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.

Development of RT-RPA-based point-of-care tests for epidemic arthritogenic alphaviruses

“…We subjected our RPA products to same-day sanger sequencing and were subsequently able to take it a step further and conduct a phylogenetic analysis of the obtained sequences with success. Short amplicon sequencing from PCR products to characterize the phylogenetics of viral isolates has been done before 54 , and while it is important to acknowledge that short amplicon sequencing is limited in its ability to fully characterize viral evolutionary dynamics, the ability of RPA products to be analyzed in a similar manner opens up the possibility of conducting molecular epidemiological studies in a more rapid and inexpensive manner, which would supplement diagnostic efforts and help better monitor outbreaks.…”