A novel Gaussia luciferase immunoprecipitation assay for the detection of Getah virus antibodies in pigs.

BackgroundAfrican swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV) are still prevalent in many regions of China. Co-infections make it difficult to distinguish their clinical symptoms and pathological changes. Therefore, a rapid and specific method is needed for the differential detection of these pathogens.ObjectivesThe aim of this study was to develop a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) for the simultaneous differential detection of ASFV, CSFV, and PRRSV.MethodsThree pairs of primers and TaqMan probes targeting the ASFV p72 gene, CSFV 5′ untranslated region, and PRRSV ORF7 gene were designed. After optimizing the reaction conditions, including the annealing temperature, primer concentration, and probe concentration, multiplex qRT-PCR for simultaneous and differential detection of ASFV, CSFV, and PRRSV was developed. Subsequently, 1,143 clinical samples were detected to verify the practicality of the assay.ResultsThe multiplex qRT-PCR assay could specifically and simultaneously detect the ASFV, CSFV, and PRRSV with a detection limit of 1.78 × 100 copies for the ASFV, CSFV, and PRRSV, but could not amplify the other major porcine viruses, such as pseudorabies virus, porcine circovirus type 1 (PCV1), PCV2, PCV3, foot-and-mouth disease virus, porcine parvovirus, atypical porcine pestivirus, and Senecavirus A. The assay had good repeatability with coefficients of variation of intra- and inter-assay of less than 1.2%. Finally, the assay was used to detect 1,143 clinical samples to evaluate its practicality in the field. The positive rates of ASFV, CSFV, and PRRSV were 25.63%, 9.36%, and 17.50%, respectively. The co-infection rates of ASFV+CSFV, ASFV+PRRSV, CSFV+PRRSV, and ASFV+CSFV+PRRSV were 2.45%, 2.36%, 1.57%, and 0.17%, respectively.ConclusionsThe multiplex qRT-PCR developed in this study could provide a rapid, sensitive, specific diagnostic tool for the simultaneous and differential detection of ASFV, CSFV, and PRRSV.

Multiplex PCR and multiplex RT-PCR for inclusive detection of major swine DNA and RNA viruses in pigs with multiple infections

In recent years, African swine fever (ASF) has caused a devastating blow to the swine industry globally. Since no effective vaccine is available, strict biosafety measures and rapid diagnosis are the most effective strategies for ASF control. ASFV p30 is one of the most antigenic viral proteins that have been widely used in the field for serological diagnosis of ASF infection. In this study, we developed a luciferase immunoprecipitation system (LIPS) assay for the detection of ASFV antibodies in pig serum using Gaussia luciferase (GLuc)-tagged p30 as a diagnostic antigen. The optimal GLuc-p30 input of 107 luminance units (LU) and optimal serum dilution factor of 1/100 were set to achieve the highest P/N ratio. Based on 87 ASFV-positive and negative pig sera, the cutoff value of the S/N ratio could be set between 2.298 and 30.59 to achieve 100% sensitivity and 100% specificity. Moreover, the diagnostic sensitivity of this LIPS is comparable to that of a commercial enzyme-linked immunosorbent assay (ELISA) and the specificity of LIPS is even superior to the tested ELISA. In conclusion, we have established a LIPS assay for ASFV antibody detection, which could be a potential method for ASFV diagnosis in laboratories and farms.

