Development of an enzyme-linked immunosorbent assay using a monoclonal antibody to a dominant epitope in non-structural protein 4 of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) caused by the PRRS virus (PRRSV) is one of the most severe swine diseases causing great economic losses for the international swine industry. Non-structural protein 4 (NSP4) is critical to the life cycle of PRRSV and contains dominant B cell epitopes. This study prepared a monoclonal antibody against Nsp4, and 2D11, which contained the sequence 138KQGGGIVTRPSGQFCN153, was confirmed as the epitope. A 2D11-based double antibody sandwich enzyme-linked immunosorbent assay (dasELISA) was next developed with a cut value of 0.1987. A total of 1354 pig serum samples were detected by dasELISA and compared to a commercial ELISA kit (N-coated iELISA), resulting in a positive coincidence rate of 98.8% and negative coincidence rate of 96.9%. A total of 119 sera were positive by dasELISA while negative by iELISA. Higher positive rates by dasELISA were found in pig farms where PRRSV antibody levels varied widely. These results indicated that the dasELISA was a useful tool to detect PRRSV antibody in clinical samples.

Development and application of a blocking ELISA based on a N protein monoclonal antibody for the antibody detection against porcine reproductive and respiratory syndrome virus 2.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most widespread illnesses in the world's swine business. To detect the antibodies against PRRSV-2, a blocking enzyme-linked immunosorbent assay (B-ELISA) was developed, utilizing a PRRSV-2 N protein monoclonal antibody as the detection antibody. A checkerboard titration test was used to determine the optimal detection antibody dilution, tested pig serum dilution and purified PRRSV coated antigen concentration. After analyzing 174 negative pig sera and 451 positive pig sera, a cutoff value of 40 % was selected to distinguish between positive and negative sera using receiver operating characteristic curve analysis. The specificity and sensitivity of the assay were evaluated to equal 99.8 % and 96 %, respectively. The method had no cross-reaction with PCV2, PRV, PPV, CSFV, PEDV, TGEV, and PRRSV-1 serum antibodies, and the coefficients of variation of intra-batch and inter-batch repeatability experiments were both <10 %. A total of 215 clinical serum samples were tested, and the relative coincidence rate with commercial ELISA kit was 99.06 %, and the kappa value was 0.989, indicating that these two detection results exhibited high consistency. Overall, the B-ELISA should serve as an ideal method for large-scale serological investigation of PRRSV-2 antibodies in domestic pigs.

An intercomparison study of ELISAs for the detection of porcine reproductive and respiratory syndrome virus - evaluating six conditionally dependent tests.

Latent class analysis is a widely used statistical method for evaluating diagnostic tests without any gold standard. It requires the results of at least two tests applied to the same individuals. Based on the resulting response patterns, the method estimates the test accuracy and the unknown disease status for all individuals in the sample. An important assumption is the conditional independence of the tests. If tests with the same biological principle are used, the assumption is not fulfilled, which may lead to biased results. In a recent publication, we developed a method that considers the dependencies in the latent class model and estimates all parameters using frequentist methods. Here, we evaluate the practicability of the method by applying it to the results of six ELISA tests for antibodies against the porcine reproductive and respiratory syndrome (PRRS) virus in pigs that generally follow the same biological principle. First, we present different methods of identifying suitable starting values for the algorithm and apply these to the dataset and a vaccinated subgroup. We present the calculated values of the test accuracies, the estimated proportion of antibody-positive animals and the dependency structure for both datasets. Different starting values led to matching results for the entire dataset. For the vaccinated subgroup, the results were more dependent on the selected starting values. All six ELISA tests are well suited to detect antibodies against PRRS virus, whereas none of the tests had the best values for sensitivity and specificity simultaneously. The results thus show that the method used is able to determine the parameter values of conditionally dependent tests with suitable starting values. The choice of test should be based on the general fit-for-purpose concept and the population under study.

The Ability of Porcine Reproductive and Respiratory Syndrome Virus Isolates to Induce Broadly Reactive Neutralizing Antibodies Correlates With In Vivo Protection.

