Veterinary Sciences

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18 pages, 4693 KB

Open AccessArticle

Mn²⁺-Mediated Antiviral Activity Through Both the cGAS-STING-IFN and ROS-Apoptosis Pathways in Porcine Alveolar Macrophage Cells

by Wanglong Zheng, Yajing Chang, Anjing Liu, Chenyang Zhang, Weilin Hao, Tianna Chen, Qing Lu, Zhiyu Wang, Wei Wang, Nanhua Chen and Jianzhong Zhu

Vet. Sci. 2026, 13(4), 396; https://doi.org/10.3390/vetsci13040396 - 17 Apr 2026

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Abstract

Manganese ions (Mn²⁺) are an essential trace element within organisms spanning the entire tree of life. It has reported that Mn²⁺ exerts strong immunocompetence effects and exhibits antiviral effects against various human and animal viruses, including DNA and RNA viruses. [...] Read more.

Manganese ions (Mn²⁺) are an essential trace element within organisms spanning the entire tree of life. It has reported that Mn²⁺ exerts strong immunocompetence effects and exhibits antiviral effects against various human and animal viruses, including DNA and RNA viruses. Recently, Mn²⁺ has been found to be involved in the activation of the innate immune DNA-sensing cyclic GMP-AMP synthase (cGAS) stimulator of interferon genes (STING) pathway and subsequent antiviral function. However, the antiviral mechanism of Mn²⁺ remains unclear. In the current study, the results suggest that the cGAS-STING pathway is essential for Mn²⁺ to promote interferon (IFN) signaling, but it is not essential for triggering antiviral functions. After knocking out the STING or interferon regulatory factor 3 (IRF3) gene, Mn²⁺ still retains its antiviral activity against herpes simplex virus type 1 (HSV-1) and vesicular stomatitis virus (VSV). Furthermore, the results from transcriptomic analysis indicate that Mn²⁺ can induce a significant change in the apoptotic process in STING⁻/⁻ 3D4/21 cells. Mn²⁺ can induce cell apoptosis through the oxidative stress pathway, and inhibiting the apoptotic signal could suppress Mn²⁺-mediated antiviral activity in STING⁻/⁻ 3D4/21 cells. Additionally, dual knockout of IRF3 and caspase3, resulting in concurrent loss of IFN and apoptotic signals, eliminates the antiviral effects of Mn²⁺. In summary, the current study suggests that Mn²⁺ could exert antiviral effects not only through the cGAS-STING-IFN pathway but also via the reactive oxygen species (ROS)-apoptosis pathway. Full article

(This article belongs to the Special Issue Diagnosis, Epidemiology, and Vaccine Development of Swine Viral Diseases—2nd Edition)

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16 pages, 8737 KB

Open AccessArticle

Preparation and Epitope Identification of Monoclonal Antibodies Against African Swine Fever Virus pE120R

by Juan Li, Miaomiao Ye, Peng Gao, Yajin Qu, Quanlin Li, Qiongqiong Zhou, Yongning Zhang, Lei Zhou, Xinna Ge, Xin Guo, Jun Han and Hanchun Yang

Vet. Sci. 2026, 13(4), 358; https://doi.org/10.3390/vetsci13040358 - 7 Apr 2026

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Abstract

The capsid protein pE120R of African swine fever virus (ASFV) is highly immunogenic and is thought to play an important role in viral replication, yet its molecular characteristics and functions during infection remain poorly understood. Here, we generated two monoclonal antibodies (mAbs), 1C11 [...] Read more.

