Rapid genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) using MinION nanopore sequencing. 2023

Leonardo Cardia Caserta, and Jianqiang Zhang, and Pablo Piñeyro, and Diego G Diel
Department of Population Medicine and Diagnostic Sciences, Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY, United States of America.

The global distribution and constant evolution are challenges for the control of porcine reproductive and respiratory syndrome virus (PRRSV), one of the most important viruses affecting swine worldwide. Effective control of PRRSV benefits from genotyping, which currently relies on Sanger sequencing. Here we developed and optimized procedures for real-time genotyping and whole genome sequencing of PRRSV directly from clinical samples based on targeted amplicon- and long amplicon tiling sequencing using the MinION Oxford Nanopore platform. Procedures were developed and tested on 154 clinical samples (including lung, serum, oral fluid and processing fluid) with RT-PCR Ct values ranging from 15 to 35. The targeted amplicon sequencing (TAS) approach was developed to obtain sequences of the complete ORF5 (main target gene for PRRSV genotyping) and partial ORF4 and ORF6 sequences of both PRRSV-1 and PRRSV-2 species. After only 5 min of sequencing, PRRSV consensus sequences with identities to reference sequences above 99% were obtained, enabling rapid identification and genotyping of clinical PRRSV samples into lineages 1, 5 and 8. The long amplicon tiling sequencing (LATS) approach targets type 2 PRRSV, the most prevalent viral species in the U.S. and China. Complete PRRSV genomes were obtained within the first hour of sequencing for samples with Ct values below 24.9. Ninety-two whole genome sequences were obtained using the LATS procedure. Fifty out of 60 sera (83.3%) and 18 out of 20 lung samples (90%) had at least 80% of genome covered at a minimum of 20X sequence depth per position. The procedures developed and optimized in this study here are valuable tools with potential for field application during PRRSV elimination programs.

UI MeSH Term Description Entries
D002681 China A country spanning from central Asia to the Pacific Ocean. Inner Mongolia,Manchuria,People's Republic of China,Sinkiang,Mainland China
D005838 Genotype The genetic constitution of the individual, comprising the ALLELES present at each GENETIC LOCUS. Genogroup,Genogroups,Genotypes
D000081414 Nanopore Sequencing A sequencing protocol that drives nucleic acids (DNA or RNA) in an electric field through NANOPORES allowing single molecule sequence analysis. Nanopore Sequencings,Sequencing, Nanopore
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D013552 Swine Any of various animals that constitute the family Suidae and comprise stout-bodied, short-legged omnivorous mammals with thick skin, usually covered with coarse bristles, a rather long mobile snout, and small tail. Included are the genera Babyrousa, Phacochoerus (wart hogs), and Sus, the latter containing the domestic pig (see SUS SCROFA). Phacochoerus,Pigs,Suidae,Warthogs,Wart Hogs,Hog, Wart,Hogs, Wart,Wart Hog
D059248 Chemoradiotherapy Treatment that combines chemotherapy with radiotherapy. Concurrent Chemoradiotherapy,Concomitant Chemoradiotherapy,Concomitant Radiochemotherapy,Concurrent Radiochemotherapy,Radiochemotherapy,Synchronous Chemoradiotherapy,Chemoradiotherapies,Chemoradiotherapies, Concomitant,Chemoradiotherapies, Concurrent,Chemoradiotherapies, Synchronous,Chemoradiotherapy, Concomitant,Chemoradiotherapy, Concurrent,Chemoradiotherapy, Synchronous,Concomitant Chemoradiotherapies,Concomitant Radiochemotherapies,Concurrent Chemoradiotherapies,Concurrent Radiochemotherapies,Radiochemotherapies,Radiochemotherapies, Concomitant,Radiochemotherapies, Concurrent,Radiochemotherapy, Concomitant,Radiochemotherapy, Concurrent,Synchronous Chemoradiotherapies
D019316 Porcine respiratory and reproductive syndrome virus A species of ARTERIVIRUS causing reproductive and respiratory disease in pigs. The European strain is called Lelystad virus. Airborne transmission is common. Lelystad Virus,PRRSV,Swine Infertility and Respiratory Syndrome Virus,Porcine Reproductive and Respiratory Syndrome Virus,Swine Infertility Respiratory Syndrome Virus

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