Porcine reproductive and respiratory syndrome (PRRS) is considered one of the most relevant diseases of swine. The condition is caused by PRRS virus (PRRSV), an extremely variable virus of the Arteriviridae family. Its heterogeneity can be responsible, at least partially, of the poor cross-protection observed between PRRSV isolates. Neutralizing antibodies (NAs), known to play a role in protection, usually poorly recognize heterologous PRRSV isolates, indicating that most NAs are strain-specific. However, some pigs develop broadly reactive NAs able to recognize a wide range of heterologous isolates. The aim of this study was to determine whether PRRSV isolates that induce broadly reactive NAs as determined in vitro are able to confer a better protection in vivo. For this purpose two in vivo experiments were performed. Initially, 40 pigs were immunized with a PRRSV-1 isolate known to induce broadly reactive NAs and 24 additional pigs were used as controls. On day 70 after immunization, the pigs were divided into eight groups composed by five immunized and three control pigs and exposed to one of the eight different heterologous PRRSV isolates used for the challenge. In the second experiment, the same experimental design was followed but the pigs were immunized with a PRRSV-1 isolate, which is known to generate mostly strain-specific NAs. Virological parameters, specifically viremia and the presence of challenge virus in tonsils, were used to determine protection. In the first experiment, sterilizing immunity was obtained in three groups, prevention of viremia was observed in two additional groups, although the challenge virus was detected occasionally in the tonsils of immunized pigs, and partial protection, understood as a reduction in the frequency of viremia compared with controls, was recorded in the remaining three groups. On the contrary, only partial protection was observed in all groups in the second experiment. The results obtained in this study confirm that PRRSV-1 isolates differ in their ability to induce cross-reactive NAs and, although other components of the immune response might have contributed to protection, pigs with cross-reactive NAs at the time of challenge exhibited better protection, indicating that broadly reactive NAs might play a role in protection against heterologous reinfections.

A modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine protects late-term pregnancy gilts against a heterologous PRRSV-2 challenge.

The objective of this study was to evaluate the efficacy of a modified-live virus (MLV) porcine reproductive and respiratory syndrome virus (PRRSV) vaccine against a heterologous PRRSV-2 challenge in late-term pregnancy gilts under experimental conditions. Eighteen gilts were randomly assigned to vaccinated-challenged, unvaccinated-challenged, and unvaccinated-unchallenged groups (n = 6 gilts per group). Pregnant gilts in the vaccinated-challenged and unvaccinated-unchallenged groups were able to carry their pregnancies to full term and farrowed after 114 to 115 days of gestation. In contrast, pregnant gilts in the unvaccinated-challenged group did not reach full term and farrowed early, after 104 to 108 days of gestation. Pregnant gilts vaccinated with the PRRSV-2 MLV vaccine exhibited a reduction in PRRSV-2 viremia. At the time of challenge with PRRSV-2, vaccinated gilts had relatively low levels of neutralizing antibody titers (≤ 1:16 titer), whereas the number of interferon-γ-secreting cells (IFN-γ-SC) was consistently at protective levels (IFN-γ-SC, ≥ 150 per million). Induction of cell-mediated immunity, as measured by PRRSV-2-specific IFN-γ-SC, correlated with a reduction in PRRSV-2 viremia. Duration of immunity was a minimum of 19 wk. Taken together, the results presented here suggest that vaccination of gilts with a PRRSV-2 MLV vaccine can protect against a heterologous PRRSV-2 challenge and improve the reproductive performance of late-term pregnancy gilts.

