Pathogenicity of genotype 2.1 classical swine fever virus isolated from Japan in 2019 in pigs.

Classical swine fever (CSF) re-emerged in Japan in 2018 for the first time in 26 years. The disease has been known to be caused by a moderately pathogenic virus, rather than the highly pathogenic virus that had occurred in the past. However, the underlying pathophysiology remains unknown. This study conducted an experimental challenge on specific pathogen-free (SPF) pigs in a naïve state for 2, 4, and 6 weeks and confirmed the disease state during each period by clinical observation, virus detection, and pathological necropsy. We revealed the pathological changes and distribution of pathogens and virus-specific antibodies at each period after virus challenge. These results were comprehensively analyzed and approximately 70% of the pigs recovered, especially at 4- and 6-week post-virus challenge. This study provides useful information for future countermeasures against CSF by clarifying the pathogenicity outcomes in unvaccinated pigs with moderately pathogenic genotype 2.1 virus.

Enhancement of systemic virus-specific T lymphocyte responses in pigs supplemented with algae-derived ß-glucan.

Algae-derived ß-glucan has been widely used as a feed additive in the swine industry. The supplementation of ß-glucan aims to improve growth performance and modulate the immunity of pigs. However, the potential effects of supplementing ß-glucan from algae on immune responses in pigs-specifically antigen-specific immunity-must be determined. In this study, the effects of algae-derived ß-glucan supplementation on growth performance, virus neutralising antibody and virus-specific T lymphocytes responses were investigated in pigs. Piglets (n=112 per treatment) were assigned to three treatments including non-supplemented group (control), ß-glucan 100 g/ton supplemented group (BG100), and ß-glucan 200 g/ton supplemented group (BG200). In this study, production performance of pigs was not found to be different between the experimental groups. Pigs supplemented with ß-glucan exhibited high levels of classical swine fever virus (CSFV)-specific producing T lymphocytes and neutralising antibody titer, compared to the control group. Interestingly, supplementation of ß-glucan significantly enhanced porcine reproductive and respiratory syndrome virus (PRRSV)-specific interferon-gamma (IFN-γ) producing T lymphocytes, including CD4+, CD8+, and CD4+CD8+ T lymphocyte subpopulations. Moreover, PRRS modified live vaccine (MLV) viremia was reduced in earlier for ß-glucan-supplemented pigs compared to the control group. The findings indicate that the algae-derived ß-glucan possesses biological potential as an immunomodulatory substance to enhance antiviral immunity, which may contribute to disease resistance in pigs.

Ferritin Nanoparticle Delivery of the E2 Protein of Classical Swine Fever Virus Completely Protects Pigs from Lethal Challenge.

Classical swine fever (CSF), caused by the classical swine fever virus (CSFV), results in significant economic losses to the swine industry in many countries. Vaccination represents the primary strategy to control CSF and the CSFV E2 protein is known as the major protective antigen. However, the E2 protein expressed or presented by different systems elicits distinct immune responses. In this study, we established a stable CHO cell line to express the E2 protein and delivered it using self-assembled ferritin nanoparticles (NPs). Subsequently, we compared the adaptive immune responses induced by the E2-ferritin NPs and the monomeric E2 protein produced by the CHO cells or a baculovirus expression system. The results revealed that the NP-delivered E2 protein elicited higher titers of neutralizing antibodies than did the monomeric E2 protein in pigs. Importantly, only the NP-delivered E2 protein significantly induced CSFV-specific IFN-γ-secreting cells. Furthermore, all the pigs inoculated with the E2-ferritin NPs were completely protected from a lethal CSFV challenge infection. These findings demonstrate the ability of the E2-ferritin NPs to protect pigs against the lethal CSFV challenge by eliciting robust humoral and cellular immune responses.

Label-Free Detection of African Swine Fever and Classical Swine Fever in the Point-of-Care Setting Using Photonic Integrated Circuits Integrated in a Microfluidic Device.

