A fast and robust method for full genome sequencing of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Type 1 and Type 2

Kvisgaard, Lise Kirstine; Hjulsager, Charlotte Kristiane; Fahnøe, Ulrik; Breum, Solvej Østergaard; Aït-Ali, Tahar; Larsen, Lars Erik

doi:10.1016/j.jviromet.2013.07.019

Cited by 27 publications

(19 citation statements)

References 32 publications

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“…The genome of PRRSV-2/Hungary/102/2012 was amplified in five overlapping parts (similarly to Kvisgaard et al, 2013b) using the Phusion II HotStart PCR kit (Thermo Scientific, Waltham, MA, USA), in 25 l final volume with 1 l of cDNA template, in 1× GC buffer in the presence of 4% DMSO. Amplification was performed using the following gradient PCR program: 98 • C 1 , 35 × [98 • C 20 , 54-72 • C (gradient ramp: 2 • C) 20 , 72 • C 5 ], 72 • C 5 .…”

Section: Rna Isolation and Cdna Synthesismentioning

confidence: 99%

Full genome sequence analysis of a wild, non-MLV-related type 2 Hungarian PRRSV variant isolated in Europe

et al. 2015

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Section: Rna Isolation and Cdna Synthesismentioning

confidence: 99%

Full genome sequence analysis of a wild, non-MLV-related type 2 Hungarian PRRSV variant isolated in Europe

et al. 2015

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“…More recently next-generation sequencing (NGS) of fragments generated by long range RT-PCR has been used to characterise multiple PRRSV genomes [6]. However, this approach relies upon prior knowledge of the target sequence and the assumption that the PCR primer binding sites are non-variable.…”

Section: Resultsmentioning

confidence: 99%

“…This full length Olot/91 contains 8 SNPs and a 3 nt insertion when compared with the published partial Olot/91 sequence. However, this PAM-passaged virus only shares 96.8% similarity with the MARC-145 cell-adapted PRRSV Olot/91 strain (Genbank:KC862570) [6]. The differences may represent the molecular basis for the adaption to growth on MARC-145 cells which are of African Green Monkey origin [17].…”

Section: Resultsmentioning

confidence: 99%

Genomic variation in macrophage-cultured European porcine reproductive and respiratory syndrome virus Olot/91 revealed using ultra-deep next generation sequencing

Brown

Wilson

et al. 2014

Virol J

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BackgroundPorcine Reproductive and Respiratory Syndrome (PRRS) is a disease of major economic impact worldwide. The etiologic agent of this disease is the PRRS virus (PRRSV). Increasing evidence suggest that microevolution within a coexisting quasispecies population can give rise to high sequence heterogeneity in PRRSV.FindingsWe developed a pipeline based on the ultra-deep next generation sequencing approach to first construct the complete genome of a European PRRSV, strain Olot/9, cultured on macrophages and then capture the rare variants representative of the mixed quasispecies population. Olot/91 differs from the reference Lelystad strain by about 5% and a total of 88 variants, with frequencies as low as 1%, were detected in the mixed population. These variants included 16 non-synonymous variants concentrated in the genes encoding structural and nonstructural proteins; including Glycoprotein 2a and 5.ConclusionUsing an ultra-deep sequencing methodology, the complete genome of Olot/91 was constructed without any prior knowledge of the sequence. Rare variants that constitute minor fractions of the heterogeneous PRRSV population could successfully be detected to allow further exploration of microevolutionary events.

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“…The key interests addressed in this study included whether sequencing can detect PRRSV strains to identify an outbreak as occurring due to the introduction of a new strain or recirculation of a previous outbreak, whether sequencing can generate whole genome information to aid in further understanding of PRRSV epidemiology, and whether sequencing can detect and differentiate multiple strains in a single sample to investigate outbreaks that occur in vaccinated herds or co-infection of multiple field-strains. Previously, PRRSV whole genomes have been generated using Sanger and Illumina sequencing technologies [10,47,72]. While both sequencing technologies can generate a whole PRRSV genome with more than 99.9% accuracy, the raw reads produced are usually less than 1500 bp.…”

