Rapid differential detection of classical and highly pathogenic North American Porcine Reproductive and Respiratory Syndrome virus in China by a duplex real-time RT-PCR

Since the emergence of highly pathogenic North American Porcine Reproductive and Respiratory Syndrome virus (H-US-PRRSV) in 2006, the classical North American PRRSV (C-US-PRRSV) and H-US-PRRSV isolates have coexisted in Chinese swine herds. A duplex real-time RT-PCR assay using minor groove binder (MGB) probes for differential detection of the two US PRRSV isolates was developed. The specificity, sensitivity, reproducibility, and interference test of this assay were validated. The sensitivity of the assay was 3.2 TCID₅₀/ml or 38 RNA copies/μl for C-US-PRRSV and 0.4 TCID₅₀/ml or 14 RNA copies/μl for H-US-PRRSV. Both assays were 10 times more sensitive than the current methods. A total of 302 clinical samples were tested by duplex real-time RT-PCR and conventional RT-PCR assays, and the results showed over 98.7% agreement. In addition, the new assay can be completed in less than 2 h. This duplex real-time RT-PCR assay is a promising tool for rapid differential detection and epidemiology of US PRRSV in China.     

A multiplex RT-PCR for rapid and simultaneous detection of porcine teschovirus, classical swine fever virus, and porcine reproductive and respiratory syndrome virus in clinical specimens

A multiplex RT-PCR (mRT-PCR) assay was developed and evaluated for its ability to detect multiple viral infections of swine simultaneously. One pair of primers was selected carefully for each of the following three RNA viruses: porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and porcine teschovirus (PTV). Each target produced a specific amplicon with a size of 451bp (PRRSV), 343bp (CSFV), or 163bp (PTV). The sensitivity of the mRT-PCR using purified plasmid constructs containing the specific viral target fragments was 2.02 x 102, 2.90 x 103, and 6.16 x 103 copies for PRRSV, CSFV, and PTV, respectively. Among 69 clinical samples from Heilongjiang, Jilin, and Henan provinces, co-infection by PRRSV and CSFV was 4.4%, co-infection by PRRSV and PTV was 11.6%, co-infection by PTV and CSFV was 13.0%, and co-infection by the three viruses was 8.7%. In conclusion, the mRT-PCR should be useful for routine molecular diagnosis and epidemiology.     

Evaluation of a real-time RT-PCR assay using minor groove binding probe for specific detection of Chinese wild-type classical swine fever virus

A one-step real-time RT-PCR assay using a minor groove binding probe was developed for the specific detection of Chinese wild-type classical swine fever virus (CSFV). The assay detected wild-type CSFV strains representing different genotypes, but did not amplify viral RNA from the Hog Cholera Lipinized Virus (HCLV) vaccine-strain and other porcine viruses. The assay had a detection limit of 10 copies/reaction or 3.0 median tissue culture infective dose/reaction. In comparison to the sequencing nested RT-PCR assay, the sensitivity and specificity of the assay were 98.3% and 94.3%, respectively, when testing 515 veterinary samples. Wild-type CSFV RNA was detected in nasal swabs 2-4 days before detection in serum samples from pigs exposed to infection by contact, and 2-4 days prior to the onset of clinical disease. HCLV RNA remained undetectable in nasal swabs and serum samples from vaccinated pigs. In conclusion, the novel assay described in this study provides a rapid and sensitive method for differentiating between wild-type and the HCLV-strain of CSFV. It could be used for monitoring in CSF outbreak areas or as a screening method for CSFV eradication strategies.     

Recombination between North American strains of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV), a recently discovered arterivirus swine pathogen, was shown to undergo homologous recombination. Co-infection of MA-104 cells with two culture-adapted North American PRRSV strains resulted in recombinant viral particles containing chimeric ORF 3 and ORF 4 proteins. Nucleotide sequence analysis of cloned recombinant PCR products, encompassing 1182 bases of the 15.4 kb viral genome, revealed six independent recombination events. Recombinant products persisted in culture for at least three passages, indicating continuous formation of recombinant viruses, growth of recombinant viruses in competition with parental viruses, or both. The frequency of recombination was estimated from <2% up to 10% in the 1182 b fragment analyzed, which is similar to recombination frequencies observed in coronaviruses. An apparent example of natural ORF 5 recombination between naturally occurring wild type viruses was also found, indicating that recombination is likely an important genetic mechanism contributing to PRRSV evolution.     