BackgroundConcurrent infection with porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) is known as one of the major causes for porcine respiratory disease complex (PRDC). Dual infection with PCV2 and PRRSV is consistently to have more severe clinical presentations and pulmonary lesions than infection with PCV2 alone or PRRSV alone. However, it is not known if dual infections with PCV2 and PRRSV in different infection order may lead to different clinical symptoms in the host. To mimic the possible field conditions, swine alveolar macrophages (AMs) were inoculated with PCV2 and PRRSV in vitro simultaneously or with one virus 18 h earlier than the other. The cell viability, cytopathic effects, antigen-containing rates, phagocytotic and microbial killing capabilities, cytokine profiles (IL-8, TNF-α, and IFN-α) and FasL transcripts were determined, analyzed, and compared to prove the hypothesis.ResultsA marked reduction in PRRSV antigen-containing rate, cytopathic effect, and TNF-α expression level was revealed in AMs inoculated with PCV2 and PRRSV simultaneously and in AMs inoculated with PCV2 first then PRRSV 18 h later, but not in AMs inoculated with PRRSV first then PCV2 18 h later. Transient decrease in phagocytosis but constant reduction in microbicidal capability in AMs in the group inoculated with PCV2 alone and constant decrease in phagocytosis and microbicidal capability in AMs in all PRRSV-inoculated groups were noted. The levels of IL-8, TNF-α, IFN-α, and FasL transcripts in AMs in all groups with dual inoculation of PCV2 and PRRSV were significantly increased regardless of the infection orders as compared with infection by PCV2 alone or PRRSV alone.ConclusionsSwine AMs infected with PCV2 first then PRRSV later or infected with PCV2 and PRRSV simultaneously displayed marked reduction in PRRSV antigen-containing rate, cytopathic effect, and TNF-α expression level. The different inoculation orders of PCV2 and PRRSV in AMs leading to different results in viral antigen positivity, cytopathology, and cytokine profile may explain, at least partially, the underlying mechanism of the enhanced pulmonary lesions in PRDC exerted by dual infection with PCV2 and PRRSV and the variable clinical manifestations of PRDC-affected pigs in the field.

Serologic and molecular survey for major viral pathogens in grazing hybrid wild boars in Northeast China

Development of an EvaGreen-based multiplex real-time PCR assay with melting curve analysis for simultaneous detection and differentiation of six viral pathogens of porcine reproductive and respiratory disorder

Since its emergence in China in 2018, African swine fever virus (ASFV) has posed a severe threat to the pig farming industry due to its high transmissibility and mortality rate. The clinical signs of ASFV infection often overlap with those caused by other swine viruses such as classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), and porcine circovirus type 2 (PCV2), making timely and precise diagnosis a considerable challenge. To address this, we established a TaqMan-based multiplex real-time quantitative PCR (qPCR) assay capable of simultaneously detecting ASFV, CSFV, PRRSV, PRV, and PCV2. Specific primer-probe sets were developed targeting conserved genomic regions: the ASFV P72 gene, CSFV 5’UTR region, PRRSV ORF6, PCV2 cap gene, and PRV gB gene. After thorough optimization, the assay demonstrated robust analytical performance, exhibiting strong target specificity with no cross-detection of non-target pathogens. The detection threshold was determined to be 10 copies/μL per virus, indicating high assay sensitivity. Repeatability analysis revealed low variability, with intra- and inter-assay coefficient of variation values remaining below 2.3%. When applied to 95 clinical samples, the multiplex assay yielded results that were fully consistent with those obtained using commercially available singleplex qPCR kits. In conclusion, the multiplex TaqMan qPCR method developed in this study is characterized by high specificity, sensitivity, and reproducibility. It provides a reliable and efficient diagnostic tool for the simultaneous detection and differential diagnosis of ASFV and other clinically similar viral infections in swine, thereby offering robust technical support for swine disease surveillance and control.

