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

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1132 ◽  
Author(s):  
Shaoyuan Tan ◽  
Cheryl M.T. Dvorak ◽  
Michael P. Murtaugh
Keyword(s):  
Rna Sequencing ◽  
Bioinformatic Analysis ◽  
Whole Genome Sequence ◽  
Effective Control ◽  
Clinical Samples ◽  
Diagnostic Process ◽  
Respiratory Syndrome Virus ◽  
Swine Industry ◽  
High Genetic Diversity ◽  
Prrsv Strain

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 104 to 106 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.

scholarly journals Multiplex RT-PCR assay to differentiate genotypes of porcine reproductive and respiratory syndrome virus in swine

2019 ◽  
Vol 18 (06) ◽  
pp. 8-13
Author(s):  
Phat X. Dinh
Keyword(s):  
Real Time ◽  
Clinical Samples ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Swine Industry ◽  
Diagnostic Challenge ◽  
Nsp2 Gene ◽  
Prrs Virus ◽  
Pcr Products

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases to swine industry worldwide. Due to the heterogeneity of field isolates, accurate detection of the PRRS virus is a diagnostic challenge. Recently, co-infection with NA-PRRSV, EU-PRRSV and HP-PRRSV isolates continuously increases in many countries, resulting in a significant impact on PRRSV diagnostics and disease control on farms. To facilitate rapid diagnosis and reliable discrimination of NA-PRRSV, EU-PRRSV and HP-PRRSV, a multiplex RT-PCR assay was established with three pairs of primers targeting highly conservative regions of nsp2 gene with predicted multiplex RT-PCR products of 364 bp, 161 bp and 259 bp, respectively. The primer pairs were optimized to be highly specific for PRRSV genotypes and were able to detect the target gene at the limit of 102 copies/μL for each gene. Clinical samples were used to evaluate this multiplex RT-PCR in parallel with a commercial real-time RT-PCR kit. Results showed over 95.2% (20/21 samples) agreement between the mRT-PCR and the real-time RT-PCR kit. Hence, it indicated that this multiplex RT-PCR could be useful for rapid and deferential diagnosis of NA-PRRSV, EU-PRRSV and HP-PRRSV in swine farms.

scholarly journals Whole-Genome Sequencing of Porcine Reproductive and Respiratory Syndrome Virus from Field Clinical Samples Improves the Genomic Surveillance of the Virus

2020 ◽  
Vol 58 (11) ◽  
Author(s):  
Christian Lalonde ◽  
Chantale Provost ◽  
Carl A. Gagnon
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Full Length ◽  
Clinical Samples ◽  
Respiratory Syndrome Virus ◽  
Whole Genome ◽  
Serum Samples ◽  
Diagnostic Laboratory ◽  
Prrsv Strain ◽  
Orf5 Gene

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) is a major economic concern worldwide. There are currently large data sets available about the ORF5 gene of the virus, with thousands of sequences available, but little data are currently available on the full-length genome of PRRSV. We hypothesized that whole-genome sequencing (WGS) of the PRRSV genome would allow better epidemiological monitoring than ORF5 gene sequencing. PRRSV PCR-positive serum, oral fluid, and tissue clinical samples submitted to the diagnostic laboratory for routine surveillance or diagnosis of PRRSV infection in Québec, Canada, swine herds were used. The PRRSV reverse transcription-quantitative PCR Cq values of the processed samples varied between 11.5 and 34.34. PRRSV strain genomes were isolated using a poly (A)-tail method and were sequenced with a MiSeq Illumina sequencer. Ninety-two full-length PRRSV genomes were obtained from 88 clinical samples out of 132 tested samples, resulting in a PRRSV WGS success rate of 66.67%. Three important deletions in ORF1a were found in most wild-type (i.e., not vaccine-like) strains. The importance of these deletions remains undetermined. Two different full-length PRRSV genomes were found in four different samples (three serum samples and one pool of tissues), suggesting a 4.55% PRRSV strain coinfection prevalence in swine. Moreover, six PRRSV whole genomes (6.52% of PRRSV strains) were found to cluster differently than they did under the ORF5 classification method. Overall, WGS of PRRSV enables better strain classification and/or interpretation of results in 9.10% of clinical samples than ORF5 sequencing, as well as allowing interesting research avenues.