L'objectif de la présente étude était d'évaluer dans des conditions expérimentales l'efficacité d'un vaccin à virus vivant modifié (MLV) du virus du syndrome reproducteur et respiratoire porcin (PRRSV) contre une infection défi avec un PRRSV-2 hétérologue chez des cochettes en fin de gestation. Dix-huit cochettes furent assignées de manière aléatoire à un des groupes suivants: vaccinées-infectées, non-vaccinées-infectées et non-vaccinées-non-infectées (n = 6 cochettes par groupe). Les cochettes gestantes dans les groupes vaccinées-infectées et non-vaccinées-non-infectées furent en mesure de mener leur gestation à terme et ont mis-bas après 114 à 115 jours de gestation. À l'opposé, les cochettes gestantes du groupe (témoin) non-vaccinées-infectées ne se sont pas rendues à terme et ont mis-bas plus tôt, après 104 à 108 jours de gestation. Les cochettes gestantes vaccinées avec le vaccin PRRSV-2 MLV ont montré une réduction de la virémie à PRRSV-2. Au moment de l'infection-défi avec le PRRSV-2, les cochettes vaccinées avaient des titres relativement bas d'anticorps neutralisants (titre ≤ 1:16), alors que le nombre de cellules secrétant de l'interféron-γ (IFN-γ-SC) était constamment à des niveaux de protection (IFN-γ-SC, ≥ 150 par million). L'induction de l'immunité à médiation cellulaire, telle que mesurée par l'IFN-γ-SC spécifique à PRRSV-2, corrélait avec une réduction de la virémie à PRRSV-2. La durée de l'immunité était d'un minimum de 19 sem. Pris dans son ensemble, les résultats présentés ici suggèrent que la vaccination des cochettes avec un vaccin PRRSV-2 MLV peut protéger contre une infection-défi avec un PRRSV-2 hétérologue et améliorer les performances de reproduction des cochettes en fin de gestation.(Traduit par Docteur Serge Messier).

[Comparison between oral fluid samples and pooled serum samples for the detection of antibodies against Porcine Reproductive and Respiratory Syndrome Virus in weaning pig herds]. / Vergleich von Speichelproben und ­gepoolten Serumproben für den ­Nachweis von Antikörpern gegen das Porzine Reproduktive und Respiratorische ­Syndrom Virus in ­Aufzuchtbeständen.

INTRODUCTION: Monitoring of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in pig farms is performed usually by testing for antibodies against PRRSV in serum samples. A new method is the detection of PRRSV antibodies in porcine saliva. In this study serum samples and saliva were collected in nine farms suspicious for PRRSV and tested for the presence of PRRSV antibodies. In total 220 serum and 41 saliva samples were taken from pigs at the age of 8 weeks (± 1 week). One saliva and one pooled serum sample (1:5) were tested from each pen. In total 11 (Cut-off 0.4/0.3) or 14 (Cut-off 0.2) serum samples and 23 saliva out of 41 pens were positive for PRRSV antibodies. Cohen`s Kappa testing showed a moderate agreement (κ = 0.446). Saliva samples compared to pooled serum samples were very sensitive, the specificity was 60 and 67, respectively.

INTRODUCTION: La surveillance du virus du syndrome reproducteur et respiratoire porcin (PRRSV) dans les élevages de porcs est généralement effectuée en recherchant des anticorps contre le PRRSV dans des échantillons de sérum. Une nouvelle méthode est la détection des anticorps anti-­PRRSV dans la salive porcine. Dans cette étude, des échantillons de sérum et de salive ont été prélevés dans neuf exploitations suspectes de PRRSV et testés quant à la présence d'anticorps PRRSV. Au total, 220 échantillons de sérum et 41 échantillons de salive ont été prélevés sur des porcs à l'âge de 8 semaines (± 1 semaine). De chaque boxe, un échantillon de salive et un échantillon de sérums regroupé (1: 5) ont été testés. Au total, 11 échantillons de sérum (seuil 0,4/0,3) ou 14 (seuil 0,2) et 23 de salive sur 41 boxes étaient positifs quant aux anticorps anti-PRRSV. Le test Kappa de Cohen a montré une corrélation modérée (κ = 0,446). Les échantillons de salive étaient très sensibles par rapport aux échantillons de sérum regroupés, la spécificité n'était toutefois que de 60 respectivement 67.

PCV2 co-infection does not impact PRRSV MLV1 safety but enhances virulence of a PRRSV MLV1-like strain in infected SPF pigs.

