Search Results (156)

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

Pseudorabies virus (PRV), a major swine pathogen, causes severe neurological, respiratory, and reproductive disorders, resulting in substantial economic losses to the global swine industry. Previous studies have shown that the gD glycoprotein of PRV has an effective protective effect. In this study, we constructed a plasmid DNA vaccine (pVAX1-GD-Fc) encoding a gD protein fused with pig IgG Fc and evaluated the adjuvant effects of porcine cGAS, the universal STING complex mimic (UniSTING), or IFN-α in mice. The mice were immunized three times (days 0, 14, and 21) with pVAX1-GD-Fc in the presence or absence of an adjuvant, followed by lethal challenge with PRV-HLJ8 3 days after the final immunization. The results revealed that the pVAX1-GD-Fc group exhibited 20% mortality (1/5 mice) on day 7 postchallenge, and all adjuvanted groups achieved 100% survival during the 14-day observation period. Flow cytometric analysis of splenocytes one week after the second immunization revealed significantly greater CD8+ T cell proportions in the adjuvant groups than in both the mock and pVAX1-GD-Fc-only control groups (p < 0.01). Furthermore, T cell proliferation assays demonstrated a significantly increased stimulation index in the adjuvant-treated mice, confirming enhanced cellular immunity. These findings demonstrate that cGAS, UniSTING, and IFN-α can serve as effective vaccine adjuvants to rapidly enhance cellular immune responses to PRV, highlighting their potential application in veterinary vaccines. Full article

Background: Pseudorabies virus (PRV), a critical porcine herpesvirus, induces severe diseases in both livestock and wildlife, imposing an incalculable burden and economic losses in livestock production. In this study, we investigated the evolutionary mechanisms and host adaptation strategies of the PRV gB gene through genomic alignment. The gB gene is highly conserved in PRV, and its encoded gB protein exhibits functional interchangeability across different herpesvirus species. Notably, the gB protein elicits the production of both complement-dependent and complement-independent neutralizing antibodies in animals, while also being closely associated with syncytium formation. Methods: Phylogenetic analysis and codon usage pattern analysis were performed in this study. A total of 110 gB gene sequences were analyzed, which were collected from [2011 to 2024] across the following regions: [Fujian, Shanxi, Guangxi, Guangdong, Chongqing, Henan, Shaanxi, Heilongjiang, Sichuan, Jiangsu, Jilin, Huzhou, Shandong, Hubei, Jiangxi, Beijing, Shanghai, Chengdu (China)], [Budapest, Szeged (Hungary)], [Tokyo (Japan)], [London (United Kingdom)], [Athens (Greece)], [Berlin (Germany)], and [New Jersey (United States)]. Results: The gB gene of PRV employs an evolutionary “selective optimization” strategy to maintain a dynamic balance between ensuring functional expression and evading host immune pressure, with this core trend strongly supported by its codon usage bias and mutation characteristics. First, the gene exhibits significant codon usage bias [Effective Number of Codons (ENC) = 27.94 ± 0.1528], driven primarily by natural selection rather than mere mutational pressure. Second, phylogenetic analysis shows that the second codon position of gB has the highest mutation rate (1.0586)—a feature closely linked to its antigenic variation and immune escape capabilities, further reflecting adaptive evolution against host immune pressure. Additionally, ENC-GC3 plot analysis reveals the complex regulatory mechanisms underlying codon bias formation, providing molecular evidence for the “selective optimization” strategy and clarifying PRV’s core evolutionary path to balance functional needs and immune pressure over time. Conclusions: Our study findings deepen our understanding of the evolutionary mechanisms of PRV and provide theoretical support for designing vaccines and assessing the risk of cross-species transmission. Full article

Aujeszky’s disease, or pseudorabies, is a viral infection caused by Suid herpesvirus 1 (pseudorabies virus), with swine as its natural host. Although eradicated in domestic pigs in many European countries, PRV remains endemic in wild boar populations, posing a risk to other species, including carnivores. In this study, we report eight fatal cases of PRV infection in hunting dogs from Epirus and Thessaly, Greece, all of which followed direct contact with hunted wild boars. Postmortem brain samples tested positive for PRV via PCR targeting the glycoprotein C (gC) gene. Partial sequencing and phylogenetic analysis of the amplified gC fragments revealed genetic divergence among the examined isolates. The Epirus-derived strains formed a distinct cluster, closely related to previously reported Greek strains from the region of Central Macedonia as well as to the French strain FRA 527 and the German isolate GER614BW. In contrast, the two Thessaly sequenced isolates were phylogenetically distant from all other Greek strains, potentially representing an independently evolving lineage, and clustered more closely with the Kaplan strain. These findings underscore the persistent threat of PRV transmission from wild to domestic species and highlight the genetic heterogeneity of PRV strains circulating in Greece. Veterinary practitioners should consider PRV in the differential diagnosis of encephalitic symptoms in hunting dogs. Enhanced molecular surveillance and public awareness are critical to mitigating the risks posed by this emerging threat. Full article

