Search Results (112)

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

Host metabolic reprogramming is a critical strategy employed by many viruses to support their replication, and the key metabolic enzyme plays important roles in virus infection. This study investigates the role of pyruvate kinase M2 (PKM2), a glycolytic enzyme with non-canonical functions, in the replication of classical swine fever virus (CSFV). Using PK-15 cells and piglet models, we demonstrate that CSFV infection upregulates PKM2 expression both in vitro and in vivo, creating a proviral environment. knockdown of PKM2 by siRNA reduced CSFV proliferation, while PKM2 overexpression significantly increased virus propagation, which was evaluated by viral protein synthesis, genome replication, and progeny virion production. A direct interaction between PKM2 and CSFV NS5B protein was identified by co-immunoprecipitation and GST-pulldown assays, and PKM2 affected NS5B polymerase activity in a dual-luciferase reporter assay, with PKM2 depletion reducing RdRp function by 50%. Temporal analysis of the first viral replication cycle confirmed PKM2-dependent enhancement of CSFV RNA synthesis. These findings establish PKM2 as a proviral host factor that directly binds NS5B to potentiate RdRp activity, thereby bridging metabolic adaptation and viral genome replication. This study provides new evidence of a glycolytic enzyme physically interacting and enhancing viral polymerase function, offering new information about CSFV–host interaction. Full article

A chimeric pestivirus KD26_E2LOM strain can induce antibodies that can be partially distinguished from antibodies from classical swine fever virus (CSFV) infection. The chimeric pestivirus vaccine strain was created using bovine viral diarrhea virus as the backbone; however, the entire BVDV E2 gene region was replaced with the E2 gene, which encodes the major target for neutralizing antibodies against CSFV. Pigs were vaccinated once or twice with the chimeric pestivirus KD26_E2LOM strain, and protective efficacy was evaluated after subsequent challenge with virulent CSFV. Pigs inoculated with the chimeric pestivirus KD26_E2LOM strain did not have a high temperature or leukopenia, and CSFV neutralizing antibodies (>64-fold) were observed from 28 days postvaccination (dpv). In addition, the level of anti-CSFV E2 antibody positivity was >0.8 (s/p value) from 30 dpv, and there were no antibody-positive individuals among the sentinel pigs. In control pigs, CSF antigen was detected in blood, nasal, and fecal samples at 5, 7, 10, 14, and 21 days postchallenge (dpc) and in several organs; however, no CSFV was detected in the organs of pigs vaccinated with the chimeric pestivirus KD26_E2LOM strain, and no virus shedding or CSF antigen was detected on any dpc. Thus, the chimeric pestivirus KD26_E2LOM strain protects pigs against horizontal transmission of virulent CSFV; however, this strain may have only partial potential for the differential detection of CSFV E^rns antibodies. Full article

Nipah virus (NiV) is a severe zoonotic pathogen that substantially threatens public health. Pigs are the natural hosts of NiV and can potentially transmit this disease to humans. Establishing a rapid, sensitive, and accurate point-of-care detection method is critical in the timely identification of infected pig herds. In this study, we developed an NiV detection method based on reverse transcription–recombinase polymerase amplification (RT-RAA) and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 13a (Cas13a) system for the precise detection of NiV. The highly conserved region of the NiV gene was selected as the detection target. We first designed eleven pairs of RT-RAA primers, and the optimal primer combination and reaction temperature were identified on the basis of RT-RAA efficiency. Additionally, the most efficient crRNA sequence was selected on the basis of the fluorescence signal intensity. The results revealed that the optimal reaction temperature for the developed method was 37 °C. The detection limit was as low as 1.565 copies/μL. Specificity testing revealed no cross-reactivity with nucleic acids from six common swine viruses, including Seneca virus A (SVA), foot-and-mouth disease virus (FMDV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), African swine fever virus (ASFV), and pseudorabies virus (PRV). A validation test using simulated clinical samples revealed a 100% concordance rate. The detection results can be visualized via a fluorescence reader or lateral flow strips (LFSs). Compared with conventional detection methods, this RT-RAA-CRISPR/Cas13a-based method is rapid and simple and does not require scientific instruments. Moreover, the reagents can be freeze-dried for storage, eliminating the need for cold-chain transportation. This detection technology provides a convenient and efficient new tool for the point-of-care diagnosis of NiV and for preventing and controlling outbreaks. Full article