Co-infection by a type 1 modified live vaccine-like strain (MLV1-like) of porcine reproductive and respiratory syndrome virus (PRRSV) and a type 2 porcine circovirus (PCV2) was identified on a French pig farm with post-weaning multisystemic wasting syndrome (PMWS). An in vivo experiment was set up to characterize the virulence level of the MLV1-like strain compared with the parental MLV1 strain, and to assess the impact of PCV2 co-infection on the pathogenicity of both PRRSV strains. Six groups of six pigs each were inoculated only with either one of the two PRRSV strains or with PCV2, or co-inoculated with PCV2 and MLV1 or PCV2 and MLV1-like strains. Six contact pigs were added to each inoculated group to assess viral transmission. The animals were monitored daily for 35 days post-inoculation for clinical symptoms. Blood and nasal swabs were sampled twice a week, and tissue samples were collected during necropsy for viral quantification. Compared to MLV1-infected pigs, animals infected with the MLV1-like strain had increased PRRSV viremia and nasal shedding, a higher viral load in the tonsils, and lymph node hypertrophy at microscopic level. PCV2 co-infection did not influence clinical, virologic or transmission parameters for MLV1, but co-infected MLV1-like/PCV2 pigs had the most severe lung lesions, the highest viremia in contact animals and the highest transmission rate. Our study demonstrated that the MLV1 strain tested was safe when co-inoculated with PCV2 in piglets. However, co-infection by the MLV1-like strain and PCV2 resulted in increased virulence compared with that due to a single infection.

Recombinant Porcine Interferon Alpha Enhances Immune Responses to Killed Porcine Reproductive and Respiratory Syndrome Virus Vaccine in Pigs.

In this study, the immunoadjuvant effects of recombinant porcine interferon alpha (rPoIFNα) on the killed virus vaccine (KV) of porcine reproductive and respiratory syndrome virus (PRRSV) in pigs were investigated. The experimental pigs were divided into six groups, including normal control group, rPoIFNα control group, PRRSV KV control group, KV+40,000 U rPoIFNα immunization group, KV+400,000 U rPoIFNα immunization group, and KV+4,000,000 U rPoIFNα immunization group. The experimental pigs were boosted immunized on the 28th day after the initial immunization, and the heparinized blood and serum samples were collected at different time points of these two immunizations to detect and evaluate the immune responses of pigs after immunization by ELISA assay, neutralization assay, flow cytometry, and so on. The results showed that the proportion of the levels of PRRSV-specific antibodies, neutralizing antibodies, stimulation index, IL-4, IFN-γ, and lymphocytes within the groups immunized with KV+rPoIFNα were significantly higher than that group immunized with KV alone. The humoral and cellular immune responses in pigs were markedly enhanced by rPoIFNα after the coadministration with KV vaccine. Therefore, we tentatively think that rPoIFNα is a potential immune promoter with prospects for future applications in the pig industry.

Fluorescence resonance energy transfer combined with asymmetric PCR for broad and sensitive detection of porcine reproductive and respiratory syndrome virus 2.

With its ever-increasing viral genetic diversity, accurate diagnosis of porcine reproductive and respiratory syndrome virus (PRRSV) infection is indispensable for PRRSV control. Here, a sensitive graphene oxide (GO)-based FRET method was developed to detect PRRSV-2 based on the ability of GO to quench fluorophore by fluorescence resonance energy transfer (FRET). Using primers and a fluorophore-labeled ssDNA probe targeting a conserved region between the PRRSV M gene and 3'UTR, asymmetric PCR specifically amplified viral ssDNA that could anneal with probe to generate dsDNA only in the presence of virus. Upon exonuclease III treatment to release the probe fluorophore, which degrades dsDNA with blunt ends or recessed 3´-termini, the ssDNA annealed with other probe to generate enhanced fluorescence. This GO-based FRET assay specifically detected both classical and highly pathogenic PRRSV, with analytical sensitivity approaching 10 copies/µL, similar to that of real-time PCR but greater than that of conventional reverse transcription PCR (RT-PCR). Consistent with real-time RT-PCR detection, the assay developed here exhibited high diagnostic sensitivity for virus detection of sera from experimentally and naturally infected pigs. Thus, this novel GO-based FRET assay combined with asymmetric PCR detection is sensitive and specific and will be valuable for future PRRSV diagnosis.

