Evaluation of infection status in Chinese swine with porcine reproductive and respiratory syndrome virus by nested RT-PCR targeting nsp2 gene

2016 ◽  
Vol 44 ◽  
pp. 55-60 ◽  
Author(s):  
Minxia Zhang ◽  
Xinan Li ◽  
Xinna Cai ◽  
Yajin Qu ◽  
Dongfang Hu ◽  
...  
Keyword(s):  
Respiratory Syndrome Virus ◽  
Infection Status ◽  
Rt Pcr ◽  
Nsp2 Gene ◽  
Pcr Targeting

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 Molecular Characterization of the Nsp2 and ORF5 (ORF5a) Genes of PRRSV Strains in Nine Provinces of China During 2016–2018

2021 ◽  
Vol 8 ◽  
Author(s):  
Baishuang Yin ◽  
Shanshan Qi ◽  
Wanli Sha ◽  
Hongyu Qin ◽  
Liming Liu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Economic Losses ◽  
Respiratory Syndrome Virus ◽  
Recombination Analysis ◽  
Rt Pcr ◽  
Nsp2 Gene ◽  
Virulent Strains ◽  
Positive Rate ◽  
Wild Strains

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a highly contagious disease and brings huge economic losses to commercial pork production worldwide. PRRSV causes severe reproductive failure in sows and respiratory distress in piglets. To trace the evolution of PRRSV in pigs with respiratory diseases in some regions of China, 112 samples were collected from nine provinces in China during 2016–2018. All samples were detected by RT-PCR and analyzed by the Nsp2/ORF5 (ORF5a)-genes-phylogeny. Sequence analysis and recombination analysis were conducted on the Nsp2/ORF5 (ORF5a) genes of the identified strain in the study. The RT-PCR result shown that the positive rate of PRRSV was 50.89% (57/112). Phylogenetic analysis showed that the identified PRRSV strains were all NA genotype and belonged to lineage 1, 3, and 8. The Nsp2 gene of identified PRRSV strains exhibited nucleotide homologies of 53.0 ~ 99.8%, and amino acid homologies of 46.8 ~ 99.7%. The ORF5 gene of identified PRRSV strains exhibited nucleotide homologies of 82.4 ~ 100%, and amino acid homologies of 79.6 ~ 100%. Sequence analysis revealed that a discontinuous 30-amino-acid deletion (positions 481 and 533–561) and a 131-amino-acid discontinuity deletion (positions 323–433, 481, and 533–551) in Nsp2 of PPRSV isolates; all identified strains in this study may be wild strains, and most identified strains may be highly virulent strains. Sequence analysis of ORF5 and ORF5a revealed that the mutation sites of GP5 were mainly concentrated in the signal peptide and epitopes region, while the mutation sites of ORF5a were mainly concentrated in the transmembrane and the intramembrane region. The recombination analysis indicated that there may be multiple recombination regions in identified strains, and the recombination pattern was more complex. This study showed that the prevalent PRRSV strain in some regions of China was still HP-PRRSV, while NADC30 strain also occupied a certain proportion; different types of PRRSV strains showed different patterns and variation in China. This study suggested that the monitoring of PRRSV prevalence and genetic variation should be further strengthened.

scholarly journals Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 615
Author(s):  
Allen Wing-Ho Chu ◽  
Cyril Chik-Yan Yip ◽  
Wan-Mui Chan ◽  
Anthony Chin-Ki Ng ◽  
Dream Lok-Sze Chan ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Nucleic Acid Extraction ◽  
Suitable Alternative ◽  
Pcr Inhibitors ◽  
Pcr Targeting

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

Validation and comparison of different end point and real time RT-PCR assays for detection and genotyping of porcine reproductive and respiratory syndrome virus

2014 ◽  
Vol 201 ◽  
pp. 79-85 ◽  
Author(s):  
Michele Drigo ◽  
Giovanni Franzo ◽  
Ilaria Belfanti ◽  
Marco Martini ◽  
Alessandra Mondin ◽  
...  
Keyword(s):  
Real Time ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
End Point ◽  
Pcr Assays

Clinical diagnosis of early dengue infection by novel one-step multiplex real-time RT-PCR targeting NS1 gene

