PMID- 37220122
OWN - NLM
STAT- MEDLINE
DCOM- 20230525
LR  - 20230605
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 18
IP  - 5
DP  - 2023
TI  - Rapid genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) 
      using MinION nanopore sequencing.
PG  - e0282767
LID - 10.1371/journal.pone.0282767 [doi]
LID - e0282767
AB  - 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.
CI  - Copyright: (c) 2023 Caserta et al. This is an open access article distributed under 
      the terms of the Creative Commons Attribution License, which permits unrestricted 
      use, distribution, and reproduction in any medium, provided the original author 
      and source are credited.
FAU - Caserta, Leonardo Cardia
AU  - Caserta LC
AD  - Department of Population Medicine and Diagnostic Sciences, Animal Health 
      Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, 
      NY, United States of America.
FAU - Zhang, Jianqiang
AU  - Zhang J
AD  - Department of Veterinary Diagnostic and Production Animal Medicine, College of 
      Veterinary Medicine, Iowa State University, Ames, Iowa, United States of America.
FAU - Pineyro, Pablo
AU  - Pineyro P
AD  - Department of Veterinary Diagnostic and Production Animal Medicine, College of 
      Veterinary Medicine, Iowa State University, Ames, Iowa, United States of America.
FAU - Diel, Diego G
AU  - Diel DG
AUID- ORCID: 0000-0003-3237-8940
AD  - Department of Population Medicine and Diagnostic Sciences, Animal Health 
      Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, 
      NY, United States of America.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20230523
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
SB  - IM
MH  - Animals
MH  - Swine
MH  - Genotype
MH  - *Nanopore Sequencing
MH  - *Porcine respiratory and reproductive syndrome virus
MH  - Chemoradiotherapy
MH  - China
PMC - PMC10205005
COIS- DGD and LCC have filed a provisional patent application on the PRRSV TAS and LATS 
      assays developed in this study (US serial no. 63/413,744).
EDAT- 2023/05/23 19:09
MHDA- 2023/05/25 06:42
PMCR- 2023/05/23
CRDT- 2023/05/23 13:34
PHST- 2023/02/21 00:00 [received]
PHST- 2023/04/24 00:00 [accepted]
PHST- 2023/05/25 06:42 [medline]
PHST- 2023/05/23 19:09 [pubmed]
PHST- 2023/05/23 13:34 [entrez]
PHST- 2023/05/23 00:00 [pmc-release]
AID - PONE-D-23-05139 [pii]
AID - 10.1371/journal.pone.0282767 [doi]
PST - epublish
SO  - PLoS One. 2023 May 23;18(5):e0282767. doi: 10.1371/journal.pone.0282767. 
      eCollection 2023.