A multiplex PCR for rapid and simultaneous detection of porcine circovirus type 2, porcine parvovirus,porcine pseudorabies virus,and porcine reproductive and respiratory syndrome virus in clinical specimens

A multiplex PCR (mPCR) assay was developed and evaluated for its ability to simultaneously detect multiple viral infections of swine. Specific primers were designed for each of the following four DNA or RNA viruses: porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), pseudorabies virus (PRV), and porcine reproductive and respiratory syndrome virus (PRRSV). Each target produced a specific amplicon with a size of 353 bp (PCV2), 271 bp (PPV), 194 bp (PRV), or 434 bp (PRRSV). The assay was sensitive and specific in detecting each target agent in composite cell cultures and clinical specimens. Results from mPCR were confirmed by PCR for individual viruses and by virus isolation. In conclusion, the mPCR has the potential to be useful for routine molecular diagnosis and epidemiology.     

A multiplex DNA suspension microarray for simultaneous detection and differentiation of classical swine fever virus and other pestiviruses

An oligonucleotide suspension microarray (Luminex microsphere system) was developed for detection and differentiation of animal pestiviruses: classical swine fever virus (CSFV), bovine viral diarrhea virus types 1 and 2 (BVDV1 and BVDV2), and border disease virus (BDV). Species-specific and pestivirus-common oligonucleotide probes were designed to the 5' UTR region and conjugated to individual color-coded Luminex carboxy beads (probe beads). Target pestivirus sequences were amplified by asymmetric PCR using a biotinylated reverse primer and a forward and reverse primer ratio of 1:5. The biotinylated products were hybridized to eight probe beads in a multiplex assay and analyzed using streptavidin conjugated to a fluorescent reporter molecule. The assay was able to detect and differentiate all 40 strains of CSFV, BVDV1, BVDV2 and BDV tested. The analytical sensitivity was determined to be 0.2-10 TCID50/ml. The major advantages of the DNA-microsphere suspension microarray, as a low density array, are its ease of handling and ability to simultaneously detect and type multiple infectious agents.     

A multiplex nested RT-PCR for the detection and differentiation of wild-type viruses from C-strain vaccine of classical swine fever virus

A multiplex nested RT-PCR (RT-nPCR) was developed for the detection and differentiation of classical swine fever virus (CSFV). A fragment of 447 or 343 bp was amplified from the genomic RNA of C-strain or virulent Shimen strain, respectively, and two fragments of 447 and 343 bp were simultaneously amplified from the mixed samples of C-strain and Shimen. When detecting several wild-type isolates representative of different subgroups (1.1, 2.1, 2.2, and 2.3) circulating in Mainland China and samples from pigs experimentally infected with Shimen strain, the RT-nPCR resulted in an amplification pattern similar to Shimen. No amplification was achieved for uninfected cells, or cells infected with bovine viral diarrhea virus (BVDV), and other viruses of porcine origin. The RT-nPCR was able to detect as little as 0.04 pg of CSFV RNA. The restrictive fragment length polymorphism (RFLP) demonstrated unique patterns of wild-type viruses and C-strain. Among the 133 field samples, 42 were tested to contain wild-type viruses and 18 showing presence of C-strain. The RT-nPCR can be used to detect and differentiate pigs infected with wild-type CSFV from those vaccinated with C-strain vaccine, thus minimizing the risk of culling vaccinates during outbreaks.     

A SYBR Green-based real-time RT-PCR assay for simple and rapid detection and differentiation of highly pathogenic and classical type 2 porcine reproductive and respiratory syndrome virus circulating in China

SYBR Green coupled to melting curve analysis has been suggested to detect RNA viruses showing high genomic variability. Here, a SYBR Green-based real-time RT-PCR assay was developed for simultaneous detection and differentiation of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and classical type 2 PRRSV (C-PRRSV). The different strains were identified by their distinctive melting temperatures: 82.98 ± 0.25 °C and 85.95 ± 0.24 °C for HP-PRRSVs or 82.74 ± 0.26 °C for C-PRRSVs. Specificity was tested using nine other viral and bacterial pathogens of swine. The detection limit was 1 TCID₅₀ for HP- or C-PRRSV. Furthermore, the detection results for samples from an animal trial with HP- or C-PRRSV infections showed that the SYBR Green-based real-time RT-PCR was more sensitive than the conventional RT-PCR. Additionally, an analysis of 319 field samples from North China, Central China and Northeast China showed that HP- and C-PRRSVs co-circulated in pig herds. Thus, the SYBR Green-based real-time RT-PCR, which can be performed within one hour, is a rapid, sensitive and low-cost diagnostic tool for rapid differential detection and routine surveillance of HP- and classical type 2 PRRSVs in China.     