The capsid protein pE120R of African swine fever virus (ASFV) is highly immunogenic and is thought to play an important role in viral replication, yet its molecular characteristics and functions during infection remain poorly understood. Here, we generated two monoclonal antibodies (mAbs), 1C11 and 3G7, against ASFV pE120R and characterized their specificity and utility. Epitope mapping showed that 1C11 recognized the linear epitope ¹⁰⁹KKHLFP¹¹⁴, whereas 3G7 recognized ¹¹²LFPKL¹¹⁶. These antibodies enabled analysis of pE120R expression and localization during ASFV infection, demonstrating that pE120R is expressed at a late stage and partially co-localizes with the structural protein p54 in viral factories. Together, these results provide valuable immunological tools for further investigation of pE120R in ASFV replication and pathogenesis. Full article

(This article belongs to the Special Issue Diagnosis, Epidemiology, and Vaccine Development of Swine Viral Diseases—2nd Edition)

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11 pages, 1065 KB

Open AccessArticle

Integrated Analysis of the mTOR Signaling Pathway Mediated by the ORF3 Protein of Swine Hepatitis E Virus in HepG2 Cells via a circRNA–miRNA Network

by Jiya Li, Shengping Wu, Lingjie Wang, Xin Cao, Yulong Yin, Leli Wang and Hanwei Jiao

Vet. Sci. 2026, 13(4), 350; https://doi.org/10.3390/vetsci13040350 - 3 Apr 2026

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Abstract

Background: The ORF3 protein of swine hepatitis E virus (HEV-4) is a key virulence factor involved in viral assembly, egress, and host signaling regulation. The mammalian target of rapamycin (mTOR) pathway plays a pivotal role in autophagy, metabolism, and immunity, and is often [...] Read more.

Background: The ORF3 protein of swine hepatitis E virus (HEV-4) is a key virulence factor involved in viral assembly, egress, and host signaling regulation. The mammalian target of rapamycin (mTOR) pathway plays a pivotal role in autophagy, metabolism, and immunity, and is often modulated by viruses to promote replication. However, it remains unknown whether HEV-4 ORF3 modulates the mTOR pathway via circular RNAs (circRNAs). Methods: Using an adenovirus-mediated ORF3 overexpression system in HepG2 cells, we integrated circRNA and transcriptome high-throughput sequencing data, followed by KEGG enrichment analysis to identify mTOR-associated differentially expressed genes. A circRNA–miRNA regulatory network was constructed using bioinformatics tools, and the expression changes of m6A-related genes, including YTHDF3, were evaluated. Results: ORF3 overexpression significantly activated the mTOR pathway (p < 0.05) and led to the identification of 20 mTOR-related circRNAs (e.g., circRNA5142). These circRNAs regulated downstream autophagy and lipid metabolism genes by sponging miRNAs such as hsa-let-7d-5p and hsa-miR-132-3p. Altered YTHDF3 expression indicated possible m6A-dependent epitranscriptomic regulation of the mTOR pathway. Conclusions: Our integrated analysis suggests that HEV-4 ORF3 may modulate the mTOR pathway through a circRNA–miRNA network, perturbing host autophagy and metabolic balance, which may contribute to viral immune evasion. Targeting the ORF3-mediated circRNA-mTOR regulatory axis represents a promising therapeutic approach and provides a theoretical basis for novel anti-HEV-4 strategies. Full article

(This article belongs to the Special Issue Diagnosis, Epidemiology, and Vaccine Development of Swine Viral Diseases—2nd Edition)

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15 pages, 3099 KB

Open AccessArticle

Integrated Bioinformatics Analysis Reveals the Impact of SHEV ORF3-Related LncRNA Network on Bile Secretion Pathway (ko 04976) in HepG2 Cells

by Hanwei Jiao, Jiya Li, Shengping Wu, Lingjie Wang, Yu Zhao, Yulong Yin, Xin Cao and Leli Wang

Vet. Sci. 2026, 13(3), 276; https://doi.org/10.3390/vetsci13030276 - 16 Mar 2026

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Abstract

(1) Background: Swine hepatitis E (SHE) is an emerging zoonotic disease caused by the swine hepatitis E virus (SHEV). The open reading frame 3 (ORF3) protein is a recognized virulence factor of SHEV. Jaundice, the typical clinical sign of SHE, primarily results from [...] Read more.