Postweaning multisystemic wasting syndrome (PMWS), also named as porcine circovirus associated disease (PCVAD), is an important emerging disease in swine in recent years. Porcine circovirus type 2 (PCV2) has been demonstrated to be the cause associated with PMWS. PCV2 co-infection with other pathogen, i.e. porcine reproductive and respiratory syndrome virus (PRRSV), is common and exacerbates the severity of PMWS. In addition to the implement of high level of biosecurity of farms, the use of the PCV2 vaccine is an effective strategy to control PCVAD. Sugar cane extract (SCE) is a native immunomodulator and can reduce the severity of infection. The objective of the present study was to evaluate the efficacy of the novel E. coli expressed PCV2/ORF2 subunit vaccines. The first experiment we evaluated the efficacy of PCV2 vaccines on conventional pigs under experimental control with less PRRSV exposure and the effects of PCV2 maternal antibodies on pigs experimentally infected with PCV2. Experimental pigs received double vaccinations of PCV2 subunit vaccine at 3 and 6 weeks of age, and then were inoculated with PCV2 inocula at 9 weeks of age. The clinical symptoms, pathological lesions, PCV2 loading and PCV2 antibody profile were evaluated. The results displayed that all experimental pigs after PCV2 inoculation presented mild clinical signs and respiratory lesions. Mean days of fever during 4 weeks inoculation after PCV2 challenge were slightly lower in vaccination group than in non-vaccination group. In contrast, mean daily weight gain were slightly higher in vaccination group (0.58±0.10 kg/day) than in non-vaccination group (0.51±0.10 kg/day). However, there was no significant difference in PCV2 loading between vaccination and non-vaccination group, but was significantly higher than PCV2 inoculated group at 5 weeks of age. Experiments above shows mild efficacy of the novel PCV2 subunit vaccine on these vaccinated pigs and high maternal antibodies offer more protection for piglets. The second experiment the evaluation of the efficacy of vaccines was repeated, that experimental pigs were naturally exposure to PRRSV infection. Moreover, effects of SCE on PRRSV or PCV2 infection were also included. The results displayed that most pigs suffered from PRRSV and PCV2 co-infection with more severe clinical signs and pulmonary lesions. The efficacy of the PCV2 vaccine on vaccinated pigs was not supported under PRRSV and PCV2 co-infection. However, pigs fed with SCE presented milder clinical signs, lesions, and PCV2 load in tissues. Therefore, SCE administration might have an immunostimulating effects on porcine immunity to reduce the pulmonary impairment against PCV2 and PRRSV co-infection. Taken together, co-infections with PCV2 and PRRSV can induce severe pulmonary lesions and clinical signs. The efficacy of the novel PCV2 subunit vaccine remains further elucidation. With the implement of high level of hygiene, increasing maternal antibody to confer more protection to piglets by means of boosting sows with PCV2 antigen, and feeding SCE in appropriate time, could be considered as another strategy in the control of PCVAD.

Detection of porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, swine influenza virus and Aujeszky's disease virus in cases of porcine proliferative and necrotizing pneumonia (PNP) in Spain

The objective of the present study was to determine the effects of porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) vaccinations in an experimental PCV2-PRRSV challenge model, based on virological (viremia), immunological (neutralizing antibodies [NAs], gamma interferon-secreting cells [IFN-γ-SCs], and CD4(+) CD8(+) double-positive cells), and pathological (lesions and antigens in lymph nodes and lungs) evaluations. A total of 72 pigs were randomly divided into 9 groups (8 pigs per group): 5 vaccinated and challenged groups, 3 nonvaccinated and challenged groups, and a negative-control group. Vaccination against PCV2 induced immunological responses (NAs and PCV2-specific IFN-γ-SCs) and reduced PCV2 viremia, PCV2-induced lesions, and PCV2 antigens in the dually infected pigs. However, vaccination against PCV2 did not affect the PRRSV immunological responses (NAs and PRRSV-specific IFN-γ-SCs), PRRSV viremia, PRRSV-induced lesions, or PRRSV antigens in the dually infected pigs. Vaccination against PRRSV did not induce immunological responses (PRRSV-specific IFN-γ-SCs) or reduce PRRSV viremia, PRRSV-induced lesions, or PRRSV antigens in the dually infected pigs. In addition, vaccination against PRRSV increased PCV2 viremia, PCV2-induced lesions, and PCV2 antigens in the dually infected pigs. In summary, vaccination against PCV2 reduced PCV2 viremia, PCV2-induced lesions, and PCV2 antigens in the dually infected pigs. However, vaccination against PRRSV increased PCV2 viremia, PCV2-induced lesions, and PCV2 antigens in the dually infected pigs. Therefore, the PCV2 vaccine decreased the potentiation of PCV2-induced lesions by PRRSV in dually infected pigs. In contrast, the PRRSV vaccine alone did not decrease the potentiation of PCV2-induced lesions by PRRSV in dually infected pigs.