Simultaneous detection of Classical swine fever virus and North American genotype Porcine reproductive and respiratory syndrome virus using a duplex real-time RT-PCR

Classical swine fever and porcine reproductive and respiratory syndrome are both notifiable diseases of the World Organization for Animal Health (OIE). The two diseases exhibit indistinguishable clinical symptoms and sometimes co-exist in swine herds. In this study, a duplex real-time RT-PCR for simultaneous detection of Classical swine fever virus (CSFV) and North American (NA) genotype Porcine reproductive and respiratory syndrome virus (PRRSV) based on two differently labeled TaqMan probes was developed and evaluated. The detection limit of the assay was 3.2 TCID(50) or 13 RNA copies for CSFV and 1.8 TCID(50) or 10 RNA copies for PRRSV, about 50 times more sensitive than conventional RT-PCRs. The duplex real-time RT-PCR was capable of specifically detecting different subgroups of wild-type CSFV and different strains of NA-genotype PRRSV, whereas a number of non-CSFV/PRRSV porcine viruses and bovine pestivirus were tested negative. Out of 155 field samples, 16 were tested positive for CSFV, 73 were positive for PRRSV, and 13 were co-infected with the two viruses. These results were 99.4% in agreement with those using conventional RT-PCRs. Therefore, the assay provides sensitive and simultaneous detection and differentiation of CSFV and PRRSV.     

Multiplex method for simultaneous serological detection of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2

Porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) are major contributors to the porcine respiratory disease complex (PRDC). Routine serological diagnosis and surveillance play an important role in the prevention of PRDC, as it is a leading cause of economic losses to the swine industry. We herein describe an advanced microsphere-based immunoassay that permits the simultaneous detection of antibodies to PCV2 and PRRSV, thereby reducing the time and effort involved in testing. Recombinant PRRSV nucleoprotein antigen and the PCV2 capsid antigen were coupled to fluorophore-dyed beads with distinct spectral addresses. Weekly serum samples from 72 pigs that were experimentally exposed to either PCV2, PRRSV, or both PCV2 and PRRSV were used to validate the microbead assay (MBA) in comparison with the "gold standard" enzyme-linked immunosorbent assays. The kinetics of the PCV2- and PRRSV-specific antibody responses measured by the microbead assay were comparable to those of the standard assays; Spearman's rank correlations were 0.72 (P < 0.001) for PRRSV and 0.80 (P < 0.001) for PCV2. Diagnostic sensitivity and specificity were determined using field sera whose positive or negative status was determined by the standard tests. The diagnostic sensitivity and specificity were both 98% for PCV2 and were 91% and 93%, respectively, for PRRSV (kappa coefficients, 0.85 and 0.67 for PCV2 and PRRSV, respectively). Multiplexing did not interfere with assay performance or diagnostic sensitivity. Therefore, the described study demonstrates proof of concept for the development of more versatile and economical microbead array-based multiplex serological test panels for veterinary use.     

Development and preliminary application of an immunochromatographic strip for rapid detection of infection with porcine reproductive and respiratory syndrome virus in swine

A "strip test" to detect porcine reproductive and respiratory syndrome virus (PRRSV) was established using a monoclonal antibody (MAb) 2D7 conjugated with colloidal gold. Two MAbs binding to protein N at different epitopes, 2D7 and 1G7 were obtained. In the test, samples of PRRSV bound to colloidal gold-conjugated MAb 2D7. The complex was then captured by MAb 1G7 at the test line (T) on the nitrocellulose membrane, presenting a purple band. If the sample did not contain PRRSV or if the quantity of PRRSV was less than that required for the kit, only the control line (C), in which goat anti-mouse antibody was added as the capture antibody, was present. Results from the sensitivity test of the kit demonstrated that the lowest detected quantity of PRRSV is 2.9 × 10(3)TCID(50)/ml. In clinical trials, the specificity and the sensitivity of this kit are 98.1% and 88.4%, respectively, compared with RT-PCR. Furthermore, this kit was found to be efficient in three areas of China and appears to have better results in practical applications than in empirical studies. In summary, this kit has not only high rates of specificity and sensitivity but also has the beneficial features such as efficiency, convenience and speed.     