(1) Background: Swine hepatitis E (SHE) is an emerging zoonotic disease caused by the swine hepatitis E virus (SHEV). The open reading frame 3 (ORF3) protein is a recognized virulence factor of SHEV. Jaundice, the typical clinical sign of SHE, primarily results from disruptions in bile production, secretion, and excretion. However, the mechanism by which SHEV ORF3 influences bile metabolism remains unclear. (2) Methods: Building on our previous work involving adenovirus-mediated overexpression of genotype IV SHEV ORF3 in HepG2 cells and subsequent high-throughput lncRNA/transcriptome sequencing, this study performed KEGG enrichment analysis on differentially expressed lncRNAs. Candidate lncRNAs were validated via qRT-PCR. Cis-regulated target genes were predicted by integrating differentially expressed mRNA data. Furthermore, AlphaFold 3.0 was employed to analyze the molecular binding sites between lncRNA UBC (MSTRG.6881.4) and its target, UBC protein. (3) Results: We identified three lncRNAs associated with the bile secretion pathway (ko 04976) in HepG2 cells expressing genotype IV SHEV ORF3, which were further confirmed by qRT-PCR: lncRNA UBC (MSTRG.6881.4), lncRNA UBC (MSTRG.6881.9), and lncRNA UBC (MSTRG.6881.12). Bioinformatics prediction suggested six lncRNA-mRNA regulatory networks involved these lncRNAs and two downregulated UBC mRNA transcripts (ENST00000540700 and ENST00000536769). Molecular docking indicated that nucleotides 395U and 41C of lncRNA UBC (MSTRG.6881.4) could potentially bind to residues 82Lys, 88Thr, and 90Thr of the UBC protein, with predicted binding energies ranging from −4.73 to −0.75 kcal/mol. (4) Conclusions: The successful identification of bile secretion-related lncRNAs, coupled with the prediction of their regulatory networks and molecular interaction sites, has advanced our understanding of SHEV ORF3 function and the pathogenesis of SHEV infection. Full article

(This article belongs to the Special Issue Diagnosis, Epidemiology, and Vaccine Development of Swine Viral Diseases—2nd Edition)

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17 pages, 940 KB

Open AccessArticle

Integrated Transcriptomics Reveals a SHEV ORF3-Mediated circRNA Network That Disrupts Riboflavin Metabolism and Activates the ko05212 Pathway

by Weihao Luo, Jiya Li, Shengping Wu, Lingjie Wang, Yulong Yin, Xin Cao, Leli Wang and Hanwei Jiao

Vet. Sci. 2026, 13(3), 253; https://doi.org/10.3390/vetsci13030253 - 9 Mar 2026

Cited by 1 | Viewed by 391

Abstract

The Swine hepatitis E virus (SHEV) ORF3 protein is pivotal in pathogenesis, yet its regulation of host metabolic homeostasis via endogenous RNA networks remains unclear. This study aimed to elucidate how the SHEV ORF3-mediated circRNA-miRNA network modulates riboflavin metabolism and triggers the aberrant [...] Read more.

The Swine hepatitis E virus (SHEV) ORF3 protein is pivotal in pathogenesis, yet its regulation of host metabolic homeostasis via endogenous RNA networks remains unclear. This study aimed to elucidate how the SHEV ORF3-mediated circRNA-miRNA network modulates riboflavin metabolism and triggers the aberrant activation of the ko05212 pathway, while also evaluating their physical interactions using AlphaFold 3 structural simulations. To achieve this, high-throughput RNA sequencing, KEGG pathway analysis, and AlphaFold 3 structural simulations were employed to elucidate the circRNA-miRNA-mRNA regulatory network and potential physical interactions. Transcriptomics revealed a “dual activation” of Riboflavin metabolism and Pancreatic cancer pathways. Specifically, we identified an “ENPP Isozyme Switch,” where upregulated hsa_circ_0077855 sponges miR-181a-2-3p, relieving repression of the metabolic enzyme ENPP3 and proto-oncogene KRAS. Furthermore, AlphaFold 3 simulations yielded an extremely low interface predicted Template Modeling score (ipTM = 0.08), refuting direct physical binding, and ORF3 was found to suppress the m6A eraser FTO, suggesting host epigenetic instability. Consequently, SHEV ORF3 induces metabolic remodeling through a dual “epigenetic-post-transcriptional” mechanism: disrupting m6A homeostasis via FTO suppression and constructing a pathogenic ceRNA network via the ENPP3/miR-181a/KRAS axis. These findings highlight the critical role of non-coding RNAs in driving the virus-induced “pre-pathological state”. Full article