scholarly journals WGS- versus ORF5-Based Typing of PRRSV: A Belgian Case Study

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2419
Author(s):  
Frank Vandenbussche ◽  
Elisabeth Mathijs ◽  
Marylène Tignon ◽  
Tamara Vandersmissen ◽  
Ann Brigitte Cay
Keyword(s):  
Genetic Diversity ◽  
Large Scale ◽  
Control Strategies ◽  
Added Value ◽  
Respiratory Syndrome Virus ◽  
Serum Samples ◽  
Swine Industry ◽  
High Genetic Diversity ◽  
Rate Analysis ◽  
Phylogenetic Cluster

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most widespread and economically devastating diseases in the swine industry. Typing circulating PRRSV strains by means of sequencing is crucial for developing adequate control strategies. Most genetic studies only target the highly variable open reading frame (ORF) 5, for which an extensive database is available. In this study, we performed whole-genome sequencing (WGS) on a collection of 124 PRRSV-1 positive serum samples that were collected over a 5-year period (2015–2019) in Belgium. Our results show that (nearly) complete PRRSV genomes can be obtained directly from serum samples with a high success rate. Analysis of the coding regions confirmed the exceptionally high genetic diversity, even among Belgian PRRSV-1 strains. To gain more insight into the added value of WGS, we performed phylogenetic cluster analyses on separate ORF datasets as well as on a single, concatenated dataset (CDS) containing all ORFs. A comparison between the CDS and ORF clustering schemes revealed numerous discrepancies. To explain these differences, we performed a large-scale recombination analysis, which allowed us to identify a large number of potential recombination events that were scattered across the genome. As PRRSV does not contain typical recombination hot-spots, typing PRRSV strains based on a single ORF is not recommended. Although the typing accuracy can be improved by including multiple regions, our results show that the full genetic diversity among PRRSV strains can only be captured by analysing (nearly) complete genomes. Finally, we also identified several vaccine-derived recombinant strains, which once more raises the question of the safety of these vaccines.

scholarly journals Pathogenic Characterization of European Genotype Porcine Reproductive and Respiratory Syndrome Virus Recently Isolated in Mainland China

2017 ◽  
Vol 11 (1) ◽  
pp. 83-89 ◽  
Author(s):  
Sun Ming ◽  
Ma Yongying ◽  
Liu Bohua ◽  
Lu Huiying ◽  
Deng Xiaoyu ◽  
...  
Keyword(s):  
Mainland China ◽  
Economic Loss ◽  
Respiratory Syndrome Virus ◽  
Type I ◽  
Swine Industry ◽  
Virulent Type ◽  
Prrsv Strain ◽  
European Genotype ◽  
And Control

Background:Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen in pig that causes tremendous economic loss in the global swine industry. PRRSV is divided into the European and North American genotypes, with virulence ranging from apathogenic-moderately virulent to highly pathogenic. The emergence of new highly virulent type 1 strains and coexistence of the two genotypes complicate the differential diagnosis, disease prevention, and control of PRRSV. Although the emergence of a novel type 1 PRRSV strain in mainland China was first confirmed in 2011, there is no information available concerning the pathogenesis of this strain.Objectives:We sought to determine the pathogenesis of a newly emerged Chinese type 1 PRRSV strain HLJB1.Methods:Pigs were infected with HLJB1 and characterized using serological and histopathological tests.Results:HLJB1 infection induced transient chemosis, reddened conjunctiva, skin cyanosis, mild transient pyrexia, dyspnea, and tachypnea between 7 and 13 days post-infection. Gross pneumonic lesions were characterized by multifocal, tan-mottled areas. Lymph nodes and spleen were enlarged. Characteristic microscopic lesions consisted of pulmonary consolidation and alveolar septal thickening with red blood cell infiltration, depletion of splenic lymphocytes, and hyperplasia and activation of macrophage. No pigs infected with HLJB1 died during the experiment.Conclusion:Our findings indicate that Chinese type I PRRSV strain HLJB1 caused classic PRRSV-specific lesions. As it caused lower viremia in pigs compared with other classic type 1 isolates, HLJB1 is less virulent than other type I strains.