Development of duplex real-time RT-PCR for detection of influenza virus A and B

Influenza viruses continue to be accountable for winter outbreaks. The potential for developing complications is higher in certain risk groups, such as children, the elderly and individuals with chronic medical conditions. We have presented a duplex real-time RT-PCR for detection of influenza A and B. The sensibility of our method was estimated at 0.01TCID(50)/ml for detection of influenza A and 0.1TCID(50)/ml for influenza B. Any other viruses with respiratory tract tropism were detected with our method. After that, we tested our duplex RT-PCR on 119 samples (nasal aspirates and liquids of broncho-alveolar washes) collected in the CHRU of Lille between November 2005 and January 2006 from patients aged one month to 81 years. Conventional methods such as direct immunofluorescence (IF) assay and/or cell culture were applied on these samples. Four samples were positive for influenza A virus detection and, in particular, one liquid of broncho-alveolar wash for which the direct IF assay was negative. Thus, our method is adapted to diagnosis of flu infections. Nevertheless, our duplex RT-PCR will be tested during a flu outbreak.     

Porcine reproductive and respiratory syndrome virus induces apoptosis through a mitochondria-mediated pathway

As with a number of other viruses, Porcine reproductive and respiratory syndrome virus (PRRSV) has been shown to induce apoptosis, although the mechanism(s) involved remain unknown. In this study we have characterized the apoptotic pathways activated by PRRSV infection. PRRSV-infected cells showed evidence of apoptosis including phosphatidylserine exposure, chromatin condensation, DNA fragmentation, caspase activation (including caspase-8, 9, 3), and PARP cleavage. DNA fragmentation was dependent on caspase activation but blocking apoptosis by a caspase inhibitor did not affect PRRSV replication. Upregulation of Bax expression by PRRSV infection was followed by disruption of the mitochondria transmembrane potential, resulting in cytochrome c redistridution to the cytoplasm and subsequent caspase-9 activation. A crosstalk between the extrinsic and intrinsic pathways was demonstrated by dependency of caspase-9 activation on active caspase-8 and by Bid cleavage. Furthermore, in this study we provide evidence of the possible involvement of reactive oxygen species (ROS)-mediated oxidative stress in apoptosis induced by PRRSV. Our data indicated that cell death caused by PRRSV infection involves necrosis as well as apoptosis. In summary, these findings demonstrate mechanisms by which PRRSV induces apoptosis and will contribute to an enhanced understanding of PRRSV pathogenesis.     

Porcine reproductive and respiratory syndrome virus: an update on an emerging and re-emerging viral disease of swine

Recognized in the late 1980s in North America and Europe the syndrome that caused reproductive and respiratory problems in swine was initially called "mystery swine disease" and is now termed "porcine reproductive and respiratory syndrome (PRRS)". In the early 1990 s an arterivirus, referred to as PRRS virus (PRRSV), was determined to be the etiologic agent of this disease. Since then research has progressed substantially. Most recently "porcine high fever disease" was reported in China starting in 2006 with PRRSV being a critical virus associated with high morbidity and mortality (20%) associated with this syndrome which in 2010 is still causing severe pathology in pigs in China, with spread to Vietnam and Cambodia. This volume contains a series of reviews that highlight the virus, its pathogenesis, epidemiology, immunology, vaccinology and host genetic control. This paper provides a brief historical review of PRRS and the associated PRRSV. It presents areas of research gaps that inhibit current progress towards PRRS elimination through production of effective vaccines and current plans for PRRS elimination or eradication programs. It is hoped that this discussion will stimulate further collaboration between researchers and swine veterinarians throughout the world to provide answers that enhance our understanding of PRRS and PRRSV in an effort to eliminate this economically important disease.     

Simultaneous detection of West Nile and Japanese encephalitis virus RNA by duplex TaqMan RT-PCR

West Nile virus (WNV) and Japanese encephalitis virus (JEV) are important mosquito-borne viruses of the Flaviviridae family, associated with encephalitis, mainly in humans and horses. WNV is also pathogen for many bird species. The incidence of human and animal WNV infections in Europe has risen, mostly in recent years, and JEV was detected in 2011 in mosquitoes collected in Italy and may emerge in Europe in the same way as other flaviviruses had emerged recently (USUTU and Bagaza virus) and should be regarded as a potential threat to public health.     