Swine viral diseases have the capacity to cause significant losses and affect the sector's sustainability, a situation further exacerbated by the lack of antiviral drugs and the limited availability of effective vaccines. In this context, a novel point-of-care (POC) diagnostic device incorporating photonic integrated circuits (PICs), microfluidics and information, and communication technology into a single platform was developed for the field diagnosis of African swine fever (ASF) and classical swine fever (CSF). The device targets viral particles and has been validated using oral fluid and serum samples. Sensitivity, specificity, accuracy, precision, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) were calculated to assess the performance of the device, and PCR was the reference method employed. Its sensitivities were 80.97% and 79%, specificities were 88.46% and 79.07%, and DOR values were 32.25 and 14.21 for ASF and CSF, respectively. The proposed POC device and PIC sensors can be employed for the pen-side detection of ASF and CSF, thus introducing novel technological advancements in the field of animal diagnostics. The need for proper validation studies of POC devices is highlighted to optimize animal biosecurity.

IDO1 promotes CSFV replication by mediating tryptophan metabolism to inhibit NF-κB signaling.

Tryptophan metabolism plays a crucial role in facilitating various cellular processes essential for maintaining normal cellular function. Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the conversion of tryptophan (Trp) into kynurenine (Kyn), thereby initiating the degradation of Trp. The resulting Kyn metabolites have been implicated in the modulation of immune responses. Currently, the role of IDO1-mediated tryptophan metabolism in the process of viral infection remains relatively unknown. In this study, we discovered that classical swine fever virus (CSFV) infection of PK-15 cells can induce the expression of IDO1, thereby promoting tryptophan metabolism. IDO1 can negatively regulate the NF-κB signaling by mediating tryptophan metabolism, thereby facilitating CSFV replication. We found that silencing the IDO1 gene enhances the expression of IFN-α, IFN-ß, and IL-6 by activating the NF-κB signaling pathway. Furthermore, our observations indicate that both silencing the IDO1 gene and administering exogenous tryptophan can inhibit CSFV replication by counteracting the cellular autophagy induced by Rapamycin. This study reveals a novel mechanism of IDO1-mediated tryptophan metabolism in CSFV infection, providing new insights and a theoretical basis for the treatment and control of CSFV.IMPORTANCEIt is well known that due to the widespread use of vaccines, the prevalence of classical swine fever (CSF) is shifting towards atypical and invisible infections. CSF can disrupt host metabolism, leading to persistent immune suppression in the host and causing significant harm when co-infected with other diseases. Changes in the host's metabolic profiles, such as increased catabolic metabolism of amino acids and the production of immunoregulatory metabolites and their derivatives, can also influence virus replication. Mammals utilize various pathways to modulate immune responses through amino acid utilization, including increased catabolic metabolism of amino acids and the production of immunoregulatory metabolites and their derivatives, thereby limiting viral replication. Therefore, this study proposes that targeting the modulation of tryptophan metabolism may represent an effective approach to control the progression of CSF.

Self-Replicating RNA Derived from the Genomes of Positive-Strand RNA Viruses.

Self-replicating RNA derived from the genomes of positive-strand RNA viruses represents a powerful tool for both molecular studies on virus biology and approaches to novel safe and effective vaccines. The following chapter summarizes the principles how such RNAs can be established and used for design of vaccines. Due to the large variety of strategies needed to circumvent specific pitfalls in the design of such constructs the technical details of the experiments are not described here but can be found in the cited literature.

Six underreported viral diseases of domesticated and wild swine in Africa: Implications and perspectives.

Pig production is increasing annually in Africa as it is recognized as a significant source of income, livelihood and food security, particularly in rural communities. Understanding the circulating swine pathogens is crucial for the success of this emerging industry. Although there is extensive data available on the African swine fever virus due to its devastating impact on pig production, knowledge about the presence of other viral swine pathogens on the continent is still extremely limited. This review discusses what is currently known about six swine pathogens in Africa: classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine circovirus-2, porcine circovirus-3, porcine parvovirus-1, and pseudorabies virus. Gaps in our knowledge are identified and topics of future focus discussed.

Classical swine fever virus non-structural protein 4A recruits dihydroorotate dehydrogenase to facilitate viral replication.