2015 ◽  
Vol 65 ◽  
pp. 11-19 ◽  
Author(s):  
Je-Hyoung Kim ◽  
Chom-Kyu Chong ◽  
Mangalam Sinniah ◽  
Jeyaindran Sinnadurai ◽  
Hyun-Ok Song ◽  
...  
Keyword(s):  
Real Time ◽  
Clinical Diagnosis ◽  
Dengue Infection ◽  
Rt Pcr ◽  
One Step ◽  
Ns1 Gene ◽  
Pcr Targeting

scholarly journals Lack of evidence of porcine reproductive and respiratory syndrome virus (PRRSV) infection in domestic swine in Brazil

2004 ◽  
Vol 34 (2) ◽  
pp. 449-455 ◽  
Author(s):  
Janice Reis Ciacci-Zanella ◽  
Cristiano Trombetta ◽  
Ildara Vargas ◽  
Denise Euclydes Mariano da Costa
Keyword(s):  
Genetic Material ◽  
Elisa Test ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
Serum Samples ◽  
Viral Isolation ◽  
Culture Cells ◽  
Commercial Kits ◽  
Swine Herds ◽  
Domestic Swine

This report describes the first prevalence of antibodies and experimental inoculation of suspected samples of porcine reproductive and respiratory syndrome virus (PRRSV) from ELISA positive pigs from swine herds in Brazil. Based on the hypothesis that this agent is present in swine herds worldwide, the objective of this work was to establish a diagnostic methodology and to investigate the occurrence of PRRSV in Brazilian swine herds. Fifty-four swine herds, the total number which imported genetic material (live pigs or swine semen) from countries where PRRS was endemic from 1990 to December 2000, from eight Brazilian States all included in this study. The sampling used was such as to detect a prevalence of infection of 5%, with a confidence level of 95%. A total of 3785 serum samples were tested for PRRSV antibodies by ELISA. Following the ELISA test, which was performed with two different commercial kits, all serum positive pigs were retested, examined and additional materials were collected. Viral isolation in permissive tissue culture cells and swine bioassays were performed. Additionally, reverse transcriptase polymerase chain reaction (RT-PCR) and nested RT-PCR were also performed. We could not demonstrate the presence of PRRSV or RNA of PRRSV by viral isolation or RT-PCR (or nested RT-PCR), respectively in all of the analyzed samples. Furthermore, the pigs inoculated with PRRSV suspicion samples did not seroconvert nor produce characteristic PRRS lesions in the swine bioassay. Thus, our results indicate no evidence of PRRSV in the samples analyzed from swine herds in this study.

scholarly journals Detection and genetic identification of pestiviruses in Brazilian lots of fetal bovine serum collected from 2006 to 2014

2018 ◽  
Vol 38 (3) ◽  
pp. 387-392 ◽  
Author(s):  
Francielle L. Monteiro ◽  
Juliana F. Cargnelutti ◽  
Patrícia Braunig ◽  
Aurea V. Folgueras-Flatschart ◽  
Nathália C. Santos ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Genetic Identification ◽  
Nucleotide Sequencing ◽  
Rt Pcr ◽  
Diarrhea Virus ◽  
Fetal Bovine ◽  
Viral Diarrhea Virus ◽  
Pcr Targeting

ABSTRACT: The present study performed a genetic identification of pestiviruses contaminating batches of fetal bovine serum (FBS) produced in Brazil from 2006 to 2014. Seventy-three FBS lots were screened by a RT-PCR targeting the 5’untranslated region (UTR) of the pestivirus genome. Thirty-nine lots (53.4%) were positive for pestivirus RNA and one contained infectious virus. Nucleotide sequencing and phylogenetic analysis of the 5’UTR revealed 34 lots (46.6%) containing RNA of bovine viral diarrhea virus type 1 (BVDV-1), being 23 BVDV-1a (5’ UTR identity 90.8-98.7%), eight BVDV-1b (93.9-96.7%) and three BVDV-1d (96.2- 97.6%). Six lots (8.2%) contained BVDV-2 (90.3-100% UTR identity) being two BVDV-2a; three BVDV-2b and one undetermined. Four FBS batches (5.5%) were found contaminated with HoBi-like virus (98.3 to 100%). Five batches (6.8%) contained more than one pestivirus. The high frequency of contamination of FBS with pestivirus RNA reinforce the need for systematic and updated guidelines for monitoring this product to reduce the risk of contamination of biologicals and introduction of contaminating agents into free areas.