scholarly journals Structural comparison of CD163 SRCR5 from different species sheds some light on its involvement in porcine reproductive and respiratory syndrome virus-2 infection in vitro

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Hongfang Ma ◽  
Rui Li ◽  
Longguang Jiang ◽  
Songlin Qiao ◽  
Xin-xin Chen ◽  
...  
Keyword(s):  
Scavenger Receptor ◽  
Host Cells ◽  
Respiratory Syndrome Virus ◽  
Swine Industry ◽  
Prrs Virus ◽  
Rich Domain ◽  
Serious Disease ◽  
The One ◽  
And Control

AbstractPorcine reproductive and respiratory syndrome (PRRS) is a serious disease burdening global swine industry. Infection by its etiological agent, PRRS virus (PRRSV), shows a highly restricted tropism of host cells and has been demonstrated to be mediated by an essential scavenger receptor (SR) CD163. CD163 fifth SR cysteine-rich domain (SRCR5) is further proven to play a crucial role during viral infection. Despite intense research, the involvement of CD163 SRCR5 in PRRSV infection remains to be elucidated. In the current study, we prepared recombinant monkey CD163 (moCD163) SRCR5 and human CD163-like homolog (hCD163L1) SRCR8, and determined their crystal structures. After comparison with the previously reported crystal structure of porcine CD163 (pCD163) SRCR5, these structures showed almost identical structural folds but significantly different surface electrostatic potentials. Based on these differences, we carried out mutational research to identify that the charged residue at position 534 in association with the one at position 561 were important for PRRSV-2 infection in vitro. Altogether the current work sheds some light on CD163-mediated PRRSV-2 infection and deepens our understanding of the viral pathogenesis, which will provide clues for prevention and control of PRRS.

scholarly journals Molecular Identification of Mushroom Species in Italy: An Ongoing Project Aimed at Reinforcing the Control Measures of an Increasingly Appreciated Sustainable Food

2020 ◽  
Vol 13 (1) ◽  
pp. 238
Author(s):  
Alice Giusti ◽  
Enrica Ricci ◽  
Laura Gasperetti ◽  
Marta Galgani ◽  
Luca Polidori ◽  
...  
Keyword(s):  
Selection Process ◽  
Winning Strategy ◽  
Control Measures ◽  
Effective Control ◽  
Mushroom Poisoning ◽  
Clinical Samples ◽  
Similar Species ◽  
Mushroom Species ◽  
Ongoing Research ◽  
Genotypic Analysis

Proper investment in mushroom production (farming and wild mushroom picking activities) may represent a winning strategy for many countries, including Italy, to better face the problems of food security and environmental impact, and to break away from imports, enhancing the local products. However, the risk related to the consumption of poisoning species requires governments to implement or reinforce effective control measures to protect consumers. Mushroom identification by phenotype observation is hardly applicable if morphologically-similar species, non-whole specimens, or clinical samples are involved. Genotypic analysis is a valid alternative. An ongoing research project involving the Experimental Zooprophylactic Institute of Lazio and Tuscany, the regional Mycological Inspectorate, the Tuscany Mycological Groups Association, and the Department of Veterinary Sciences of the University of Pisa aims to reinforce the collaboration among institutions for the management of mushroom poisoning. The core’s project aims to develop an internal genetic database to support the identification of wild and cultivated mushroom species in the Italian territory. The database will include Internal Transcribed Spacer (ITS) sequences retrieved from official databases (the NCBI GenBank and the BOLD system) which are considered to be reliable, after a proper selection process, and sequences from specimens collected directly and identified by expert mycologists. Once it is validated, the database will be available and further implementable by the official network of national laboratories.

scholarly journals Prediction of seasonal patterns of porcine reproductive and respiratory syndrome virus RNA detection in the U.S. swine industry