Determination of antibody induction by highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) vaccine: A comparison of two ELISA kits.

Two commercial porcine reproductive and respiratory syndrome virus (PRRSV) antibody ELISA kits (IDEXX and LSI) are currently in extensive use. To determine which kit is more suitable for the evaluation of HP-PRRSV vaccine efficacy, the two kits were used to test 546 serum samples. The agreement between the results was unsatisfactory, with a kappa statistic of 0.681 and a linear correlation coefficient of 0.665. In tests of samples from experimentally vaccinated and PRRSV-negative herds, IDEXX-ELISA identified antibody-positive conversion earlier and showed a higher specificity compared to LSI-ELISA. The serological profile obtained by neutralization testing was closer to that obtained by IDEXX-ELISA than by LSI-ELISA in the late immunization period. The findings reveal that IDEXX-ELISA is the more suitable for the evaluation of antibody response to HP-PRRSV vaccine and for guiding vaccination strategies.

Perturbation of Thymocyte Development Underlies the PRRS Pandemic: A Testable Hypothesis.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes immune dysregulation during the Critical Window of Immunological Development. We hypothesize that thymocyte development is altered by infected thymic antigen presenting cells (TAPCs) in the fetal/neonatal thymus that interact with double-positive thymocytes causing an acute deficiency of T cells that produces "holes" in the T cell repertoire allowing for poor recognition of PRRSV and other neonatal pathogens. The deficiency may be the result of random elimination of PRRSV-specific T cells or the generation of T cells that accept PRRSV epitopes as self-antigens. Loss of helper T cells for virus neutralizing (VN) epitopes can result in the failure of selection for B cells in lymph node germinal centers capable of producing high affinity VN antibodies. Generation of cytotoxic and regulatory T cells may also be impaired. Similar to infections with LDV, LCMV, MCMV, HIV-1 and trypanosomes, the host responds to the deficiency of pathogen-specific T cells and perhaps regulatory T cells, by "last ditch" polyclonal B cell activation. In colostrum-deprived PRRSV-infected isolator piglets, this results in hypergammaglobulinemia, which we believe to be a "red herring" that detracts attention from the thymic atrophy story, but leads to our second independent hypothesis. Since hypergammaglobulinemia has not been reported in PRRSV-infected conventionally-reared piglets, we hypothesize that this is due to the down-regulatory effect of passive maternal IgG and cytokines in porcine colostrum, especially TGFß which stimulates development of regulatory T cells (Tregs).

Development of luciferase-linked antibody capture assay based on luciferase immunoprecipitation systems for antibody detection of porcine reproductive and respiratory syndrome virus.

BACKGROUND: Early detection of porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine is necessary to control this devastating disease. By monitoring host serum antibodies to viral antigens, early virus detection within herds is feasible. In this study, recombinant antigens were generated using recombinant DNA techniques to fuse PRRSV structural protein (N) or nonstructural protein 1α (nsp1α) with the Rellina luciferase gene. Next, fused genes were cloned into plasmids and transfected into HEK-293 T cells for transient expression. Upon co-incubation of lysates with pig sera, antigen-antibody complexes formed that bound to Protein-G coated onto microplates. By further measurement of luminance value, a modified form of Luciferase Immunoprecipitation Systems, namely luciferase-linked antibody capture assay (LACA) was developed for detection of PRRSV-specific antibodies. RESULTS: Known anti-PRRSV antibody-positive or -negative serum samples (125 and 122 samples, respectively) were used to validate the LACA and compared it with IDEXX PRRS ×3 ELISA. Based on the result, N-Rluc and nsp1α-Rluc LACA results were 95.3 and 94.4% in agreement with IDEXX ELISA, suggested a similar specificity of LACA to IDEXX ELISA. Moreover, when both LACA and IDEXX ELISA were used to evaluate sequential serum samples obtained from PRRSV experimentally infected pigs, the PRRSV-specific antibody response was detectable as early as 3 days post-inoculation (dpi) using N-Rluc LACA, but undetectable until 7 dpi using IDEXX ELISA, suggesting an improved sensitivity of LACA. Meanwhile, antibodies specific for nsp1α were detected at higher levels overall, but were undetectable until 10 dpi. Furthermore,. Notably, one IDEXX ELISA positive result was not confirmed by LACA or IFA and was thus considered a false-positive result. CONCLUSION: The LACA exhibited similar specificity but improved sensitivity to that of the commercial IDEXX PRRS ×3 ELISA kit for detection of PRRSV-specific antibodies in pig serum. Importantly, LACA could be adapted for detecting antibodies against other PRRSV targets, such as nsp1α, to achieve earlier detection of PRRSV infection.

