Rapid Identification of ASFV, CSFV and FMDV from Mongolian Outbreaks with MinION Short Amplicon Sequencing

Abstract

African swine fever virus (ASFV), classical swine fever virus (CSFV), and foot-and-mouth disease virus (FMDV) cause important transboundary animal diseases (TADs) that have a significant economic impact. The rapid and unequivocal identification of these pathogens and distinction from other animal diseases based on clinical symptoms in the field is difficult. Nevertheless, early pathogen detection is critical in limiting their spread and impact as is the availability of a reliable, rapid, and cost-effective diagnostic test. The purpose of this study was to evaluate the feasibility to identify ASFV, CSFV, and FMDV in field samples using next generation sequencing of short PCR products as a point-of-care diagnostic. We isolated nucleic acids from tissue samples of animals in Mongolia that were infected with ASFV (2019), CSFV (2015), or FMDV (2018), and performed conventional (RT-) PCR using primers recommended by the Terrestrial Animal Health Code of the World Organization for Animal Health (WOAH). The (RT-) PCR products were then sequenced in Mongolia using the MinION nanopore portable sequencer. The resulting sequencing reads successfully identified the respective pathogens that exhibited 91-100% nucleic acid similarity to the reference strains. Phylogenetic analyses suggest that the Mongolian virus isolates are closely related to other isolates circulating in the same geographic region. Based on our results, sequencing short fragments derived by conventional (RT-) PCR is a reliable approach for rapid point-of-care diagnostics for ASFV, CSFV, and FMDV even in low-resource countries.

Keywords: African swine fever virus; MinION; classical swine fever virus; foot-and-mouth disease virus; point-of-care diagnostics; short amplicon sequencing; transboundary animal diseases.

Figure 1

Agarose gel electrophoresis (2% agarose) of (RT)-PCR amplified products using virus-specific primer sets. The amplicons used for the MinION sequencing are in the respective red squares for ASF (a), CSF (b), and FMD (c). Lane M, 100 bp DNA size marker.

Figure 2

Relative distribution of reads in sequence data generated by nanopore sequencing of DNA from (RT-) PCR products of three pathogens, ASFV (a), CSFV (b), and FMDV (c). The pie-charts were created using Microsoft Office Excel 2021.

Figure 3

Phylogenetic trees of ASFV (a), CSFV (b), and FMDV (c) field isolates based on partial p72 (a), domain II of IRES (internal ribosomal entry site; (b)) and 5′ UTR (c), respectively. The Molecular Evolutionary Genetics Analysis (MEGA) Version 11.0.13 was used to create the phylogenetic trees. Genotype (GT) and serotype (ST) clusters are differentiated in each panel by color: (a) ASFV GT-I (yellow) and GT-II (blue); (b) CSF GT-2.1 (blue), GT-2.2 (pink) and GT-2.3 (yellow); (c) FMD ST-O (blue), ST-A (yellow) and ST-Asia-1 (As1; (pink).

Figure 4

Timeline for pathogen identification. Shown are the steps from tissue sample collection and preparation to sequence data analysis. The tools in biorender.com were used to make the figure.