Section: Discussionmentioning

confidence: 99%

“…Since then, studies have been performed for other viruses, confirming the potential of MinION technology to aid in the detection of infectious viral agents [45,46]. PRRSV whole genome sequencing (WGS) has been carried out previously using traditional Sanger sequencing [10] and next-generation short-read sequencing platforms [47]. Incorporating a bioinformatic approach, we systematically evaluated and standardized third-generation long-read MinION DRS for PRRSV whole genome generation and determined its ability to detect the viral strain present, its analytical sensitivity for strain-level diagnosis of clinical samples, and its feasibility for differentiation of co-existing multiple strains in a single sample.…”

mentioning

confidence: 99%

Rapid, Unbiased PRRSV Strain Detection Using MinION Direct RNA Sequencing and Bioinformatics Tools

Tan

Dvorak

Murtaugh

2019

Viruses

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Prompt detection and effective control of porcine reproductive and respiratory syndrome virus (PRRSV) during outbreaks is important given its immense adverse impact on the swine industry. However, the diagnostic process can be challenging due to the high genetic diversity and high mutation rate of PRRSV. A diagnostic method that can provide more detailed genetic information about pathogens is urgently needed. In this study, we evaluated the ability of Oxford Nanopore MinION direct RNA sequencing to generate a PRRSV whole genome sequence and detect and discriminate virus at the strain-level. A nearly full length PRRSV genome was successfully generated from raw sequence reads, achieving an accuracy of 96% after consensus genome generation. Direct RNA sequencing reliably detected the PRRSV strain present with an accuracy of 99.9% using as few as 5 raw sequencing reads and successfully differentiated multiple co-infecting strains present in a sample. In addition, PRRSV strain information was obtained from clinical samples containing 10 4 to 10 6 viral copies or more within 6 hours of sequencing. Overall, direct viral RNA sequencing followed by bioinformatic analysis proves to be a promising approach for identification of the viral strain or strains involved in clinical infections, allowing for more precise prevention and control strategies during PRRSV outbreaks.2 of 18 against a new introduction from outside the farm indicates a need for enhanced biosecurity, while a re-break of a resident strain suggests better strategies for an elimination or vaccination program are needed. Another big challenge for PRRS control is that PRRSV vaccine is not completely effective at preventing and controlling infection due to the high genetic diversity of the virus, thus outbreaks still occur in vaccinated herds [13][14][15][16]. A diagnostic method which can provide genetic information about the strain causing infection would allow for identification of potential reasons for vaccination failure, such as limited cross-protection due to high genetic divergence from vaccine [17], or in the case of genetic similarity to vaccine, perhaps a reversion to virulence (escape mutation) of the vaccine itself [18]. In addition to the clinical challenges mentioned above, PRRSV has widely divergent genetic lineages and is a rapidly evolving pathogen with novel variants which seem to be more divergent and virulent than those in the past [10,[19][20][21]. The continuous emergence of new virulent strains causes unexpected devastating outbreaks, such as the severe outbreaks of HP-PRRSV in China and MN184 and NADC30 outbreaks in the United States [22][23][24][25]. The increasing incidence of co-infections of multiple strains further complicates PRRS diagnosis and control [26]. Hence, diagnostic tools that can provide more genetic information are extremely important for investigation, prevention, and control strategies for PRRSV outbreaks.Prompt detection of pathogens during an outbreak is essential for efficient disease control. Real-time quantitative...

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A fast and robust method for full genome sequencing of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Type 1 and Type 2

Cited by 27 publications

References 32 publications

Full genome sequence analysis of a wild, non-MLV-related type 2 Hungarian PRRSV variant isolated in Europe

Full genome sequence analysis of a wild, non-MLV-related type 2 Hungarian PRRSV variant isolated in Europe

Genomic variation in macrophage-cultured European porcine reproductive and respiratory syndrome virus Olot/91 revealed using ultra-deep next generation sequencing

Rapid, Unbiased PRRSV Strain Detection Using MinION Direct RNA Sequencing and Bioinformatics Tools

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