Search Results (171)

Cell cultures are an essential tool for laboratory diagnosis of porcine viral infections. However, interpreting the results requires considering the species and tissue origin of cell lines as well as the specific virus replication characteristics (cytopathic effect). This guide discusses the development of techniques for the primary isolation of viruses from biological material and provides recommendations for culturing viruses in different cell types. According to the World Organization for Animal Health, laboratory diagnosis should aim to isolate the virus in cell culture. We have studied the evolution of virus isolation methods for various diseases affecting pigs, including African swine fever virus (ASFV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (Aujeszky’s disease, PRV), rotaviruses (RV), teschoviruses (PTVs), swine pox virus (SwPV), swine influenza A virus (IAVs), parvovirus (PPV), coronaviruses, circoviruses (PCVs), diseases with vesicular syndrome, and others. During our analysis of the literature and our own experience, we found that the porcine kidney (PK-15) cell line is the most suitable for isolating most viral porcine pathogens. For ASFV and PRRSV, the porcine alveolar macrophages (PAMs) continue to remain the primary model for isolation. These findings can serve as a starting point for virological reference laboratories to select optimal conditions for cultivating, obtaining field isolates, and strain adaptation. Full article

Background/Objectives: Classical swine fever (CSF) is listed by the World Organisation for Animal Health as a highly devastating and contagious pig disease, causing severe economic losses to the swine industry. In spite of the successful elimination of CSF in Taiwan, preparedness against potential reintroduction remains essential. The live attenuated vaccines have been effective in disease control, but are not capable of a viable strategy that differentiates infected from vaccinated animals (DIVA). Subunit vaccines are recognized for their safety and ability to induce protective immunity against infectious diseases. Methods: In this study, the recombinant CSF virus (CSFV) E2 proteins were formulated with a CpG motif as an adjuvant to produce the E2-CpG subunit vaccine. Its efficiency in specific-pathogen-free (SPF) pigs was compared with a commercially available E2 subunit vaccine (Bayovac^® CSF-E2; Bayer Taiwan Co., Ltd., Taipei City, Taiwan). Results: Significantly higher titers of anti-E2 antibodies were induced in pigs immunized with a single dose of the E2-CpG vaccine, particularly the reduced E-0.5A formulation, than those immunized with a dose of the commercialized E2 subunit vaccine adjusted to double dosage. This designed subunit vaccine showed high efficacy in protection against clinical symptoms and significant pathological alterations in pigs after a highly virulent CSFV (genotype 1.1) challenge. Viral shedding was not detected in vaccinated pigs before completion of the challenge study, and the viral load in their spleens remained undetectable. Conclusions: These results could support the potential of the E2-CpG vaccine as a cost-effective, single-dose subunit vaccine capable of inducing robust CSFV-specific immunity and providing 100% protection against lethal CSFV challenges. Full article

Background: Classical swine fever virus (CSFV) is a highly contagious and fatal disease in pigs and wild boars. While hunting and bait vaccination are effective for CSFV eradication, additional strategies are needed to control wild boar populations. This study aimed to develop an oral vaccine, Flc-LOM-GnRHx3, by inserting gonadotropin-releasing hormone (GnRH) epitopes into the Flc-LOM clone. Methods: The Flc-LOM-GnRHx3 strain was rescued from CPK cells and propagated to high titers in MDBK cells. Male boars (20 weeks old) received three doses (10^5.0 TCID₅₀/ml/dose) of Flc-LOM-GnRHx3 either orally or intramuscularly at 2-week intervals. Anti-CSFV E2 antibodies were detected via immunofluorescence and Western blotting. Results: Both vaccination routes induced anti-GnRH antibodies and reduced testosterone levels. Testis size and weight were slightly lower than controls, with seminiferous tubule and spermatid deformities observed in 52.5% of intramuscularly vaccinated pigs and 20.8% of orally vaccinated pigs. Conclusions: Flc-LOM-GnRHx3 demonstrates potential as a dual-function oral vaccine that can eradicate CSFV and impair reproductive capacity in wild boars, offering a novel approach for integrated disease control and population management. Full article

