title: High-throughput whole genome sequencing of Porcine reproductive and respiratory syndrome virus from cell culture materials and clinical specimens using next-generation sequencing technology.
abstract: Next-generation sequencing (NGS) technologies have increasingly played crucial roles in biological and medical research, but are not yet in routine use in veterinary diagnostic laboratories. We developed and applied a procedure for high-throughput RNA sequencing of Porcine reproductive and respiratory syndrome virus (PRRSV) from cell culture-derived isolates and clinical specimens. Ten PRRSV isolates with known sequence information, 2 mixtures each with 2 different PRRSV isolates, and 51 clinical specimens (19 sera, 16 lungs, and 16 oral fluids) with various PCR threshold cycle (Ct) values were subjected to nucleic acid extraction, cDNA library preparation (24-plexed), and sequencing. Whole genome sequences were obtained from 10 reference isolates with expected sequences and from sera with a PRRSV real-time reverse transcription PCR Ct ≤ 23.6, lung tissues with Ct ≤ 21, and oral fluids with Ct ≤ 20.6. For mixtures with PRRSV-1 and -2 isolates (57.8% nucleotide identity), NGS was able to distinguish them as well as obtain their respective genome sequences. For mixtures with 2 PRRSV-2 isolates (92.4% nucleotide identity), sequence reads with nucleotide ambiguity at numerous sites were observed, indicating mixed infection; however, individual virus sequences could only be separated when 1 isolate identity and sequence in the mixture is known. The NGS approach described herein offers the prospect of high-throughput sequencing and could be adapted to routine workflows in veterinary diagnostic laboratories, although further improvement of sequencing outcomes from clinical specimens with higher Ct values remains to be investigated.
authors.affiliation: Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA (Zhang, Zheng, Chen, Bade, Yoon, Harmon, Gauger, Main, Li).|Laboratory of Aquatic Bioinformatics, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China (Xia).
authors.forename: Jianqiang
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA (Zhang, Zheng, Chen, Bade, Yoon, Harmon, Gauger, Main, Li).|Laboratory of Aquatic Bioinformatics, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China (Xia).
authors.forename: Ying
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA (Zhang, Zheng, Chen, Bade, Yoon, Harmon, Gauger, Main, Li).|Laboratory of Aquatic Bioinformatics, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China (Xia).
authors.forename: Xiao-Qin
authors.initials: XQ
authors.lastname: Xia
authors.affiliation: Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA (Zhang, Zheng, Chen, Bade, Yoon, Harmon, Gauger, Main, Li).|Laboratory of Aquatic Bioinformatics, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China (Xia).
authors.forename: Qi
authors.initials: Q
authors.lastname: Chen
authors.affiliation: Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA (Zhang, Zheng, Chen, Bade, Yoon, Harmon, Gauger, Main, Li).|Laboratory of Aquatic Bioinformatics, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China (Xia).
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Bade
authors.affiliation: Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA (Zhang, Zheng, Chen, Bade, Yoon, Harmon, Gauger, Main, Li).|Laboratory of Aquatic Bioinformatics, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China (Xia).
authors.forename: Kyoung-Jin
authors.initials: KJ
authors.lastname: Yoon
authors.affiliation: Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA (Zhang, Zheng, Chen, Bade, Yoon, Harmon, Gauger, Main, Li).|Laboratory of Aquatic Bioinformatics, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China (Xia).
authors.forename: Karen M
authors.initials: KM
authors.lastname: Harmon
authors.affiliation: Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA (Zhang, Zheng, Chen, Bade, Yoon, Harmon, Gauger, Main, Li).|Laboratory of Aquatic Bioinformatics, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China (Xia).
authors.forename: Phillip C
authors.initials: PC
authors.lastname: Gauger
authors.affiliation: Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA (Zhang, Zheng, Chen, Bade, Yoon, Harmon, Gauger, Main, Li).|Laboratory of Aquatic Bioinformatics, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China (Xia).
authors.forename: Rodger G
authors.initials: RG
authors.lastname: Main
authors.affiliation: Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA (Zhang, Zheng, Chen, Bade, Yoon, Harmon, Gauger, Main, Li).|Laboratory of Aquatic Bioinformatics, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China (Xia).
authors.forename: Ganwu
authors.initials: G
authors.lastname: Li
mesh_terms: D000818:Animals; D016679:Genome, Viral; D059014:High-Throughput Nucleotide Sequencing / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000662:veterinary; D019318:Porcine Reproductive and Respiratory Syndrome / Q000097:blood / Q000175:diagnosis* / Q000821:virology; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000302:isolation & purification*; D011237:Predictive Value of Tests; D012367:RNA, Viral / Q000032:analysis; D013552:Swine
chemical_list: D012367:RNA, Viral
keywords: Cell culture isolates; Porcine reproductive and respiratory syndrome virus; clinical specimens; lung; next-generation sequencing; oral fluid; serum