(This article belongs to the Special Issue Diagnosis, Epidemiology, and Vaccine Development of Swine Viral Diseases—2nd Edition)

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18 pages, 10247 KB

Open AccessArticle

African Swine Fever Virus R238L and R298L Disrupt Lung Cell Collagen Formation and Cell Adhesion Pathway by Targeting Transcription Factors Containing zf-C2H2 Domain

by Siqi Niu, Fanghong Zhang, Jingchun Wen, Yiyun Wang, Alegria Agostinho Francisco, Beneque Alberto Anzol, Min Yao, Guoping Liu, Jianwu Wang and Tinghua Huang

Vet. Sci. 2026, 13(3), 236; https://doi.org/10.3390/vetsci13030236 - 28 Feb 2026

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Abstract

The regulatory mechanisms of collagen formation and cell adhesion pathways during African Swine Fever Virus (ASFV) infection remain poorly understood. This study aims to investigate whether ASFV manipulates these pathways by targeting host transcriptional regulators. Through weighted Kendall correlation analysis of transcription factor [...] Read more.

The regulatory mechanisms of collagen formation and cell adhesion pathways during African Swine Fever Virus (ASFV) infection remain poorly understood. This study aims to investigate whether ASFV manipulates these pathways by targeting host transcriptional regulators. Through weighted Kendall correlation analysis of transcription factor binding sites (TFBSs) in differentially expressed genes (DEGs) from the lung tissue of ASFV-recovered pigs, we identified SP2 and KLF6 as key transcription factors (TFs) associated with collagen synthesis and cell adhesion, respectively. Domain–domain interaction prediction, followed by Förster resonance energy transfer (FRET) assays, confirmed that the ASFV proteins R238L and R298L directly bind to the zf-C2H2 domains of SP2 and KLF6. Furthermore, overexpression of R238L and R298L in HeLa and 3D4/21 cells significantly downregulated SP2 and KLF6 target genes involved in these pathways. Our findings reveal a novel mechanism by which ASFV proteins R238L and R298L interfere with host transcription factors SP2 and KLF6, potentially disrupting collagen matrix integrity and cell adhesion to facilitate viral pathogenesis. Full article

(This article belongs to the Special Issue Diagnosis, Epidemiology, and Vaccine Development of Swine Viral Diseases—2nd Edition)

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16 pages, 1946 KB

Open AccessArticle

Indirect ELISA Using Multi-Antigenic Dominants of VP1, VP2, and VP3 Recombinant Protein to Detect Antibodies Against Senecavirus A in Pigs

by Zenglin Wang, Dexin Li, Yufang Li, Yunjing Zhang, Junhua Deng, Liying Hao, Kegong Tian and Xiangdong Li

Vet. Sci. 2026, 13(1), 90; https://doi.org/10.3390/vetsci13010090 - 15 Jan 2026

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Abstract

Senecavirus A (SVA) is an emerging pathogen that poses a significant threat to the global swine industry. With the advent of SVA vaccines, there is a growing need to develop serological diagnostic methods for evaluating vaccine-induced immunity. This study successfully established an indirect [...] Read more.