Classical swine fever (CSF) is a highly contagious and lethal disease caused by classical swine fever virus (CSFV), and it is also a notifiable disease according to the World Organization for Animal Health. Owing to the continuous growth of the international trade in pigs and pig products, pig farming has become the pillar industry of the global livestock industry and is the most important source of animal protein for mankind. As a single-stranded RNA virus, CSFV can avoid being recognized and cleared by the host immune system through a variety of immune evasion strategies so that it persists in the host body and causes multisystemic pathology. CSF has also become one of the most serious infectious diseases affecting the pig industry, resulting in considerable economic losses to the pig industry. Therefore, understanding the main immune evasion mechanism of CSFV is very important for the prevention and control of CSF infection. This article reviews the main immune evasion mechanisms of CSFV, including the suppression of nonspecific immune responses; evasion of adaptive immune responses; and the regulation of host cell apoptosis and cell autophagy. CSFV affects type I interferon regulatory signals; the JAK-STAT signaling pathway; the RIG-I and NF-κB signaling pathways; immune cell function; the mitochondrial apoptosis pathway; and the endoplasmic reticulum stress apoptosis pathway; the PI3K-Akt signaling mediated AMPK-mTOR macroautophagy pathway through its structural proteins E^rns and E1 and E2; and the nonstructural proteins N^pro, NS4B, and NS5A to achieve immune evasion. As our understanding of CSFV immune strategies continues to deepen, we believe that this understanding will provide new strategies for the development of new vaccines and novel diagnostic methods in the future. Full article

African swine fever virus (ASFV) and classical swine fever virus (CSFV) are important transboundary animal diseases (TADs) affecting swine. ASFV is a large DNA virus with a genome size of 170–190+ kilobases (kB) belonging to the family Asfarviridae, genus Asfivirus. CSFV is a single-stranded RNA virus with a genome size of approximately 12 kB, belonging to the family Flaviviridae, genus Pestivirus. Outbreaks involving either one of these viruses result in similar disease syndromes and significant economic impacts from: (i) high morbidity and mortality events; (ii) control measures which include culling and quarantine; and (iii) export restrictions of swine and pork products. Current detection methods during an outbreak provide minimal genetic information on the circulating virus strains/genotypes that are important for tracing and vaccine considerations. The increasing availability and reduced cost of next-generation sequencing (NGS) allow for the establishment of NGS protocols for the rapid identification and complete genetic characterization of outbreak strains during an investigation. NGS data provides a better understanding of viral spread and evolution, facilitating the development of novel and effective control measures. In this study, panels of primers spanning the genomes of ASFV and CSFV were independently developed to generate approximately 10 kB and 6 kB amplicons, respectively. The primer panels consisted of 19 primer pairs for ASFV and 2 primer pairs for CSFV, providing whole genome amplification of each pathogen. These primer pools were further optimized for batch pooling and thermocycling conditions, resulting in a total of 5 primer pools/reactions used for ASFV and 2 primer pairs/reactions for CSFV. The ASFV primer panel was tested on viral DNA extracted from blood collected from pigs experimentally infected with ASFV genotype I and genotype II viruses. The CSFV primer panel was tested on 11 different strains of CSFV representing the three known CSFV genotypes, and 21 clinical samples collected from pigs experimentally infected with two different genotype 1 CSF viruses. ASFV and CSFV amplicons from optimized PCR were subsequently sequenced on the Oxford Nanopore MinION platform. The targeted protocols for these viruses resulted in an average coverage greater than 1,000X for ASFV, with 99% of the genome covered, and 10,000X–20,000X for CSFV, with 97% to 99% of the genomes covered. The ASFV targeted whole genome sequencing protocol has been optimized for genotype II ASF viruses that have been responsible for the more recent outbreaks outside of Africa. The CSFV targeted whole genome sequencing protocol has universal applications for the detection of all CSFV genotypes. Protocols developed and evaluated here will be essential complementary tools for early pathogen detection and differentiation, as well as genetic characterization of these high-consequence swine viruses, globally and within the United States, should an outbreak occur. Full article

Despite intensive eradication efforts, classical swine fever (CSF) remains endemic across South America, Europe, Asia, and the Caribbean, highlighting the need for more effective surveillance and detection methods. Reverse-transcription real-time polymerase chain reaction (RRT-PCR) is the fastest, and most sensitive assay for detecting CSF virus (CSFV) genomic material. Previously, we demonstrated that spleen swabs outperformed spleen homogenates for the detection of ASFV genomic material by RRT-PCR. In this study, we compared CSFV genome detection in paired spleen homogenates and spleen swabs generated using 49 frozen and 33 fresh spleen samples collected from experimentally inoculated pigs with acute infection. The results show that the CSFV genome detection in spleen swabs is comparable to that in spleen homogenates. The study also demonstrated that the CSFV genomic material can be detected in spleen swabs during early CSFV infections, and the viruses can be successfully isolated from the swabs. The use of spleen swabs instead of spleen tissue homogenates for CSF detection will reduce labor, decrease costs associated with reporting, and increase the diagnostic throughput. Full article