Pseudorabies virus (PRV) is a highly pathogenic agent that adversely impacts swine populations, leading to considerable economic losses within the Chinese pig industry. Furthermore, the potential for PRV to transmit across species from pigs to other hosts has attracted significant attention. In light of this, it is essential to identify efficacious antiviral agents for the treatment of PRV infection. In this study, we investigated the antiviral properties of Diltiazem HCl (DTZ) against PRV infection in susceptible cell lines. Our results demonstrated that DTZ significantly inhibited PRV infection in both PK15 and Vero cells. Moreover, this chemical compound exhibited antiviral activity against both variant and classical strains of PRV, as well as herpes simplex virus type 1 (HSV-1). Time-of-addition assays showed that DTZ exerted its inhibitory effect through specific interference with the virus replication process. Subsequent transcriptomic analysis via RNA sequencing indicated that the calcium signaling pathway might be involved in the antiviral properties of DTZ against PRV infection. Specifically, treatment with EGTA or calcium ion (Ca²⁺)-free medium inhibited PRV infection; this inhibitory effect was substantially mitigated upon the reintroduction of CaCl₂. In summary, DTZ effectively suppressed PRV infection in vitro, demonstrating its potential as an antiviral agent against PRV infections. Full article

Pseudorabies virus (PRV), a highly pathogenic alphaherpesvirus, poses a potential threat to public health and safety due to its broad host range and risk of cross-species transmission. Viruses have evolved multiple strategies to exploit host factors for entry into and survival in host cells. Drebrin is an actin-binding protein that restricts rotavirus entry by inhibiting dynamin-mediated endocytosis. However, its role and mechanism in DNA virus infection, particularly in herpesviruses, remain unexplored. In this study, we investigated the role of Drebrin in PRV infection using pharmacological inhibition (BTP−2) and CRISPR-Cas9-mediated gene knockout. Both the Drebrin inhibitor BTP−2 and gene knockout significantly suppressed PRV replication. Intriguingly, Drebrin exhibited stage-specific effects on the viral life cycle: its inhibition enhanced viral internalization during early infection but impaired viral replication at later stages, suggesting that Drebrin plays a complex role in the regulation of PRV infection. PRV infection partially disrupted actin stress fibers and caused an increase in cell size. Drebrin knockout also altered the host-cell morphology, reduced the cell surface area, and induced actin cytoskeleton rearrangement, which was further modulated in PRV-infected cells. In summary, our data demonstrate that Drebrin functions as a critical host factor governing the entire PRV life cycle by regulating actin cytoskeleton reorganization. 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

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

Pseudorabies virus (PRV) can infect a wide range of animal species, including swine and rodents. Infection in pigs is associated with significant economic losses in the global pork industry and is characterized by acute, often fatal disease, as well as central nervous system (CNS) invasion, which leads to neurological manifestations. Although PRV replication has been extensively characterized in certain murine neuronal cell lines such as Neuro-2a, the mechanisms underlying PRV-induced neuroinflammatory injury and necroptosis remain largely unclear. In this study, Kunming mice and mouse astrocytes (C8-D1A) were infected with PRV-GXLB-2013 at different doses to evaluate neurological injury and inflammatory responses. Given that the NF-κB/MLKL signaling pathway was found to be activated during PRV infection, a selective MLKL inhibitor, necrosulfonamide (NSA), was applied to investigate the role of necroptosis in PRV-induced neuroinflammatory damage. Mice infected with higher viral doses showed increased mortality, severe neurological symptoms, elevated brain inflammation, and pathological changes. In C8-D1A cells, PRV infection significantly upregulated inflammatory cytokines and key components of the NF-κB/MLKL pathway. Importantly, NSA treatment markedly reduced these inflammatory responses, mitochondrial damage, and cellular necrosis. Collectively, these findings suggest that PRV infection triggers neuroinflammatory injury through the activation of necroptosis and the NF-κB/MLKL signaling pathway. This study provides novel mechanistic insights into PRV-induced neurological damage and highlights potential therapeutic targets for intervention. Full article

The immediate-early protein IE180 of pseudorabies virus (PRV) is a multifunctional regulator of viral and host gene expression. However, its role in modulating antiviral immune responses remains poorly understood. Here, we demonstrate that IE180 overexpression significantly inhibits PRV and H1N1 influenza virus replication in Hep2 and A549 cells, respectively. Mechanistically, IE180 activates the type I interferon (IFN-I) pathway by enhancing IFN-β promoter activity and IFN transcription, leading to upregulated expression of interferon-stimulated genes (ISGs). Notably, IE180 failed to suppress PRV or H1N1 replication in Vero cells, which lack functional IFN-I signaling, confirming the dependence of its antiviral function on the IFN-I pathway. Domain mapping revealed that the ICP4-Like2 domain of IE180 is critical for IFN-β activation and antiviral activity. These findings establish IE180 as a novel viral immunomodulator that activates host innate immunity to restrict viral replication, providing insights into PRV-host interactions and potential therapeutic strategies. Full article