Use of processing fluids and serum samples to characterize porcine reproductive and respiratory syndrome virus dynamics in 3 day-old pigs.

Collection of serum samples of pigs at weaning to monitor for porcine reproductive and respiratory syndrome virus (PRRSV) has become a common practice to determine PRRSV herd infection status. Diagnostic sensitivity of this practice is low in herds undergoing PRRSV elimination once prevalence of infection is near zero. Thus, the goal of this study was to characterize the dynamics of PRRSV infection in 3 day-old pigs overtime using serum and serosanguineous fluids obtained as part of castration and tail docking practices (processing fluids (PF)). Secondary goal was to estimate sensitivity and specificity of PF in the 3 day old population. A 6000 breed-to-wean sow herd was monitored every three weeks for 23 weeks after a PRRSV outbreak by collecting both PF and individual serum samples from all pigs in the selected litters. Out of the 77 litters tested, 23 (29.8%) were identified as positive using the PF and the serum samples, with a Cohen's kappa statistic of 0.81 (95% CI: 0.59-1) between the results obtained in each sample type. The sensitivity and specificity of the PF relative to the results in serum was 87% (95% CI: 66%-97%) and 94% (95% CI: 85%-99%) respectively. The percentage of PRRSV positive litters decreased over time and litters from gilts were more likely to test positive than those from older sows. Overall, the study demonstrates that PF can be a convenient and reliable specimen to monitor PRRSV infection in breeding herds.

Field evaluation of two commercial RT-rtPCR assays for porcine reproductive and respiratory syndrome virus detection using sera from ill and healthy pigs, China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious respiratory virus causing severe morbidity in pigs worldwide. Control strategies for PRRSV often rely on detecting PRRSV, culling or isolating sick pigs, disinfecting pig barns, vaccination, and monitoring for virus spread. Given the high economic impact of PRRSV on pig farms, there is a great need for rapid and reliable PRRSV detection assays. We compared the performance of 2 commercial reverse-transcription real-time PCR (RT-rtPCR) assays, the VetMAX PRRSV NA and EU reagents (ABI assay) and the PRRSV general RT-rtPCR kit (Anheal assay), for the molecular detection of PRRSV in sera collected from pigs in China. Between June and September 2015, sera were collected from 219 healthy and 104 suspected PRRSV-infected pigs on 4 farms in China. Employing blinding, the 2 assays were run by 2 laboratories (Guangzhou Animal Health Inspection Institute [GAHII] and Sun Yat-sen University [SYSU] laboratories) and compared. Although both assays detected PRRSV with 100% specificity at both laboratories, the sensitivity (95% vs. 78% at GAHII; 94% vs. 72% at SYSU Laboratory) and the reproducibility (kappa value 0.933 vs. 0.931) were slightly better for the ABI assay compared to the Anheal assay.

Assessment of abattoir based monitoring of PRRSV using oral fluids.

