASFV with 99% of the genome covered, and 10,000X-20,000X for CSFV with 97% to 99% of the genomes covered. The ASFV targeted whole genome sequencing protocol has been optimized for genotype II ASF viruses that have been responsible for the more recent outbreaks outside of Africa. The CSFV targeted whole genome sequencing protocol has universal applications for the detection of all CSFV genotypes. Protocols developed and evaluated here will be essential complementary tools for early pathogen detection and differentiation as well as genetic characterization of these high consequence swine viruses, globally and within the United States, should an outbreak occur.

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McDowell, C. D., Kwon, T., Assato, P., Mantlo, E., Trujillo, J. D., Gaudreault, N. N., Caserta, L. C., Morozov, I., Souza-Neto, J. A., Pogranichniy, R. M., Diel, D., Richt, J. A.. 2025-07-18. Targeted Whole Genome Sequencing of African Swine Fever Virus and Classical Swine Fever Virus on the MinION Portable Sequencing Platform. https://doi.org/10.1101/2025.07.16.665162

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