Mitochondria are energy producers in cells, which can affect viral replication by regulating the host innate immune signaling pathways, and the changes in their biological functions are inextricably linked the viral life cycle. In this study, we screened a library of 382 mitochondria-targeted compounds and identified the antiviral inhibitors of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in the de novo synthesis pathway of pyrimidine ribonucleotides, against classical swine fever virus (CSFV). Our data showed that the inhibitors interfered with viral RNA synthesis in a dose-dependent manner, with half-maximal effective concentrations (EC50) ranging from 0.975 to 26.635 nM. Remarkably, DHODH inhibitors obstructed CSFV replication by enhancing the innate immune response including the TBK1-IRF3-STAT1 and NF-κB signaling pathways. Furthermore, the data from a series of compound addition and supplementation trials indicated that DHODH inhibitors also inhibited CSFV replication by blocking the de novo pyrimidine synthesis. Remarkably, DHODH knockdown demonstrated that it was essential for CSFV replication. Mechanistically, confocal microscopy and immunoprecipitation assays showed that the non-structural protein 4A (NS4A) recruited and interacted with DHODH in the perinuclear. Notably, NS4A enhanced the DHODH activity and promoted the generation of UMP for efficient viral replication. Structurally, the amino acids 65-229 of DHODH and the amino acids 25-40 of NS4A were pivotal for this interaction. Taken together, our findings highlight the critical role of DHODH in the CSFV life cycle and offer a potential antiviral target for the development of novel therapeutics against CSF. IMPORTANCE: Classical swine fever remains one of the most economically important viral diseases of domestic pigs and wild boar worldwide. dihydroorotate dehydrogenase (DHODH) inhibitors have been shown to suppress the replication of several viruses in vitro and in vivo, but the effects on Pestivirus remain unknown. In this study, three specific DHODH inhibitors, including DHODH-IN-16, BAY-2402234, and Brequinar were found to strongly suppress classical swine fever virus (CSFV) replication. These inhibitors target the host DHODH, depleting the pyrimidine nucleotide pool to exert their antiviral effects. Intriguingly, we observed that the non-structural protein 4A of CSFV induced DHODH to accumulate around the nucleus in conjunction with mitochondria. Moreover, NS4A exhibited a strong interaction with DHODH, enhancing its activity to promote efficient CSFV replication. In conclusion, our findings enhance the understanding of the pyrimidine synthesis in CSFV infection and expand the novel functions of CSFV NS4A in viral replication, providing a reference for further exploration of antiviral targets against CSFV.

Porcine low-density lipoprotein receptor plays an important role in classical swine fever virus infection.

Several cellular factors have been reported to be required for replication of classical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae. However, many steps of its replication cycle are still poorly understood. The low-density lipoprotein receptor (LDLR) is involved in cell entry and post-entry processes of different viruses including other members of the Flaviviridae. In this study, the relevance of LDLR in replication of CSFV and another porcine pestivirus, Bungowannah pestivirus (BuPV), was investigated by antibody-mediated blocking of LDLR and genetically engineered porcine cell lines providing altered LDLR expression levels. An LDLR-specific antibody largely blocked infection with CSFV, but had only a minor impact on BuPV. Infections of the genetically modified cells confirmed an LDLR-dependent replication of CSFV. Compared to wild type cells, lower and higher expression of LDLR resulted in a 3.5-fold decrease or increase in viral titers already 20 h post infection. Viral titers were 25-fold increased in LDLR-overexpressing cells compared to cells with reduced LDLR expression at 72 h post infection. The varying LDLR expression levels had no clear effect on permissivity to BuPV. A decoy receptor assay using recombinant soluble LDLR provided no evidence that LDLR may function as a receptor for CSFV or BuPV. Differences in their dependency on LDLR suggest that CSFV and BuPV likely use different mechanisms to interact with their host cells. Moreover, this study reveals similarities in the replication cycles of CSFV and other members of the family Flaviviridae that are dependent on LDLR.

Exosomes secreted by CSFV-infected cells evade neutralizing antibody to activate innate immune responses and establish productive infection in recipient cells.