Pseudorabies virus (PRV) is a highly contagious pathogen in swine that can cross species barriers and infect other mammals, including humans. Given the potential for interspecies transmission and its threat to public health, understanding the molecular biology of PRV strains is essential for developing effective control measures and preparing for future pandemics. In this study, a novel PRV strain, PRV-HL-2021, was isolated from an outbreak in Heilongjiang Province, China. The viral genome was used to establish a reverse genetics system based on a fosmid library of the PRV-HL-2021 genome. This system facilitated the creation of recombinant PRV, including one expressing EGFP and another with deletions in the US9, gI, and gE genes. PRV-HL-2021 was found to be highly lethal to mice in vivo. The recombinant PRV strains, such as rPRV-US9-EGFP and rPRV-delgI/gE/US9, exhibited growth characteristics similar to the parental PRV-HL-2021 strain. The isolation and characterization of PRV-HL-2021 contribute to a better understanding of the genetic diversity of PRV strains. The developed reverse genetics system provides valuable tools for investigating viral functions, creating genetically modified PRV strains, and advancing the development of safer vaccines. These findings will enhance strategies for controlling PRV outbreaks and mitigating its impact on both animal and public health. 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

Pseudorabies virus (PRV) is a pathogen that causes severe neurological dysfunction in the host, leading to extensive neuronal damage and inflammation. Despite extensive research on the neuropathogenesis of α-herpesvirus infections, many scientific questions remain unresolved, such as the largely unknown functions of long non-coding RNAs (lncRNAs) in herpesvirus-infected nervous systems. To address these questions, we used RNA sequencing (RNA-seq) to investigate the expression profiles of lncRNAs and mRNAs in the brains of mice infected with PRV. Through bioinformatic analysis, we identified 316 differentially expressed lncRNAs and 886 differentially expressed mRNAs. We predicted the biological functions of these differentially expressed lncRNAs and mRNAs using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and the results showed that the differentially expressed transcripts were mainly involved in the innate immune response. Finally, we validated the differential expression trends of lncRNAs and mRNAs using quantitative real-time PCR (q-PCR), which were consistent with the sequencing data. To our knowledge, this is the first report analyzing the lncRNA expression profile in the central nervous system (CNS) of mice infected with PRV. Our findings provide new insights into the roles of lncRNAs and mRNAs during PRV infection of the host CNS. Full article

G-quadruplex (G4) structures have emerged as critical regulatory elements in viral genomes and represent potential targets for antiviral intervention. In this study, we identified and characterized G4 structures in the unique long (UL) region of the Pseudorabies virus (PRV) genome, highlighting their role as novel antiviral targets. Bioinformatic analysis revealed two guanine-rich regions (R1 and R2) that form stable G4 structures, as confirmed by fluorescence assays, circular dichroism (CD) spectroscopy, and immunofluorescence staining. Notably, these G4 structures exhibit a tandem repeat arrangement, a previously unreported feature in the PRV genome. Epiberberine (EPI), a natural G4-stabilizing ligand, bound to and stabilized these structures, leading to the inhibition of Taq polymerase progression. Functional assays demonstrated that EPI effectively suppressed PRV replication in vitro while having no significant impact on viral entry or release. In vivo, EPI treatment significantly improved survival rates and reduced viral loads in multiple organs, including the brain, heart, lungs, and kidneys of infected mice. These findings provide new insights into the role of G4 structures in PRV replication and demonstrate that EPI exhibits potential antiviral activity by targeting G4 structures. Full article

Pseudorabies virus (PRV) remains a critical threat for the global swine industry, with heightened attention due to the emergence of variant strains since late 2011 in China. Emergent viral variants generally undergo three to four years of adaptation to present new phenotypes. However, limited investigations have been performed on the evolution and pathogenicity of variant PRV strains in growing pigs after 2015. In this study, three PRV field strains, named SD1501, SD1701, and SD1801, were isolated and their genetic characteristics and pathogenicity on 9-week-old pigs were analyzed. Nucleotide identity and phylogenetic analyses based on the complete genome sequence, as well as major immunogenic and virulence-related genes revealed that all three isolates clustered closely with genotype II variant strains prevalent in China. The pathogenicity analysis demonstrated that the three isolates exhibited moderate pathogenicity in growing pigs with a TCID₅₀ of 10⁷. Infected pigs displayed transient fever and reduced appetite, with only one pig in each challenge group showing typical neurological symptoms and succumbing within 6 days post infection. These findings enrich the epidemiological data of PRV and provide direct evidence for the phenotypic variations caused by PRV infection, which enhances our understanding of PRV evolution in China and contributes to PRV control in the field. Full article