Various porcine reproductive and respiratory syndrome virus (PRRSV) regional elimination projects have been implemented in the U.S., but none have yet succeeded. In part, this reflects the need for efficient methods to monitor over time the progress of PRRSV status of participating herds. This study assessed the feasibility of monitoring PRRSV using oral fluids collected at the abattoir. A total of 36 pig lots were included in the study. On-farm oral fluid (n = 10) and serum (n = 10) collected within two days of shipment to the abattoir were used to establish the reference PRRSV status of the population. Oral fluids (n = 3 per lot) were successfully collected from 32 lots (89%) at the lairage. Three veterinary diagnostic laboratories (VDLs) tested the sera (VDL1 and VDL3: n = 316, VDL2: n = 315) and oral fluids (VDL1 and VDL3: n = 319, VDL2: n = 320) for PRRSV antibodies (ELISA) and RNA (rRT-PCR). Environmental samples (n = 64, 32 before and 32 after pigs were placed in lairage) were tested for PRRSV RNA at one VDL. All oral fluids (farm and abattoir) tested positive for PRRSV antibody at all VDLs. PRRSV positivity frequency on serum ranged from 92.4% to 94.6% among VDLs, with an overall agreement of 97.6%. RNA was detected on 1.3% to 1.9%, 8.1% to 17.7%, and 8.3% to 17.7% of sera, on-farm and abattoir oral fluids, respectively. Between-VDLs rRT-PCR agreement on sera and oral fluids (farm and abattoir) ranged from 97.8% to 99.0%, and 79.0% to 81.2%, respectively. Between-locations agreement of oral fluids varied from 31.3% to 50% depending on the VDL. This study reported the application of swine oral fluids collected at the abattoir for monitoring PRRSV, and describes the between-VDL agreement for PRRS testing of serum and oral fluid field samples.

Effect of imidacloprid ingestion on immune responses to porcine reproductive and respiratory syndrome virus.

Nicotine and acetylcholine cause immunosuppresion by signaling to the α7 nicotinic acetylcholine receptor (α7 nAChR) on immune cells. Neonicotinoids are nAChR agonists and widly used insecticides. We aimed to define the immunosuppressive potential of dietary exposure to the neonicotinoid imidacloprid (IMI) on the generation of innate and adaptive immune responses to porcine reproductive and respiratory syndrome virus (PRRSV). Piglets were randomized into groups based on diet and infection. Behavioral signs of illness were recorded. Urine IMI levels were measured by high performance liquid chromatography-mass spectrometry. Flow cytometry was used to determine the expression pattern of the α7 nAChR on porcine leukocytes as well as the effects of infection and treatment on circulating leukocyte populations. Serum cytokines and PRRSV-specific antibody levels were determined by ELISA. Viral RNA in lung, spleen and plasma was determined by RT-qPCR. Pigs in the treatment group had elevated urine levels of IMI. Treatment with IMI reduced body weight, caused bouts of hypothermia, increased serum IL-10 and elevated levels of virus-specific antibodies. Viral RNA levels in the spleen showed a trend toward being increased in pigs fed IMI. Our data indicates that IMI injection may modulate virus specific immune function during PRRSV infection.

Isolation of Peripheral Blood CD8 T Cells Specific to Porcine Reproductive and Respiratory Syndrome Virus Utilizing Porcine CD137 Activation Marker.

CD137 is a costimulatory molecule transiently expressed on activated T cells after mitogen or antigen stimulation that can be exploited for isolating antigen-specific T cells as reported in mouse models. By utilizing an antiporcine CD137 monoclonal antibody (mAb, clone 3B9) developed in our laboratory, we isolated virus-specific CD8ß T cells from peripheral blood of pigs experimentally infected with different porcine reproductive and respiratory syndrome virus (PRRSV) strains. Similar to mouse, porcine CD8ß T cells also express CD137 transiently upon Concavalin A stimulation while the unstimulated cells did not. Most frequently, virus-specific CD8ß T cells were isolated at low levels from peripheral blood of pigs experimentally infected with PRRSV strains VR2385, NADC20, and MN184B at 49 and 63 days postinfection. The results suggest that porcine CD137-specific mAb is a useful tool for isolating virus-specific CD8 T cells from peripheral blood and tissues of pigs after in vitro stimulation with viral antigen.