Exosomes, which are small membrane-enclosed vesicles, are actively released into the extracellular space by a variety of cells. Growing evidence indicates that exosomes derived from virus-infected cells can selectively encapsulate viral proteins, genetic materials, or even entire virions. This enables them to mediate cell-to-cell communication and facilitate virus transmission. Classical swine fever (CSF) is a disease listed by the World Organisation for Animal Health (WOAH) Terrestrial Animal Health Code and must be reported to the organisation. It is caused by classical swine fever virus (CSFV) belonging to the Flaviviridae family. Recent studies have demonstrated that extracellular vesicles originating from autophagy can facilitate the antibody-resistant spread of classical swine fever virus. However, due to the extreme difficulty in achieving a complete separation from virions, the role of exosomes during CSFV infection and proliferation remains elusive. In this study, we ingeniously chose to perform immunoprecipitation (IP) targeting the CSFV E2 protein, thereby achieving the complete removal of infectious virions. Subsequently, we discovered that the purified exosomes are shown to contain viral genomic RNA and partial viral proteins. Furthermore, exosomes secreted by CSFV-infected cells can evade CSFV-specific neutralizing antibodies, establish subsequent infection, and stimulate innate immune system after uptake by recipient cells. In summary, exosomes play a critical role in CSFV transmission. This is of great significance for in-depth exploration of the characteristics of CSFV and its complex interactions with the host.

PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation.

Viruses exploit the host cell's energy metabolism system to support their replication. Mitochondria, known as the powerhouse of the cell, play a critical role in regulating cell survival and virus replication. Our prior research indicated that the classical swine fever virus (CSFV) alters mitochondrial dynamics and triggers glycolytic metabolic reprogramming. However, the role and mechanism of PKM2, a key regulatory enzyme of glycolytic metabolism, in CSFV replication remain unclear. In this study, we discovered that CSFV enhances PKM2 expression and utilizes PKM2 to inhibit pyruvate production. Using an affinity purification coupled mass spectrometry system, we successfully identified PKM as a novel interaction partner of the CSFV non-structural protein NS4A. Furthermore, we validated the interaction between PKM2 and both CSFV NS4A and NS5A through co-immunoprecipitation and confocal analysis. PKM2 was found to promote the expression of both NS4A and NS5A. Moreover, we observed that PKM2 induces mitophagy by activating the AMPK-mTOR signaling pathway, thereby facilitating CSFV proliferation. In summary, our data reveal a novel mechanism whereby PKM2, a metabolic enzyme, promotes CSFV proliferation by inducing mitophagy. These findings offer a new avenue for developing antiviral strategies. IMPORTANCE: Viruses rely on the host cell's material-energy metabolic system for replication, inducing host metabolic disorders and subsequent immunosuppression-a major contributor to persistent viral infections. Classical swine fever virus (CSFV) is no exception. Classical swine fever is a severe acute infectious disease caused by CSFV, resulting in significant economic losses to the global pig industry. While the role of the metabolic enzyme PKM2 (pyruvate dehydrogenase) in the glycolytic pathway of tumor cells has been extensively studied, its involvement in viral infection remains relatively unknown. Our data unveil a new mechanism by which the metabolic enzyme PKM2 mediates CSFV infection, offering novel avenues for the development of antiviral strategies.

Intranasal Inoculation with Classical Swine Fever Virus Provided a More Consistent Experimental Disease Model Compared to Oral Inoculation.

The severity of disease resulting from classical swine fever virus (CSFV) infection is determined by several factors, including virus strain and host factors. The different outcomes of experimental studies in pigs with the same strain of CSFV emphasize the need to elucidate the influence of individual factors within experimental protocols. In this study, we investigated the outcome of disease after oral and intranasal inoculation with a moderately virulent CSFV strain in young pigs. To compare the two routes of inoculation, various infection parameters were examined during a period of two weeks. While all intranasally inoculated pigs (n = 5) were directly infected, this was only the case for two out of five pigs after oral inoculation. In addition, the intranasally inoculated pigs developed a more pronounced clinical disease and pathological lesions, as well as markedly more change in hematological and immunological parameters than the orally inoculated pigs. The wide variation among the orally inoculated pigs implied that statistical evaluation was markedly impaired, leaving this route of application less suitable for comparative studies on classical swine fever. Furthermore, our study provides additional details about the immunomodulatory effects of CSFV on the kinetics of CRP, TNF-α, and leukocyte sub-populations in pigs after infection with the CSFV strain Paderborn.