2020 ◽  
Vol 32 (3) ◽  
pp. 394-400
Author(s):  
Giovani Trevisan ◽  
Leticia C. M. Linhares ◽  
Bret Crim ◽  
Poonam Dubey ◽  
Kent J. Schwartz ◽  
...  
Keyword(s):  
Robust Regression ◽  
Historical Data ◽  
The United States ◽  
Respiratory Syndrome Virus ◽  
Seasonal Patterns ◽  
Test Results ◽  
Swine Industry ◽  
Cyclic Pattern ◽  
Rna Detection ◽  
Virus Rna

We developed a model to predict the cyclic pattern of porcine reproductive and respiratory syndrome virus (PRRSV) RNA detection by reverse-transcription real-time PCR (RT-rtPCR) from 4 major swine-centric veterinary diagnostic laboratories (VDLs) in the United States and to use historical data to forecast the upcoming year’s weekly percentage of positive submissions and issue outbreak signals when the pattern of detection was not as expected. Standardized submission data and test results were used. Historical data (2015–2017) composed of the weekly percentage of PCR-positive submissions were used to fit a cyclic robust regression model. The findings were used to forecast the expected weekly percentage of PCR-positive submissions, with a 95% confidence interval (CI), for 2018. During 2018, the proportion of PRRSV-positive submissions crossed 95% CI boundaries at week 2, 14–25, and 48. The relatively higher detection on week 2 and 48 were mostly from submissions containing samples from wean-to-market pigs, and for week 14–25 originated mostly from samples from adult/sow farms. There was a recurring yearly pattern of detection, wherein an increased proportion of PRRSV RNA detection in submissions originating from wean-to-finish farms was followed by increased detection in samples from adult/sow farms. Results from the model described herein confirm the seasonal cyclic pattern of PRRSV detection using test results consolidated from 4 VDLs. Wave crests occurred consistently during winter, and wave troughs occurred consistently during the summer months. Our model was able to correctly identify statistically significant outbreak signals in PRRSV RNA detection at 3 instances during 2018.

scholarly journals Traveling Wave Solutions of a Four Dimensional Reaction-Diffusion Model for Porcine Reproductive and Respiratory Syndrome with Time Dependent Infection Rate

Computation ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 30
Author(s):  
Jeerawan Suksamran ◽  
Yongwimon Lenbury ◽  
Sanoe Koonprasert
Keyword(s):  
Traveling Wave ◽  
Traveling Wave Solutions ◽  
Reaction Diffusion ◽  
Growing Pigs ◽  
Respiratory Syndrome Virus ◽  
Swine Industry ◽  
Reaction Diffusion Model ◽  
Spatial Dimensions ◽  
Tangent Method ◽  
Spatial Transmission

Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failure in sows and respiratory disease in piglets and growing pigs. The disease rapidly spreads in swine populations, making it a serious problem causing great financial losses to the swine industry. However, past mathematical models used to describe the spread of the disease have not yielded sufficient understanding of its spatial transmission. This work has been designed to investigate a mathematical model for the spread of PRRSV considering both time and spatial dimensions as well as the observed decline in infectiousness as time progresses. Moreover, our model incorporates into the dynamics the assumption that some members of the infected population may recover from the disease and become immune. Analytical solutions are derived by using the modified extended hyperbolic tangent method with the introduction of traveling wave coordinate. We also carry out a stability and phase analysis in order to obtain a clearer understanding of how PRRSV spreads spatially through time.

scholarly journals The Function of the PRRSV–Host Interactions and Their Effects on Viral Replication and Propagation in Antiviral Strategies

Vaccines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 364
Author(s):  
Jun Ma ◽  
Lulu Ma ◽  
Meiting Yang ◽  
Wei Wu ◽  
Wenhai Feng ◽  
...  
Keyword(s):  
Immune Response ◽  
Molecular Mechanisms ◽  
Immune Escape ◽  
Rna Virus ◽  
Economic Losses ◽  
Respiratory Syndrome Virus ◽  
Swine Industry ◽  
Live Virus ◽  
Virus Challenge ◽  
Host Interactions