Background/Objectives: Classical swine fever (CSF) continues to challenge global eradication efforts, particularly in endemic regions, where pregnant sows face heightened risks of vertical transmission following exposure to CSFV. Methods: This study evaluates the early protective efficacy of FlagT4G, a novel live attenuated DIVA-compatible vaccine. Pregnant sows were vaccinated at mid-gestation and challenged 14 days later with a highly virulent CSFV strain. Results: FlagT4G conferred complete clinical protection, preventing both maternal viremia and transplacental transmission. No CSFV RNA, specific antibodies, or IFN-α were detected in fetal samples from vaccinated animals. In contrast, unvaccinated sows exhibited clinical signs, high viral loads, and widespread fetal infection. Interestingly, early protection was observed even in the absence of strong humoral responses in some vaccinated sows, suggesting a potential role for innate or T-cell-mediated immunity in conferring rapid protection. Conclusions: The demonstrated efficacy of FlagT4G within two weeks of vaccination underscores its feasibility for integration into emergency vaccination programs. Its DIVA compatibility and ability to induce early fetal protection against highly virulent CSFV strains position it as a promising tool for CSF control and eradication strategies. Full article

Wild boars are recognized reservoirs of numerous viral pathogens, posing a significant risk to domestic pig populations, particularly in areas with poor biosecurity. This study assessed the prevalence and co-infection patterns of porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine cytomegalovirus (PCMV), African swine fever virus (ASFV), classical swine fever virus (CSFV), and pseudorabies virus (PRV) in wild boars from western Serbia and the Republic of Srpska (Bosnia and Herzegovina). Sixty-six spleen samples from legally hunted wild boars were analyzed by qPCR. All animals were negative for ASFV, CSFV, and PRV. The cumulative prevalence of infection with at least one of the other three viruses was 86.4% (95% CI: 76.2–92.8%). PCMV was detected in 74.2% of samples, PCV2 in 50%, and PPV in 28.8%. Co-infections were common: 42.4% of animals were positive for two viruses, and 12.1% for all three. A statistically significant association was observed between triple co-infection and sex, with higher rates in males. Subadult wild boars showed the highest PCV2 + PCMV co-infection rate (p = 0.0547). These findings highlight the need to expand molecular surveillance, particularly for PCMV, in both wild and domestic pigs, especially in regions reliant on low-biosecurity backyard farming. Full article

Background/Objectives: Porcine Circovirus Type 2 (PCV2) impairs pigs’ immune systems and increases susceptibility to co-infections, including Classical Swine Fever (CSF), a highly contagious disease listed by the World Organisation for Animal Health (WOAH) as notifiable. Therefore, swine operations in CSF-endemic regions are encouraged to immunize piglets with both PCV2 and CSFV vaccinations. Currently, there is no commercially available bivalent vaccine for PCV2/CSFV. Methods: In this study, a total of twenty 4-week-old SPF pigs were administered our formulated PCV2/CSFV bivalent subunit vaccine, containing soluble CSFV-E2 (50 µg) and PCV2-ORF2 (100 µg) antigens with a porcine-specific CpG adjuvant. After 4 weeks of vaccination, all pigs were evaluated for efficacy against PCV2 and CSFV. Results: Pigs were only immunized once and showed significantly increased neutralizing or ELISA antibody titers against both viruses four weeks post-vaccination. After viral challenges, vaccinated pigs displayed no clinical signs or lesions and had markedly reduced CSFV and PCV2 viral loads in the serum and tissues compared to controls. Conclusions: These results demonstrate that a single dose of the PCV2/CSFV bivalent subunit vaccine is safe and effective in young pigs, induces strong antibody responses, and suppresses viral replication, making it a promising tool for swine disease control and cost-effective vaccination strategies. Full article

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. Full article

Beta-sitosterol (BS), a naturally occurring phytosterol abundant in plants, has been reported to exhibit diverse biological activities, including immunomodulatory and antiviral effects. Classical swine fever virus (CSFV), a member of the Pestivirus genus, remains a persistent threat to the swine industry worldwide, causing considerable economic damage. Our research found that BS significantly enhances the replication of both the CSFV-Shimen strain and the attenuated C-strain vaccine virus in PK-15 cells. Additionally, transcriptomic profiling (RNA-Seq) identified 175 differentially expressed genes (DEGs) following BS exposure, comprising 53 upregulated and 122 downregulated genes. Further results demonstrated that treatment with β-sitosterol suppressed IκBα expression, thereby activating the NF-κB pathway, and that knockdown of endogenous IκBα significantly promoted CSFV replication. These findings contribute to a deeper understanding of how BS influences the CSFV infection process, suggesting its role as a host lipid-associated factor facilitating viral propagation. Full article