Monitoring and evaluation of provincial classical swine fever immunization implementation with an E2 subunit vaccine in Jeju Island, South Korea.

Purpose: Accidental vaccination with a live attenuated low-virulence strain of Miyagi (LOM) vaccine led to the reemergence of classical swine fever virus (CSFV) in Jeju province, South Korea in 2014. To control the continual outbreaks of LOM-derived CSFV, the provincial government launched a provincial mass vaccination project using a CSF-E2 subunit vaccine. We conducted this study to assess the herd immunity level and outcomes of E2 vaccine-based immunization in breeding and growing herds on Jeju Island during 2020-2021. Materials and Methods: A large-scale vaccination trial using the Bayovac CSF-E2 vaccine investigated its efficacy in breeding and growing herds under farm application conditions (10 CSFV-affected and three CSFV-naïve swine farms). Results: The level of herd immunity in each farm was classified into three (S1-S3) and six (G1-G6) profiles in breeding and growing herds, respectively. Immunity monitoring revealed a remarkable improvement in the herd immunity status in all farms. The majority (10/13) of farms, including CSFV-free farms, showed the S1G1 immunity profile in 2021, indicating the appropriate implementation of the advised vaccination regime. Moreover, there were significant decreases in Erns seropositivity from 100% to 50% and 25.9% to 4.3% at farm and pig levels, respectively. In particular, all farms were confirmed as CSFV free in the growing-finishing herds. Conclusion: Our large-scale trial demonstrated the effectiveness of the E2 subunit vaccine in establishing herd immunity stabilization and eliminating CSFV circulation in the affected farms and highlighted the need for a provincial vaccination policy to regain the CSF-free status on Jeju Island.

Establishment of a superinfection exclusion method for pestivirus titration using a recombinant reporter pestiviruses.

Pestiviruses are classified into two biotypes based on their cytopathogenicity. As the majority of pestivirus field isolates are noncytopathogenic, their titration requires alternative methods rather than direct observation of cytopathogenic effects, such as immunostaining using specific antibodies or interference with cytopathogenic strains. However, these methods require microscopic observation to assess virus growth, which is time- and labor-intensive, especially when handling several samples. In this study, we developed a novel luciferase-based pestivirus titration method using the superinfection exclusion phenomenon with recombinant reporter pestiviruses that possessed an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). In this method, swine kidney cells were inoculated with classical swine fever virus (CSFV) and superinfected with the reporter CSFV vGPE-/HiBiT 5 days postinoculation. Virus titer was determined based on virus growth measured in luminescence using the culture fluid 3 days after superinfection; the resultant virus titer was comparable to that obtained by immunoperoxidase staining. Furthermore, this method has proven to be applicable for the titration of border disease virus (BDV) by superinfection with both the homologous reporter BDV and heterologous reporter CSFV, suggesting that this novel virus titration method is a simple technique for automated virus detection based on the luciferase system.

Npro of classical swine fever virus enhances HMGB1 acetylation and its degradation by lysosomes to evade from HMGB1-mediated antiviral immunity.

Classical swine fever virus (CSFV) can dampen the host innate immunity by destabilizing IRF3 upon its binding with viral Npro. High mobility group box 1 (HMGB1), a non-histone nuclear protein, has diverse functions, including inflammation, innate immunity, etc., which are closely related to its cellular localization. We investigated potential mutual interactions between CSFV and HMGB1 and their effects on virus replication. We found that HMGB1 at the protein level, but not at mRNA level, was markedly reduced in CSFV-infected or Npro-expressing IPEC-J2 cells. HMGB1 in the nuclear compartment is anti-CSFV by promoting IFN-mediated innate immune response, as evidenced by overexpression of nuclear or cytoplasmic dominant HMGB1 mutant in IPEC-J2 cells stimulated with poly(I:C). However, CSFV Npro upregulates HMGB1 acetylation, a modification that promotes HMGB1 translocation into the cytoplasmic compartment where it is degraded by lysosomes. Ethyl pyruvate could downregulate HMGB1 acetylation and prevent Npro-mediated HMGB1 reduction. Inhibition of deacetylase HDAC1 with MS275 or by RNA silencing could promote Npro-mediated HMGB1 degradation. Taken together, our study elucidates the mechanism with which HMGB1 in the nuclei initiates antiviral innate immune response to suppress CSFV replication and elaborates the pathway by which CSFV uses its Npro to evade from HMGB1-mediated antiviral immunity through upregulating HMGB1 acetylation with subsequent translocation into cytoplasm for lysosomal degradation.