Identification of small non-coding RNA classes expressed in swine whole blood during HP-PRRSV infection.

It has been established that reduced susceptibility to porcine reproductive and respiratory syndrome virus (PRRSV) has a genetic component. This genetic component may take the form of small non-coding RNAs (sncRNA), which are molecules that function as regulators of gene expression. Various sncRNAs have emerged as having an important role in the immune system in humans. The study uses transcriptomic read counts to profile the type and quantity of both well and lesser characterized sncRNAs, such as microRNAs and small nucleolar RNAs to identify and quantify the classes of sncRNA expressed in whole blood between healthy and highly pathogenic PRRSV-infected pigs. Our results returned evidence on nine classes of sncRNA, four of which were consistently statistically significantly different based on Fisher's Exact Test, that can be detected and possibly interrogated for their effect on host dysregulation during PRRSV infections.

Molecular epidemiology of porcine reproductive and respiratory syndrome virus in Central China since 2014: The prevalence of NADC30-like PRRSVs.

Porcine reproductive and respiratory syndrome (PRRS), characterized by respiratory disorders in piglets and reproductive failure in sows, is still the great threat of swine industry. Recently, Emergence of the novel NADC30-like PRRS viruses (PRRSVs) has caused widespread outbreaks of PRRS. To investigate the epidemic characteristics of PRRSVs in Central China since 2014, 6372 clinical serum samples were tested by ELISA, 250 tissue samples were tested by RT-PCR, and among these, 30 ORF5 and 17 Nsp2 genes sequences were analyzed. Phylogenetic tree based on ORF5 revealed that, 17 isolates were clustered into subgroup 1, represented by the NADC30. And for the Nsp2, The strains which had a discontinuous 131-amino-acid deletion in Nsp2, called NADC30-like strains, were clustered into subgroup 2. Our data suggested that the NADC30-like PRRSV strains spread quickly and are now circulating and prevalent in Central China as well as the classical HP-PRRSV strains. In addition, amino acid variation analysis of GP5 revealed that the amino acid sequences of NADC30-like PRRSV strains underwent rapid evolution and contained extensive amino acid substitutions in important motifs, such as potential neutralization epitope and the N-glycosylation sites. In summary, our data would provide a large amount of detailed information on molecular variation and genetic diversity of PRRSV in central China.

Antigenic and Biological Characterization of ORF2-6 Variants at Early Times Following PRRSV Infection.

Genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) challenges efforts to develop effective and broadly acting vaccines. Although genetic variation in PRRSV has been extensively documented, the effects of this variation on virus phenotype are less well understood. In the present study, PRRSV open reading frame (ORF)2-6 variants predominant during the first six weeks following experimental infection were characterized for antigenic and replication phenotype. There was limited genetic variation during these early times after infection; however, distinct ORF2-6 haplotypes that differed from the NVSL97-7895 inoculum were identified in each of the five pigs examined. Chimeric viruses containing all or part of predominant ORF2-6 haplotypes were constructed and tested in virus neutralization and in vitro replication assays. In two pigs, genetic variation in ORF2-6 resulted in increased resistance to neutralization by autologous sera. Mapping studies indicated that variation in either ORF2-4 or ORF5-6 could confer increased neutralization resistance, but there was no single amino acid substitution that was predictive of neutralization phenotype. Detailed analyses of the early steps in PRRSV replication in the presence and absence of neutralizing antibody revealed both significant inhibition of virion attachment and, independently, a significant delay in the appearance of newly synthesized viral RNA. In all pigs, genetic variation in ORF2-6 also resulted in significant reduction in infectivity on MARC-145 cells, suggesting variation in ORF2-6 may also be important for virus replication in vivo. Together, these data reveal that variation appearing early after infection, though limited, alters important virus phenotypes and contributes to antigenic and biologic diversity of PRRSV.