Classical swine fever (CSF) is an infectious disease caused by classical swine fever virus (CSFV), which is endemic in many areas of China, causing serious economic losses to pig farms. Currently, 2.1 subgenotype strains are predominantly prevalent in China. Although abundant information is available on 2.1 subgenotype isolates, limited data are available on pathogenicity analysis. In this study, a CSFV strain was isolated from a pig farm in Guangdong Province, China. Whole genome sequencing showed that the strain had a genome length of 12,296 bp, and it was named GD-2024. Based on genetic evolutionary analysis, the strain was categorized into subgenotype 2.1c, and the nucleotide and amino acid homology of the strain with the representative strains of each subgenotype was in the range of 83.1–97.6% and 90.8–99.4%, respectively. Further mutation analysis revealed that the strain had three nucleotide site mutations in the 5′UTR and 3′UTR regions and two amino acid site mutations in the E2 region. The clinical pathogenicity of this strain was investigated. Infection with GD-2024 led to persistent fever and high viremia in pigs as well as inflammatory damage in multivisceral tissues. The mortality rate of infected pigs reached as high as 60%, contradicting the currently reported virulence of 2.1 strains. In summary, we have isolated and reported a subgenotype 2.1c strain with high virulence. Its genomic variation provides a basis for further analysis of virulence determinants and serves as a clinical reference and guide for the prevention and control of CSF. Full article

Background/Objectives: Control of classical swine fever virus (CSFV) in endemic countries relies on vaccination using live attenuated vaccines (LAVs). Most of these LAVs do not allow for the differentiation of vaccinated animals from infected animals (DIVA) based on their serological response. FlagT4G vaccine is a novel candidate that confers robust protective immunity early after vaccination and shows DIVA capabilities. Methods: This report presents the characterization of FlagT4G virus in terms of the stability of its genomic and attenuated phenotypes assessed by a reversion to virulence protocol, as well as its protective efficacy by determining the minimal protective dose. Results: Results presented here demonstrate that after five consecutive passages in groups of 5-week-old susceptible domestic pigs, FlagT4G virus remains genetically stable, and its attenuated phenotype remains unaltered. In terms of efficacy, FlagT4G virus induced solid protection against the intranasal challenge with 10⁵ tissue culture infectious dose (TCID₅₀) of virulent field isolate Brescia virus, even with a vaccine dose as low as 10² TCID50. Conclusions: Results presented here indicate that the FlagT4G vaccine may be a useful tool for CSFV control. Full article

Foot and mouth disease virus (FMDV) is a highly pathogenic virus that mainly infects cloven hooved animals, such as pigs. The establishment of a rapid, sensitive and accurate point-of-care detection method is critical for the timely identification and elimination of infected pigs for controlling this disease. In this study, a RT-RAA-CRISPR/Cas13a method was developed for the detection of FMDV serotype O in pigs. Six pairs of RT-RAA primers were designed based on the conserved gene sequence of FMDV serotype O, and the optimal amplification primers and reaction temperatures were screened. The CRISPR-derived RNA (crRNA) was further designed based on the optimal target band sequence and the most efficient crRNA was screened. The results revealed that FMDV-O-F4/R4 was the optimal primer set, and the optimal temperature for the RT-RAA reaction was 37 °C. Moreover, crRNA4 exhibited the strongest detection signal among the six crRNAs. The established RT-RAA-CRISPR/Cas13a method demonstrated high specificity and no cross-reactivity with other common swine pathogens such as Senecavirus A (SVA), porcine reproductive and respiratory virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), and pseudorabies virus (PRV), additionally, it was observed to be highly sensitive, with a detection limit of 19.1 copies/µL. The repeatability of this method was also observed to be good. This method could produce stable fluorescence and exhibited good repeatability when three independent experiments yielded the same results. A validation test using three types of simulated clinical samples (including swab, tissue, and serum samples) revealed a 100% concordance rate. The detection results could be visualized via a fluorescence reader or lateral flow strips (LFSs). Thus, a highly specific and sensitive RT-RAA-CRISPR/Cas13a detection method was developed and is expected to be applied for the rapid detection of FMDV serotype O in situ. Full article

The increasing spread of African swine fever (ASF) in recent years and the presence of classical swine fever (CSF) subclinical forms in endemic countries suggests that the possibility of coinfection with ASF virus (ASFV) and CSF virus (CSFV) in pigs cannot be ruled out in areas where both diseases are prevalent. Thus, rapid and reliable diagnosis through molecular testing is essential for the timely implementation of control measures to prevent the spread of these devastating swine diseases. Here, we have coupled two of the most validated PCR assays for the detection of CSFV and ASFV in a single reaction tube. The combination of the two tests for the detection of two target nucleic acids did not affect the analytical sensitivity, and the duplex RT-qPCR assay was comparable with the standard molecular techniques. The detection limits for CSFV RNA and ASFV DNA were 0.12 TCID₅₀/reaction and 0.25 TCID₅₀/reaction, respectively. The test showed high repeatability and reproducibility, the coefficient of variation was below 2%, and excellent performance was demonstrated in clinical samples. The duplex assay shows great potential to become a robust diagnostic tool for the rapid and reliable detection and differentiation of CSFV and ASFV in areas where both viruses may be circulating. Full article