Performance of a Differentiation of Infected from Vaccinated Animals (DIVA) Classical Swine Fever Virus (CSFV) Serum and Oral Fluid Erns Antibody AlphaLISA Assay.

Classical swine fever virus (CSFV) is an OIE-listed disease that requires effective surveillance tools for its detection and control. The aim of this study was to develop and evaluate the diagnostic performance of a novel CSFV Erns IgG AlphaLISA for both serum and oral fluid specimens that would likewise be compatible with the use of CSFV E2 DIVA vaccines. Test performance was evaluated using a panel of well-characterized serum (n = 760) and individual (n = 528) or pen-based (n = 30) oral fluid samples from four groups of animals: (1) negative controls (n = 60 pigs); (2) inoculated with ALD strain wild-type CSFV (n = 30 pigs); (3) vaccinated with LOM strain live CSFV vaccine (n = 30 pigs); and (4) vaccinated with live CSFV marker vaccine on commercial farms (n = 120 pigs). At a cutoff of S/P ≥ 0.7, the aggregate estimated diagnostic sensitivities and specificities of the assay were, respectively, 97.4% (95% CI 95.9%, 98.3%) and 100% for serum and 95.4% (95% CI 92.9%, 97.0%) and 100% for oral fluid. The Erns IgG antibody AlphaLISA combined DIVA capability with solid diagnostic performance, rapid turnaround, ease of use, and compatibility with both serum and oral fluid specimens.

Plasma metabonomics of classical swine fever virus-infected pigs.

Classical swine fever (CSF) is an infectious disease caused by Classical swine fever virus (CSFV), which is characterized by depression, high fever, extensive skin bleeding, leukopenia, anorexia, alternating constipation, and diarrhea. Hemorrhagic infarction of the spleen is the main characteristic pathological change following CSFV infection. Large-scale outbreaks of CSF are rare in China and are mainly distributed regionally. The clinical symptoms of CSF are not obvious, and show variation from typical to atypical symptoms, which makes diagnosis based on clinical symptoms and pathology challenging. In recent years, the incidence of CSF-immunized pig farms in China has increased and new CSFV gene subtypes have appeared, posing new challenges to the prevention and control of CSF in China. Changes in metabolites caused by viral infection reflect the pathogenic process. Metabonomics can reveal the trace metabolites of organisms; however, plasma metabonomics of CSFV-infected pigs have rarely been investigated. Therefore, we used an established pig CSFV infection model to study changes in plasma metabolites. The results showed significant differences in forty-five plasma metabolites at different time periods after CSFV infection in pigs, with an increase in twenty-five metabolites and a decrease in twenty metabolites. These changed metabolites were mainly attributed to the tricarboxylic acid cycle, amino acid cycle, sugar metabolism, and fat metabolism. Thirteen metabolic pathways changed significantly in CSFV-infected pigs, including tricarboxylic acid cycle, inositol phosphate metabolism, glycine, serine and threonine metabolism,lysine degradation, alanine, aspartate and glutamic acid metabolism, pantothenate and CoA biosynthesis, ß-alanine metabolism, lysine degradation, arginine and proline metabolism, glycerolipid metabolism, phenylalanine metabolism, arachidonic acid metabolism, linoleic acid metabolism. Among these, changes in fatty acid biosynthesis and metabolism occurred at all time periods post-infection. These results indicate that CSFV infection in pigs could seriously alter metabolic pathways.

A multiplex real-time RT-PCR system to simultaneously diagnose 16 pathogens associated with swine respiratory disease.