Senecavirus A (SVA) is an emerging pathogen that poses a significant threat to the global swine industry. With the advent of SVA vaccines, there is a growing need to develop serological diagnostic methods for evaluating vaccine-induced immunity. This study successfully established an indirect enzyme-linked immunosorbent assay (iELISA) through heterologous expression of a novel VP2-VP3-VP1 tandem recombinant protein in Escherichia coli (E. coli), which was constructed by integrating B-cell epitopes from VP1, VP2, and VP3. Comparative analysis using indirect ELISA revealed that the tandem recombinant VP2-VP3-VP1 protein and VP2 exhibited superior immunoreactivity. Consequently, the iELISAs for the tandem protein and VP2 were selected for further validation. Following optimization, the cut-off for the rVP2-VP3-VP1 iELISA was set at a sample-to-positive (S/P) ratio ≥ 0.60, while that for the rVP2 iELISA was set at ≥0.53. Analysis of kinetic sera from inactivated vaccine-immunized pigs showed that the rVP2-VP3-VP1 iELISA detected seroconversion synchronously with neutralizing antibodies, earlier than anti-VP2 antibodies. Finally, a serological survey for SVA was conducted in parts of mainland China from 2023 to 2024, with the rVP2-VP3-VP1 iELISA revealing an overall seroprevalence of 20.8%. These results indicate that the established detection method can be effectively used to evaluate SVA immunity and for epidemic surveillance. Full article

(This article belongs to the Special Issue Diagnosis, Epidemiology, and Vaccine Development of Swine Viral Diseases—2nd Edition)

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13 pages, 6834 KB

Open AccessArticle

ColorDetect RT-LAMP Assay for the Rapid, Sensitive, and Specific Detection of Porcine Abortion-Associated Pestivirus (PAAPeV)

by Xu Yang, Ying Li, Wenqi Yin, Wenjie Tang, Hui Diao, Mengjia Zhou, Hao Yang, Wenyi Fu, Lu Yong, Xu Luo, Guo Liao and Yuancheng Zhou

Vet. Sci. 2026, 13(1), 74; https://doi.org/10.3390/vetsci13010074 - 12 Jan 2026

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Abstract

The primary objective of this study was to develop and optimize a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for porcine abortion-associated pestivirus (PAAPeV)—an emerging pathogen that causes severe reproductive disorders in swine, for which no effective treatments or vaccines are currently [...] Read more.

The primary objective of this study was to develop and optimize a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for porcine abortion-associated pestivirus (PAAPeV)—an emerging pathogen that causes severe reproductive disorders in swine, for which no effective treatments or vaccines are currently available. In this study, four sets of LAMP primers were designed and screened for the colorimetric RT-LAMP assay, targeting the highly conserved 5′ untranslated region (5′UTR) of PAAPeV. Three reaction parameters, including reaction temperature, reaction duration, and inner-to-outer primer ratio, were then optimized based on cycle threshold (Ct) values, fluorescence intensity, and color changes of the endpoint products. Subsequently, the specificity and sensitivity of the optimized colordetect RT-LAMP assay were systematically validated, and its diagnostic performance was compared with that of the gold-standard reverse transcription quantitative polymerase chain reaction (RT-qPCR). The results demonstrated that the optimized assay achieved a detection limit of 2 copies/μL under the conditions of 65 °C incubation for 25 min and an inner-to-outer primer ratio of 8:1, with results amenable to naked-eye interpretation. Furthermore, this assay exhibited high specificity, showing no cross-reactivity with other known pestiviruses or prevalent swine pathogens. Clinical sample testing results showed 100% concordance between colordetect RT-LAMP and RT-qPCR. Collectively, this colordetect RT-LAMP assay represents a rapid, sensitive, and specific tool for PAAPeV RNA detection in both clinical laboratories and field settings. Full article

(This article belongs to the Special Issue Diagnosis, Epidemiology, and Vaccine Development of Swine Viral Diseases—2nd Edition)

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