scholarly journals Rapid, sensitive, full genome sequencing of Severe Acute Respiratory Syndrome Virus Coronavirus 2 (SARS-CoV-2)

2020 ◽  
Author(s):  
Clinton R Paden ◽  
Ying Tao ◽  
Krista Queen ◽  
Jing Zhang ◽  
Yan Li ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Sanger Sequencing ◽  
Respiratory Syndrome Virus ◽  
Full Genome ◽  
Miseq Sequencing ◽  
Genomic Information ◽  
Rt Pcr ◽  
Full Genome Sequencing ◽  
High Quality ◽  
Global Pandemic

AbstractSARS-CoV-2 recently emerged, resulting a global pandemic. Rapid genomic information is critical to understanding transmission and pathogenesis. Here, we describe validated protocols for generating high-quality full-length genomes from primary samples. The first employs multiplex RT-PCR followed by MinION or MiSeq sequencing. The second uses singleplex, nested RT-PCR and Sanger sequencing.

scholarly journals Monitoring porcine reproductive and respiratory syndrome virus infection status in swine herds based on analysis of antibodies in meat juice samples

2001 ◽  
Vol 32 (5) ◽  
pp. 441-453 ◽  
Author(s):  
Sten Mortensen ◽  
Bertel Strandbygaard ◽  
Anette B�tner ◽  
Niels Feld ◽  
Preben Willeberg
Keyword(s):  
Virus Infection ◽  
Respiratory Syndrome Virus ◽  
Infection Status ◽  
Meat Juice ◽  
Swine Herds

scholarly journals Sequencing and analysis of complete genome of the attenuated Hanvet1.VN strain used for vaccine production against porcine reproductive and respiratory syndrome

2018 ◽  
Vol 16 (1) ◽  
pp. 51-57
Author(s):  
Nguyễn Thị Nga ◽  
Hà Thị Thu ◽  
Nguyễn Thị Hoa ◽  
Vũ Thị Hiền ◽  
Trần Thị Thu Hiền ◽  
...  
Keyword(s):  
North American ◽  
Sanger Sequencing ◽  
Respiratory Syndrome Virus ◽  
Whole Genome ◽  
Type Ii ◽  
Vaccine Production ◽  
Rt Pcr ◽  
North American Strain ◽  
American Strain ◽  
Vietnamese Strain

The porcine reproductive and respiratory syndrome virus (PRRSV) attenuated strain Hanvet1.VN has been developed by the Pharmaceutical and Veterinary Material J.S.C (HANVET) by passaging HY-2010 strain on MARC-145 cells for 80 passages and used for PRRS vaccine production. In this study, we sequenced and analyzed the whole genome of the attenuated Hanvet1.VN strain. The total RNA was extracted from the Hanvet1.VN strain, RT-PCR was used for amplification of 15 separate segments of the whole genome. The amplified segments were cloned into the pCR2.1 vector and sequenced by Sanger sequencing. The sequences were analyzed with BioEdit and DNA Star Software. The results showed that, GP5 of the Hanvet1.VN attenuated strain had 100% identity in amino acid (aa) sequences with one of the pathogenic Vietnamese strain isolated in Quang Nam Province and had 98% identity with that of the Chinese 07NM strain. However, the identity of aa sequence of the Hanvet1.VN GP5 was much lower in the comparison with GP5 of VR2332, and it was only 87%. The MP and NP proteins were highly conserved compared with pathogenic strains circulating in Vietnam (07QN) and China (07NM) (99-100%, respectively). The other eight proteins of the Hanvet1.VN strain showed changes from 1.2% in NP1a to 3.9% in GP2 compared with the 07QN strain. However, the aa identity of all Hanvet1.VN proteins were very low when compared with proteins of PRRSV type II strain (North American strain, VR2332), ranged from 86.25% to 97.7%. Our results showed that the Hanvet1.VN attenuated vaccine strain had protective immunogenicity similar to that strain circulating in Vietnam closely related to a strain from China but different from the type II North American strain VR2332. Hence, for importing PRRSV vaccine, especially from American or Europe Countries, antigenic compatibility of the PRRSV vaccine and strains circulating in Vietnam should be concerned in PRRSV vaccine production.

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