AIMS: Swine respiratory disease (SRD) is a major disease complex in pigs that causes severe economic losses. SRD is associated with several intrinsic and extrinsic factors such as host health status, viruses, bacteria, and environmental factors. Particularly, it is known that many pathogens are associated with SRD to date, but most of the test to detect those pathogens can be normally investigated only one pathogen while taking time and labor. Therefore, it is desirable to develop rapidly and efficiently detectable methods those pathogens to minimize the damage caused by SRD. METHODS AND RESULTS: We designed a multiplex real-time RT-PCR (RT-qPCR) system to diagnose simultaneously 16 pathogens, including nine viruses and seven bacteria associated with SRD, on the basis of single qPCR and RT-qPCR assays reported in previous studies. Multiplex RT-qPCR system we designed had the same ability to single RT-qPCR without significant differences in detection sensitivity for all target pathogens at minimum to maximum genomic levels. Moreover, the primers and probes used in this system had highly specificity because the sets had not been detected pathogens other than the target and its taxonomically related pathogens. Furthermore, our data demonstrated that this system would be useful to detect a causative pathogen in the diagnosis using oral fluid from healthy pigs and lung tissue from pigs with respiratory disorders collected in the field. CONCLUSIONS: The rapid detection of infected animals from the herd using our system will contribute to infection control and prompt treatment in the field.

A Triple Gene-Deleted Pseudorabies Virus-Vectored Subunit PCV2b and CSFV Vaccine Protect Pigs against a Virulent CSFV Challenge.

Classical swine fever (CSF) remains one of the most economically significant viral diseases affecting domestic pigs and wild boars worldwide. To develop a safe and effective vaccine against CSF, we have constructed a triple gene-deleted pseudorabies virus (PRVtmv)-vectored bivalent subunit vaccine against porcine circovirus type 2b (PCV2b) and CSFV (PRVtmv+). In this study, we determined the protective efficacy of the PRVtmv+ against virulent CSFV challenge in pigs. The results revealed that the sham-vaccinated control group pigs developed severe CSFV-specific clinical signs characterized by pyrexia and diarrhea, and became moribund on or before the seventh day post challenge (dpc). However, the PRVtmv+-vaccinated pigs survived until the day of euthanasia at 21 dpc. A few vaccinated pigs showed transient diarrhea but recovered within a day or two. One pig had a low-grade fever for a day but recovered. The sham-vaccinated control group pigs had a high level of viremia, severe lymphocytopenia, and thrombocytopenia. In contrast, the vaccinated pigs had a low-moderate degree of lymphocytopenia and thrombocytopenia on four dpc, but recovered by seven dpc. Based on the gross pathology, none of the vaccinated pigs had any CSFV-specific lesions. Therefore, our results demonstrated that the PRVtmv+ vaccinated pigs are protected against virulent CSFV challenge.

Development of a quadruplex real-time quantitative RT-PCR for detection and differentiation of PHEV, PRV, CSFV, and JEV.

Porcine hemagglutinating encephalomyelitis virus (PHEV), porcine pseudorabies virus (PRV), classical swine fever virus (CSFV), and Japanese encephalitis virus (JEV) cause similar neurological symptoms in the infected pigs, and their differential diagnosis depends on laboratory testing. Four pairs of specific primers and probes were designed targeting the PHEV N gene, PRV gB gene, CSFV 5' untranslated region (5'UTR), and JEV NS1 gene, respectively, and a quadruplex real-time quantitative RT-PCR (qRT-PCR) was developed to detect and differentiate PHEV, PRV, CSFV, and JEV. The assay showed high sensitivity, with the limit of detection (LOD) of 1.5 × 101 copies/µL for each pathogen. The assay specifically detected only PHEV, PRV, CSFV, and JEV, without cross-reaction with other swine viruses. The coefficients of variation (CVs) of the intra-assay and the inter-assay were less than 1.84%, with great repeatability. A total of 1,977 clinical samples, including tissue samples, and whole blood samples collected from Guangxi province in China, were tested by the developed quadruplex qRT-PCR, and the positivity rates of PHEV, PRV, CSFV, and JEV were 1.57% (31/1,977), 0.35% (7/1,977), 1.06% (21/1,977), and 0.10% (2/1,977), respectively. These 1,977 samples were also tested by the previously reported qRT-PCR assays, and the coincidence rates of these methods were more than 99.90%. The developed assay is demonstrated to be rapid, sensitive, and accurate for detection and differentiation of PHEV, PRV, CSFV, and JEV.