Validation of two commercial real-time RT-PCR kits for rapid and specific diagnosis of classical swine fever virus

Two real-time RT-PCR kits, developed by LSI (TaqVet CSF) and ADIAGENE (Adiavet CSF), obtained an agreement to be commercialised in France, subject to conditions, defined by the French Classical Swine Fever (CSF) National Reference Laboratory. The producers were asked to introduce an internal control to check the RNA extraction efficacy. The different criteria assessed were sensitivity, "pestivirus specificity", reproducibility and ease of handling, using 189 different samples. These samples were either CSFV inactivated strains or blood/serum/organs collected from CSFV experimentally infected pigs or naturally infected wild boars. The reproducibility of the assays was confirmed by the analysis of a batch-to-batch panel control that was used for inter-laboratory tests involving nine laboratories. The two kits were also tested for the use in mass diagnostics and the results proved the kits to be suited using pools of blood, serum and tonsils. Moreover, a field evaluation, carried out on spleen samples collected from the CSF surveillance of wild boars in an area known to be infected and from domestic pigs at a slaughterhouse, confirmed the high sensitivity and specificity of the two kits. This step-by-step evaluation procedure confirmed that the two commercial CSF real-time RT-PCR kits have a higher predictive value than the current diagnostic standard, Virus Isolation.     

Immunological responses of swine to porcine reproductive and respiratory syndrome virus infection

The immunology of porcine reproductive and respiratory syndrome virus (PRRS) begins with an initial encounter of PRRSV with the pig. Regardless of the route of entry of PRRSV--via inhalation, intramuscular vaccination, insemination, or other routes--productive infection occurs predominately in alveolar macrophages of the lung. Thus, innate responses of the lung and the alveolar macrophage comprise the initial defense against PRRSV. The virus appears not to elicit innate interferon and cytokine responses characteristic of other strongly immunogenic viral pathogens, and its effects are consistent with induction of a weak adaptive immune response. Humoral and cell-mediated immunity is induced in due course, and results in clearance of virus from the circulation but not from lymphoid tissues, where the infection becomes persistent. Subsequent reexposure to PRRSV elicits an anamnestic response that is partially to completely protective. Within this unconventional picture of anti-PRRSV immunity lie a variety of unresolved issues, including the nature of protective immunity within individual pigs and among pigs in commercial populations, the efficacy of protective immunity against genetically different PRRSV isolates, the effects of developmental age, sex, genetics, and other host factors on the immune response to PRRSV, and the possible suppression of host immunity to other pathogens.     

Development of an immunochromatographic strip for rapid detection of antibodies against classical swine fever virus

The genes encoding the Erns and E2 antigen epitopes of classical swine fever virus (CSFV) were expressed as a chimeric protein in Escherichia coli BL21 by pET expression system. The antigenicity of the expressed protein CnC2 was identified by indirect enzyme-linked immunoabsorbant assay (ELISA) and immunoblot with anti-CSFV antibodies. Based on the CnC2 protein, an immunochromatographic strip was developed to evaluate the antibody titer of serum samples from swine vaccinated with CSFV vaccine rapidly. The chimeric protein used as a detector was labeled with colloidal gold. Staphylococcal protein A (SPA) and anti-CnC2 monoclonal antibodies (mAbs) were blotted onto the nitrocellulose membrane as the test and control lines, respectively. The strip assay could be performed within 5min, which did not require any special equipment or skills. Through testing sera against various strains of CSFV, the sensitivity of the strip was determined to be 97.0% (65/67) and the specificity was 100% (98/98). The strip results were consistent with those of the existing commercial ELISA kit, and their correlation coefficient was 0.935. In conclusion, the immunochromatographic strip was an acceptable method for surveying CSFV-antibody titers in pigs.     

Heterogeneity of swine lung macrophages inoculated by porcine reproductive and respiratory syndrome virus

The biological features of porcine alveolar macrophages (PAMs) and interstitial macrophages (IMs) were investigated, including morphology, nitric oxide (NO) secretion, cell viability and porcine reproductive and respiratory syndrome virus (PRRSV) mRNA expression post-inoculation with TJ-F10 or TJM-F92. Viability and NO secretion of PAMs and IMs were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Griess's assay, respectively. mRNA expression of PRRSV, inducible nitric oxide synthase (iNOS) and Arginase1 (Arg1) in PAMs and IMs were detected by quantitative real-time polymerase chain reaction technique. Our results show that PAMs were bigger and more granular than IMs and the Arg1/iNOS value was much higher in PAMs than in IMs. In addition, the vaccine strain TJM-F92 evoked higher NO production in PAMs and IMs compared with the wild type strain TJ-F10. In conclusion, our results indicate that the PAMs and IMs are heterogeneous in morphology, NO production and susceptibility to PRRSV.     