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.     

Nucleocapsid protein-based enzyme-linked immunosorbent assay for detection and differentiation of antibodies against European and North American porcine reproductive and respiratory syndrome virus

Two types of porcine reproductive and respiratory syndrome virus (PRRSV) have been reported, the European type (EU PRRSV) and the North American type (US PRRSV). We developed a dual enzyme-linked immunosorbent assay (ELISA) for the simultaneous detection and differentiation of serum antibodies directed against either of the two PRRSV types. This tandem PRRS ELISA is based on affinity-purified recombinant nucleocapsid protein expressed in Escherichia coli. Sensitivity and specificity were assessed by using the IDEXX HerdChek PRRS ELISA and the indirect immunofluorescence assay as reference tests. A total of 1571 sera originating from the United States, Europe, and two PRRS-free countries, i.e., Switzerland and New Zealand, were used for validation of the tandem PRRS ELISA. The new test performed at least as well as the reference tests in regard to sensitivity (0.94 for the US PRRS ELISA and 0.93 for the EU PRRS ELISA) and specificity (0.96 for the US PRRS ELISA and 0.99 for the EU PRRS ELISA). Positive sera were correctly differentiated in 582 of 591 cases, indicating a high differentiation capability of this dual ELISA. The robustness and repeatability of the test were assessed and found to be appropriate for diagnostic applications. Taken together, the data indicate that the tandem PRRS ELISA described here is the first differentiation ELISA for PRRSV serology based on recombinant antigen. It is convenient with respect to antigen production, and it is reliable, economical, and highly sensitive and specific. Thus, it is considered to be a powerful tool for routine diagnostics, epidemiological surveys, and outbreak investigations.     

Sequence analysis of porcine reproductive and respiratory syndrome virus of the American type collected from Danish swine herds

Summary.  Vaccine-like viruses of American type of porcine reproductive and respiratory syndrome virus (PRRSV) were detected in serum samples by RT-PCR. The viruses were analysed by nucleotide sequencing of the genomic region encoding open reading frames 2 to 7. During the ongoing study of Danish isolates of PRRSV by means of nucleotide sequencing, RT-PCR reactions and subsequent nucleotide sequencing showed the presence of American type PRRSV in Danish breeding herds. Most likely, these atypical viruses originated from boars vaccinated with live vaccine of American type (MLV RespPRRS), which were taken to artificial insemination centres and there brought together with unvaccinated boars already at the centres. The nucleotide sequences of three Danish viruses of American type PRRSV were compared to those of known PRRSV isolates. The nucleotide sequence identities of the atypical Danish isolates were between 99.2–99.5% to the vaccine virus RespPRRS and 99.0–99.3% to VR2332 which are the parental virus to the vaccine virus. Phylogenetic analysis including field isolates of American type supports the conclusion that the introduction of American type PRRSV in Denmark was due to spread of vaccine virus. Received December 24, 1997 Accepted April 15, 1998     

Simultaneous detection of three fish rhabdoviruses using multiplex real-time quantitative RT-PCR assay

Spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are three important fish rhabdoviruses, causing serious Office International des Epizooties (OIE) classified diseases in wild and farmed fish. Here, a new multiplex real-time quantitative RT-PCR (mqRT-PCR) assay was developed for simultaneous detection, identification and quantification of these three rhabdoviruses. The sets of primers and probes were targeted to conserved regions of glycoprotein (G) gene of SVCV, nucleoprotein (N) gene of IHNV and G gene of VHSV and used to amplify. The sensitivity, specificity and interference test of mqRT-PCR assay was analyzed. It was shown that the detection levels of 100 copies of SVCV, 220 copies of IHNV and 140 copies of VHSV were achieved, and there was no non-specific amplification and cross-reactivity using RNA of pike fry rhabdovirus (PFRV), infectious pancreatic necrosis virus (IPNV) and grass carp reovirus (GCRV). A total of 80 clinical fish samples were tested using the mqRT-PCR assay and the results were confirmed by antigen-capture ELISA and cell culture assay. This assay has the potential to be used for both research applications and diagnosis.     

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.