Porcine reproductive and respiratory syndrome virus (PRRSV) affects the global swine industry and causes disastrous economic losses each year. The genome of PRRSV is an enveloped single-stranded positive-sense RNA of approximately 15 kb. The PRRSV replicates primarily in alveolar macrophages of pig lungs and lymphatic organs and causes reproductive problems in sows and respiratory symptoms in piglets. To date, studies on how PRRSV survives in the host, the host immune response against viral infections, and pathogenesis, have been reported. PRRSV vaccines have been developed, including inactive virus, modified live virus, attenuated live vaccine, DNA vaccine, and immune adjuvant vaccines. However, there are certain problems with the durability and effectiveness of the licensed vaccines. Moreover, the high variability and fast-evolving populations of this RNA virus challenge the design of PRRSV vaccines, and thus effective vaccines against PRRSV have not been developed successfully. As is well known, viruses interact with the host to escape the host’s immune response and then replicate and propagate in the host, which is the key to virus survival. Here, we review the complex network and the mechanism of PRRSV–host interactions in the processes of virus infection. It is critical to develop novel antiviral strategies against PRRSV by studying these host–virus interactions and structures to better understand the molecular mechanisms of PRRSV immune escape.

Porcine reproductive and respiratory syndrome virus infection upregulates negative immune regulators and T-cell exhaustion markers

2021 ◽  
Author(s):  
Jayeshbhai Chaudhari ◽  
Chia-Sin Liew ◽  
Jean-Jack M. Riethoven ◽  
Sarah Sillman ◽  
Hiep L. X. Vu
Keyword(s):  
T Cell ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Acute Stage ◽  
Respiratory Syndrome Virus ◽  
Porcine Alveolar Macrophage ◽  
Host Immune System ◽  
T Cell Exhaustion ◽  
Swine Industry ◽  
Cell Exhaustion

Porcine alveolar macrophage (PAM) is one of the primary cellular targets for PRRSV, but less than 2% of PAMs are infected with the virus during the acute stage of infection. To comparatively analyze the host transcriptional response between PRRSV-infected PAMs and bystanders PAMs that remained uninfected but were exposed to the inflammatory milieu of an infected lung, pigs were infected with a PRRSV strain expressing green fluorescent protein (PRRSV-GFP) and GFP + (PRRSV infected) and GFP – (bystander) cells were sorted for RNA-sequencing (RNA-seq). Approximately 4.2% of RNA reads from GFP + and 0.06% reads from GFP – PAMs mapped to the PRRSV genome, indicating that PRRSV-infected PAMs were effectively separated from bystander PAMs. Further analysis revealed that inflammatory cytokines, interferon-stimulated genes, and antiviral genes were highly upregulated in GFP + as compared to GFP – PAMs. Importantly, negative immune regulators including NF-κB inhibitors (NFKBIA, NFKBID, NFKBIZ, and TNFAIP3), and T-cell exhaustion markers (PD-L1, PD-L2, IL10, IDO1, and TGFB2) were highly upregulated in GFP + cells as compared to GFP – cells. By using in situ hybridization assay, RNA transcripts of TNF and NF-κB inhibitors were detected in PRRSV-infected PAMs cultured ex vivo and lung sections of PRRSV-infected pigs during the acute stage of infection. Collectively, the results suggest that PRRSV infection upregulates expression of negative immune regulators and T-cell exhaustion markers in PAMs to modulate the host immune response. Our findings provide further insight into PRRSV immunopathogenesis. Importance PRRSV is widespread in many swine producing countries, causing substantial economic loses to the swine industry. PAM is considered the primary target for PRRSV replication in pigs. However, less than 2% of PAM from an acutely infected pigs are infected with the virus. In the present study, we utilized a PRRSV-GFP strain to infect pigs and sorted infected- and bystander- PAMs from the pigs during the acute stage of infection for transcriptome analysis. PRRSV infected PAMs showed a distinctive gene expression profile and contained many uniquely activated pathways compared to bystander PAMs. Interestingly, upregulated expression of and NF-κB signaling inhibitors and T-cell exhaustion molecules were observed in PRRSV-infected PAMs. Our findings provide additional knowledge on the mechanisms that PRRSV employs to modulate the host immune system.

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