A simple and rapid immunochromatographic strip test for detecting antibody to porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome is rapidly gaining worldwide importance as one of the most economically significant diseases of swine. The antibody of Porcine reproductive and respiratory syndrome virus (PRRSV) is detected currently by the combined use of an enzyme-linked immunosorbent assay, serum neutralization test, immunoperoxidase monolayer assay, indirect immunofluorescent antibody test. These methods are time-consuming and require specialized equipment operated by trained technicians. The purpose of this study was to evaluate a simple strip assay (based on a chromatographic and immunogold system) for specific detection of PRRSV antibody in swine sera. This "immunochromatographic strip" test uses Escherichia coli-expressed viral recombinant membrane protein antigen in combination with recombinant nucleocapsid protein as capture protein for detecting antibodies against PRRSV. In this study, the performance of this assay was evaluated with sera from both clinical samples and experimentally infected piglets. Detection by immunochromatographic strip test was compared with detection by a standard, available commercially, indirect enzyme-linked immunosorbent assay and an immunoperoxidase monolayer assay. The immunochromatographic test strip detected antibodies in sera known to contain antibodies to PRRSV in 95.7% sensitivity of samples from pigs infected experimentally and 98.6% sensitivity of clinical serum samples. For sera that did not contain antibodies to PRRSV, the specificity was 97.8% and 98.2% for clinical and experimental serum samples, respectively.     

Real-time RT-PCR assay for rapid and specific detection of classical swine fever virus: comparison of SYBR Green and TaqMan MGB detection methods using novel MGB probes

Real-time PCR is an accurate, rapid and reliable method that can be used for the detection and also for the quantitation of specific DNA molecules. The basic principle is the recurring measurement of a fluorescent signal, which is proportional to the amount of amplification product. In our trial two detection systems were tested for classical swine fever virus (CSFV) detection and for its discrimination from other pestiviruses; non-specific dsDNA-binding dye SYBR Green and specific fluorogenic TaqMan MGB probes. Real-time RT-PCR assays were evaluated for diagnostic sensitivity and specificity by different pestiviral reference and field strains. With both approaches, SYBR Green and TaqMan probes, respectively, all of the CSFV strains isolated on cell culture were detected and also clearly distinguished from other pestiviruses. However, the established one-step real-time TaqMan RT-PCR assay was shown to be more appropriate for pestivirus quantitation, it reduces the risk of contamination and is less time consuming.     

A sensitive fluorescence in situ hybridization technique for detection of porcine reproductive and respiratory syndrome virus.

A sensitive fluorescence in situ hybridization (ISH) for detecting porcine reproductive and respiratory syndrome virus (PRRSV) RNA in viral infected tissue was developed using digoxigenin-labeled RNA probes targeted on the nucleocapsid gene of PRRSV. In situ RNA/RNA hybrids were detected with an anti-digoxigenin antibody alkaline phosphatase conjugate and further revealed with Fast Red TR salt/naphthol AS-MX phosphate using a fluorescent microscope. Viral nucleic acid was readily demonstrated within macrophages, known to be the major target of PRRSV. In addition, positively stained cells were found in the salivary gland and skin tissues which have not been reported to contain PRRSV infected cells before. In conclusion, the fluorescence ISH used in this study provides a fast and sensitive means for screening virus-infected tissues in which relatively few cells are affected. This advantage will be especially beneficial for studying viral persistence and for routine diagnosis of PRRSV infection.     

Simple and rapid detection of the porcine reproductive and respiratory syndrome virus from pig whole blood using filter paper

The combination of Flinders Technology Associates filter papers (FTA cards) and real-time PCR was examined to establish a simple and rapid technique for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) from whole pig blood. A modified live PRRS vaccine was diluted with either sterilised saline or pig whole blood, and the suspensions were applied onto the FTA cards. The real-time RT-PCR detection of PRRSV was performed directly with the samples applied to the FTA card without the RNA extraction step. Six whole blood samples from at random selected piglets in the PRRSV infected farm were also assayed in this study. The expected PCR product was successfully amplified from either saline diluted or pig whole blood diluted vaccine. The same PCR ampliocon was detected from all blood samples assayed in this study. This study suggested that the combination of an FTA card and real-time PCR is a rapid and easy technique for the detection of PRRSV. This technique can remarkably shorten the time required for PRRSV detection from whole blood and makes the procedure much easier.