African swine fever (ASF), classical swine fever (CSF), and porcine reproductive and respiratory syndrome (PRRS) are highly infectious diseases of domestic pigs and wild boars. The co-infections of ASF virus (ASFV), CSF virus (CSFV), and PRRS virus (PRRSV) have been reported in different pig farms. Early differential detection and diagnosis of ASFV, CSFV, and PRRSV in the clinical samples is very important for the effective prevention and control of these diseases. A multiplex crystal digital PCR (dPCR) was developed for differential detection of ASFV, CSFV, and PRRSV in this study, targeting p72, 5' untranslated region (UTR), and ORF7 genes, respectively. The different reaction conditions were optimized, and the specificity, sensitivity, and repeatability of the assay were evaluated. The results showed that the multiplex crystal dPCR was able to accurately and differentially detect ASFV, CSFV, and PRRSV with a limit of detection of 4.69 × 10−1 copies/μl, respectively, and could not detect other porcine viruses, i.e., foot-and-mouth disease virus (FMDV), Senecavirus A (SVA), atypical porcine pestivirus (APPV), pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), and porcine parvovirus (PPV). The assay showed excellent repeatability and reproducibility, with coefficients of variation (CV) of the intra- and inter-assay from 0.09 to 1.40%, and from 0.64 to 2.26%, respectively. The 289 clinical samples from different pig herds in Guangxi province, China, were tested by the multiplex crystal dPCR and a reference multiplex real-time quantitative RT-PCR (qRT-PCR) established previously in our laboratory. The positive rates of ASFV, CSFV, and PRRSV were 30.10, 13.49, and 22.49% by the multiplex crystal dPCR, and 24.57, 8.65, and 18.34% by the multiplex qRT-PCR, with coincidence rates of 94.66, 95.16, and 95.84%, respectively. The results indicated that the established multiplex crystal dPCR was a specific, sensitive, and accurate method for the detection and quantification of ASFV, CSFV, and PRRSV. This is the first report on the multiplex dPCR for detecting ASFV, CSFV, and PRRSV.

To investigate the transition in concentration of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) and antibody for these viruses in serum, serum samples were collected from 29 pigs on weaning day and at 7, 14, 21, 28, 53, 84, and 120 days after weaning. The concentration of circulated PRRSV and PCV2 in serum was measured by real-time RT-PCR and real-time PCR, respectively. The specific antibody for PRRSV and PCV2 was measured using ELISA. PRRSV was not detected on 0 days post-weaning (dpw). The specific antibody for PRRSV began to increase as the concentration of PRRSV in serum increased, and the level of PRRSV then tended to decrease. PCV2 was detected in 12 of 28 pigs on 0 dpw. The concentration of PCV2 and the specific antibody for PCV2 showed a similar tendency to those of PRRSV. The correlation analysis suggests that a decline in the daily weight gain coincided with an increase in the PRRSV concentration. Pigs with a higher antibody titer against PRRSV or PCV2 on 0 dpw showed the lower level of PRRSV or PCV2, respectively.

Effect of an interferon-stimulated response element (ISRE) mutant of porcine circovirus type 2 (PCV2) on PCV2-induced pathological lesions in a porcine reproductive and respiratory syndrome virus (PRRSV) co-infection model

Development and application of a novel Bio–Plex suspension array system for high–throughput multiplexed nucleic acid detection of seven respiratory and reproductive pathogens in swine

Changes in peripheral blood leukocyte subpopulations in piglets co-infected experimentally with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2