Following its initial documented emergence in Hunan’s swine facilities in 2017, GETV has inflicted considerable financial damage upon China’s pork production sector. Beyond its impact on swine, GETV also poses a potential risk to other animal species and public health, primarily due to its mosquito-borne transmission. Between September 2020 and May 2021, a GETV outbreak occurred on a commercial swine farm in Sichuan Province, where affected animals exhibited movement disorders, neurological symptoms, and mild diarrhea, with severe cases resulting in mortality. To elucidate the genetic characteristics and transmission dynamics of the virus, a comprehensive investigation of this outbreak was conducted. Clinical samples from diseased or deceased swine were collected and analyzed by PCR, confirming the presence of GETV infection. Additionally, differential diagnostic assays were performed for other common swine pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), classical swine fever virus (CSFV), and atypical porcine pestivirus (APPV). All tests yielded negative results, confirming GETV as the sole etiological agent of the outbreak. The GETV-positive samples were inoculated into BHK-21 cells, leading to the isolation of a GETV strain (SC202009). Whole-genome sequence analysis revealed that SC202009 belongs to the GIII genotype of GETV, with a nucleotide sequence identity ranging from 95.1% to 99.8% compared to reference sequences in GenBank. This high level of sequence identity suggests that SC202009 is closely related to other circulating strains in the region, indicating potential local transmission networks and endemic viral circulation. The nucleotide sequence identity of the E2 gene ranged from 94.2% to 99.6%, while the E2 protein amino acid sequence identity ranged from 97.2% to 99.5%. Phylogenetic analysis further indicated that SC202009 is genetically distant from the original Malaysian prototype strain (MM2021) but shares high sequence identity with several strains isolated in Sichuan Province, including SC266, SC201807, and SC483. Notably, NMJA_F2_18-8L-NH-Cxp-Y-1-1 was isolated from Culex pipiens, suggesting a mosquito-borne transmission route. These findings provide novel insights into the regional dissemination patterns and genetic diversity of GETV in China. These results also highlight the importance of the continuous surveillance of viral genetic variations and the elucidation of transmission dynamics to develop effective control strategies and mitigate potential public health risks. Future studies should focus on the ecological and environmental factors influencing GETV transmission, particularly the role of mosquito vectors in cross-species viral spread, to enhance targeted prevention and control measures for this emerging arboviral threat. Full article

Classical swine fever (CSF) is an acute and often fatal disease caused by CSF virus (CSFV) infection. In the present study, we investigated the transcriptional profiles of peripheral blood mononuclear cells (PBMCs) in pigs infected with CSFV. The results revealed that CSFV inhibits IFNα/β production, but up-regulates the expression of signal transducer and activator of transcription 1 (STAT1); this result was verified in vitro. Interestingly, STAT1 is typically a downstream target of IFNα/β, raising the question of how CSFV can inhibit IFNα/β expression, yet up-regulate STAT1 expression. To explore this further, we observed that UV-treated CSFV induced STAT1 expression. Our results demonstrated that CSFV E^rns, E1, and E2 could up-regulate STAT1 expression within the host cell cytoplasm and facilitate its translocation into the nucleus. The E^rns, E1, and E2 proteins also separately induced the up-regulation of interleukin (IL)-10; IL-10 acts as the communicator connecting E^rns, E1, and E2 proteins to STAT1, leading to the subsequent up-regulation, phosphorylation, and nuclear translocation of STAT1. Silencing of IL-10 down-regulated STAT1 expression. Finally, MX1 and OAS1 were identified as downstream targets of the IL-10-STAT1 pathway. In summary, a novel IL-10-STAT1 pathway independent of IFNα/β induced by CSFV E^rns, E1, and E2 was identified in this study. Full article