Porcine reproductive and respiratory syndrome virus: Interlaboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction detection methods

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc (Impact Factor: 1.35). 07/2012; 24(5):855-66. DOI: 10.1177/1040638712452724
Source: PubMed


To compare the real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays used for the diagnosis of Porcine reproductive and respiratory syndrome virus (PRRSV), a Europe-wide interlaboratory ring trial was conducted. A variety of PRRSV strains including North American (NA) and European (EU) genotype isolates were analyzed by the participants. Great differences regarding qualitative diagnostics as well as analytical sensitivity were observed between the individual RT-qPCR systems, especially when investigating strains from the EU genotype. None of the assays or commercial kits used in the ring trial could identify all different PRRSV strains with an optimal analytical and diagnostic sensitivity. The genetic variability of the PRRSV strains, which is supposed to hinder the diagnostic of the RT-PCR because of mutations at the primer binding sites, was also confirmed by sequencing and subsequent phylogenetic analysis. In summary, a major problem in PRRSV diagnostics by RT-qPCR is false-negative results. To achieve maximum safety in the molecular diagnosis of PRRSV, the combined usage of different assays or kits is highly recommended.

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Available from: Paolo Bonilauri
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    • "However, national and international animal trade is exposed constantly to new strains, imposing on local laboratories to update continuously. Therefore, it is not surprising that the lack of sensitivity in diagnosis of PRRSV infection of both in-house and commercial kits has been reported by previous studies and laboratory experiences (Toplak et al., 2012; Wernike et al., 2012). "
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    ABSTRACT: The remarkable economic losses due to Porcine Reproductive and Respiratory Syndrome (PRRS) have stated the control and eradication of this disease one of the main issue of swine modern farming. The limited cross-protection of vaccine-induced immunity compelled the adoption of strict biosecurity measures that must be associated with the prompt diagnosis of infection. In our study four RT-PCR methods, a RT-PCR, a SYBR Green I and two hydrolysis probes, were compared to evaluate their respective benefits and disadvantages. One hundred and seventy samples originating from 50 farms located in northern Italy were tested with all assays and performances were evaluated using a Bayesian approach to deal with the absence of a Gold Standard. Sequencing the complete of ORF7, the segment targeted by all methods, allowed a gain of insight into the genetic variability of Italian strains and to investigate the role of mismatches on assay sensitivity. Our study evidenced that methods based only on primers-genome interaction better tolerate PRRSV genetic variability, demonstrating a greater sensitivity (Se): SYBR Green I (Se=98.4%) and RT-PCR (Se=99%) outperform both in-house (Se=71.4%) and commercial (Se=91.7%) probe-based methods. On the other hand, probe-based assays allowed an easier genotyping of PRRSV strains and implementation of the internal control system (IC). Phylogenetic analysis allowed demonstration of a presence of two clades circulating continuously in northern Italy since 1996, when their probable ancestors were collected.
    Full-text · Article · Mar 2014 · Journal of virological methods
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    • "Recently, increased rates of false-negative results were reported in several PRRSV reverse transcription polymerase chain reaction assays used commonly in veterinary diagnostic laboratories, and combined use of different assays has been recommended to improve the diagnosis for PRRSV in early stages of infection (Harmon et al., 2012; Toplak et al., 2012; Wernike et al., 2012; Gerber et al., 2013). ELISA is the method that is used most commonly for detection of antibodies against PRRSV (Okinaga et al., 2009) due to its specificity , cost-effectiveness, and simplicity. "
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    ABSTRACT: The objective of this study was to compare the ability of two commercial enzyme-linked immunosorbent assays (ELISAs) and an in-house fluorescent microbead immunoassay (FMIA) to detect IgG antibodies against porcine reproductive and respiratory syndrome virus (PRRSV) types 1 and 2 in serum and oral fluids from boars infected experimentally. Samples from uninfected control pigs and PRRSV-negative field samples were also used. Serum samples were tested by ELISAs of IDEXX-Se; HIPRA-Se; and an in-house FMIA-Se for detection of PRRSV types 1 and 2. Oral fluids were tested by ELISAs of IDEXX-SO and IDEXX-OF and HIPRA-OF for detection of PRRSV types 1 and 2. Among the sera, IDEXX-Se and HIPRA-Se had similar sensitivity and specificity (p>0.05); however, IDEXX-Se detected positive animals earlier than HIPRA-Se (p<0.05). FMIA-Se had the highest false-positive rates in known negative field samples (1/205 for IDEXX-Se, 5/205 for HIPRA-Se, and 37/205 for FMIA-Se; p<0.01). Serum and oral fluid samples had similar detection rates and antibody kinetics using the IDEXX tests. There was a higher detection rate in serum than oral fluid using the HIPRA assays. In this study, the nucleocapsid protein utilized as antigen in the FMIAs yielded a low specificity. IDEXX-Se had the earliest detection and similar sensitivity and specificity to the HIPRA-Se.
    Full-text · Article · Dec 2013 · Journal of virological methods
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    • "Only quite rare exceptions have so far been found from these subtypeprototypical nucleocapsid protein sizes (Fig. 1B, size variants marked with a star). Three individual ORF7 sequences of subtype 1, from pigs in Germany, Romania and Slovakia, show deduced nucleocapsid protein lengths of 126, 129 or 132 amino acids, respectively (Jackova et al., 2012; Wernike et al., 2012; Zaulet et al., 2012). Also, subtype 2 ORF7 sequences from one Russian (Ili) and one Belarusian pig farm (Bor) show deduced nucleocapsid protein lengths of 124 and 131 amino acids (Fig. 1B). "
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    ABSTRACT: Porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to European swine production. The existence of extensive genetic variation in endemic strains and the presence of highly virulent strains in other geographic regions pose the threat of devastating epidemic outbreaks. Here we describe the current knowledge of genetic variation in European PRRSV isolates, the implications for PRRSV evolution, and the presence of multiple genetic lineages of Type 2 (North American genotype) isolates in Europe. In Type 1 (European genotype) PRRSV, three genetic subtypes are recognized and a fourth subtype appears to be present. Type 2 PRRSV was considered to be genetically homogenous in Europe due to a unique presence of an introduced vaccine strain, but independent introductions of virulent Type 2 field viruses are now evident. In Type 1 PRRSV, only subtype 1 (Lelystad virus-like) circulates in Central and Western Europe and globally. In Eastern Europe, all subtypes are present. The subtypes of Type 1 PRRSV also exhibit length differences in the nucleocapsid protein, ranging in size from 124 to 132 amino acids depending on subtype. This size heterogeneity is unparalleled in the nucleocapsid proteins of Type 2 PRRSV or other viruses. Surprisingly, it affects the C-terminus, otherwise thought to be under strong structural constraints. Finally, divergent subtypes of Type 1 PRRSV have produced high rates of false-negative RT-PCR results in diagnostic tests, and may also degrade the reliability of serodiagnostic assays using the nucleocapsid protein antigen. In summary, the extensive genetic diversity of Type 1 PRRSV is of relevance for understanding nucleocapsid protein structure/function relationships. Further, the extensive genetic diversity of Type 1 PRRSV in Europe, and the presence of diverse Type 2 PRRSV strains, together emphasize the importance of relevant validation of PRRSV diagnostics. More extensive and systematic molecular phylogeny studies are needed to fully understand PRRSV diversity in Europe, to provide swine producers with reliable diagnostics, and to better assess the potential consequences of endemic spread and exotic introductions.
    Full-text · Article · Mar 2013 · Veterinary Microbiology
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