Plant-derived natural compounds hold significant potential in antiviral drug development, characterized by their diverse and broad-spectrum against multiple viral pathogens. Current evidence indicates that these compounds not only interfere with critical stages of the viral life cycle, but also modulate host immune responses. Antiviral potential of narciclasine against several RNA viruses has been documented, but its inhibitory activity against pseudorabies virus (PRV) remains unexplored. In this study, we systematically evaluated the antiviral activity of narciclasine in vitro and in vivo. The results demonstrated that narciclasine significantly inhibited PRV proliferation in vitro and in vivo. Meanwhile, we found that narciclasine interfered with critical stages of the PRV life cycle, such as viral replication and release. In addition, narciclasine significantly improved the survival rate of PRV-infected mice and reduced viral loads in vivo. Furthermore, narciclasine also inhibited the replication of porcine reproductive and respiratory syndrome virus (PRRSV), vesicular stomatitis virus (VSV) and porcine epidemic diarrhea virus (PEDV) in vitro, which preliminary observations suggesting that narciclasine may have a potential broad-spectrum antiviral activity. This study provided evidences for the development of narciclasine as antiviral therapeutics.

Viral infection triggers a robust DNA damage response (DDR), reshaping the host chromatin landscape to facilitate viral replication. Here, we uncover a novel mechanism by which alphaherpesviruses exploit the DDR pathway. We demonstrated that herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) induced selective degradation of class I histone deacetylases (HDAC1/2), leading to histone hyperacetylation and subsequent DDR activation. Strikingly, viral infection promoted nuclear export of HDAC1/2, followed by MDM2-mediated K63-linked polyubiquitination and proteasomal degradation in the cytoplasm. Pharmacological inhibition of either DDR signaling or HDAC1/2 nuclear export significantly affected viral replication in vitro and in vivo . Our findings reveal a unique viral strategy to hijack host epigenetic regulation for efficient replication and identify potential therapeutic targets for alphaherpesvirus infections.

Feline calicivirus (FCV) is a highly heterogeneous pathogen and a major cause of feline upper respiratory tract disease, highlighting the need for continuous surveillance of its genetic diversity and pathogenic characteristics. In this study, we isolated and comprehensively characterized a newly isolated FCV strain, HN/ZZ/2025, from cats at a feline trading market in Zhengzhou, China, and systematically evaluated its molecular features, in vitro replication characteristics, physicochemical properties, and pathogenicity in cats. Phylogenetic analysis classified HN/ZZ/2025 as genotype GI. The isolate replicated efficiently in CRFK, F81, and Fc3Tg cells, reaching peak titers of 107.18, 107.50, and 105.89 TCID50/0.1 mL, respectively, and exhibited typical calicivirus-like particles with diameters of 35–40 nm. Complete genome analysis showed that HN/ZZ/2025 shared the highest nucleotide and amino acid sequence identities of 85.7% and 62.8%, respectively, with the closely related Chinese FCV strain CH-JL4, and revealed distinct amino acid variations within the hypervariable E region of the VP1 capsid protein. Experimental infection of cats (n = 3/group) resulted in pyrexia, with rectal temperatures reaching 39.8–40.2 °C, weight loss, oral ulceration, and persistent viral RNA shedding from 1 to 21 days post-infection. Viral RNA and VP1 antigen were detected in the lungs, trachea, kidneys, and spleen, indicating systemic dissemination and broad tissue distribution. Although no mortality occurred during the observation period, the observed clinical signs, viral dissemination, and histopathological lesions indicate that HN/ZZ/2025 is capable of causing clinically evident disease in experimentally infected cats. These findings provide useful insights into the molecular epidemiology, tissue tropism, and host–virus interactions of circulating FCV strains and establish HN/ZZ/2025 as a useful isolate for future studies of FCV genetic diversity and pathogenesis.

Jumonji domain-containing protein 6 (JMJD6) has been implicated in epigenetic regulation. Here, we demonstrated that JMJD6 was upregulated during pseudorabies virus (PRV) infection and critically enhanced viral replication by promoting virion release. Mechanistically, JMJD6 suppressed PRV-induced histone H4K16 acetylation, a modification associated with chromatin relaxation and DNA damage response activation. This epigenetic modulation attenuated the cGAS-STING-mediated innate immune signaling pathway, leading to reduced interferon production and enhanced viral propagation. Furthermore, we identified METTL23 as a nuclear interactor of JMJD6 upon viral infection, revealing a cooperative role between these proteins in facilitating immune evasion. Importantly, administration of the JMJD6-specific inhibitor JMJD6-IN-1 potently activated innate immunity and restricted PRV replication in mice. Our findings unveil a novel epigenetic strategy employed by PRV to evade host antiviral responses and highlight JMJD6 as a potential therapeutic target for combating herpesvirus infections.IMPORTANCEThe ongoing conflict between viruses and host antiviral defenses is central to viral pathogenesis. Pseudorabies virus (PRV), a highly contagious alphaherpesvirus, causes severe economic losses in the global swine industry and poses an emerging zoonotic threat to humans. This study identifies the epigenetic modulator, the JMJD6, as a critical host factor exploited by PRV to evade antiviral immunity. Our work uncovers a previously unrecognized epigenetic strategy employed by herpesviruses and establishes JMJD6 as a promising target for developing broad-spectrum antivirals against PRV and related pathogenic herpesviruses.

Porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped single-stranded positive-sense RNA virus, poses a significant threat to global swine production. Despite the availability of modified live virus and inactivated vaccines, their limited efficacy and safety concerns highlight the urgent need for novel antiviral therapeutics. This study aimed to investigate the molecular mechanisms by which lycopene inhibits PRRSV replication. Initial assessments confirmed that lycopene did not adversely affect cellular viability, cell cycle progression, or apoptosis. Using fluorescence microscopy, flow cytometry, immunoblotting, quantitative real-time PCR (qRT-PCR), and viral titration assays, lycopene was shown to exhibit potent antiviral activity against PRRSV. Mechanistic studies revealed that lycopene suppresses reactive oxygen species (ROS) production, which is critical for PRRSV proliferation. Additionally, lycopene attenuated PRRSV-induced inflammatory responses, as demonstrated by immunoblotting, ELISA, and qRT-PCR assays. These findings suggest that lycopene inhibits PRRSV replication by modulating ROS levels and mitigating inflammation, offering a promising avenue for the development of antiviral therapeutics. This study provides new insights and strategies for combating PRRSV infections, emphasizing the potential of lycopene as a safe and effective antiviral agent.

This study investigated a suspected Feline calicivirus (FCV) outbreak at a veterinary facility in Zhengzhou, Henan Province, China. RT-PCR analysis confirmed the FCV presence, with subsequent CRFK cell culture propagation leading to the isolation and characterization of strain ZZ202306. Immunofluorescence and Western blot analyses validated the specificity of monoclonal antibodies targeting the FCV VP1 capsid protein. Transmission electron microscopy revealed non-enveloped virions of ~40 nm in diameter, exhibiting typical caliciviral architecture. Viral replication kinetics demonstrated exponential growth between 6 and 18 h post-inoculation, reaching a peak titer of 107.96 TCID50/0.1 mL. Genomic sequencing coupled with phylogenetic reconstruction of the VP1 gene revealed a close genetic relation to domestic Chinese strains and international variants, while maintaining distinct evolutionary divergence from other calicivirus genera.

Feline infectious peritonitis (FIP), a fatal disorder driven by the feline infectious peritonitis virus (FIPV), has no clinically approved vaccine available to date-rendering prevention and early-stage diagnosis critical to its control. In this study, a feline infection model was developed via intraperitoneal inoculation of virus-laden ascitic fluid, with infection validation conducted using clinical observation, hematological testing, biochemical profiling, imaging examinations, histopathological analysis, and immunofluorescence assay. The viral nucleocapsid N gene underwent cloning and heterologous expression in Escherichia coli, followed by purification of the recombinant protein for subsequent antibody generation. With this purified recombinant protein serving as the immunogen, three monoclonal antibodies were successfully generated and comprehensively characterized; concurrently, three highly conserved linear B-cell epitopes were pinpointed and subjected to structural characterization. Subsequently, an indirect enzyme-linked immunosorbent assay targeting the nucleocapsid N protein was developed and optimized. This assay exhibited high sensitivity, specificity, and reproducibility, demonstrating 98 % concordance with immunofluorescence assay outcomes when tested on 50 clinical feline specimens. In summary, this study presents the development of a reliable FIP infection model, yields novel monoclonal antibodies, and delivers a robust serological assay for feline coronavirus detection. These findings provide valuable tools to inform future diagnostic protocols and control strategies for FIP.

Bovine enterovirus (BEV), a non-enveloped and icosahedral virus with the positive-stranded RNA genome, belongs to the Enterovirus genus, Picornaviridae family. The structural protein VP1 of BEV plays a pivotal role in viral genotyping and immunogenicity. Its immunodominant epitopes not only serve as primary sites triggering antibody production, but also constitutes crucial targets for vaccine development. This study presents the precise mapping of linear B-cell epitopes on the recombinant VP1 (rVP1) protein of bovine enterovirus (BEV). In this study, we expressed and purified BEV rVP1 protein and validated its immunogenicity in serum from mice immunized with inactivated whole-virus. Through hybridoma technology, the mAb 14D9 specifically targeting the BEV Sichuan/SQ/20 strain was successfully developed. Specific binding of mAb 14D9 to Sichuan/SQ/20 strain was confirmed by Western blot and immunofluorescence. The minimal epitope 16KDTVESHHSIST27 recognized by mAb 14D9 was determined by serial terminal truncation of the rVP1 protein. By combining bioinformatics prediction with experimental validation, we confirmed that the N-terminal 16-27aa region of VP1 constitutes an immunodominant epitope. The preparation of mAb 14D9 and identification of its corresponding linear epitope not only provides crucial insights for investigating the antigenic structure and biological functions of the BEV VP1 protein, but also helps to develop the serological diagnostic methods and subunit vaccines targeting BEV.

Glucosylceramide synthase (UGCG) is a key enzyme that catalyzes the initial glycosylation step in the biosynthesis of glycosphingolipids (GSLs) derived from glucosylceramide. UGCG is closely associated with various cellular processes, including the cell cycle, angiogenesis, multidrug resistance, and pathogen invasion. In this study, a short hairpin RNA (shRNA) library designed to target key genes involved in the sphingolipid metabolic pathway was utilized to elucidate their roles in Pseudorabies Virus (PRV). Those findings confirm a significant association between sphingolipid metabolism and PRV infection. In addition, this study demonstrated that the knockdown UGCG expression or inhibition of its activity significantly suppresses PRV infection. This suppression is accompanied by reduced expression of autophagy-related proteins that are induced by PRV infection, blockade of autophagic flux, and significant activation of the STING signaling pathway induced by PRV infection. Through extensive investigation, this research revealed that inhibition of UGCG affects the expression of lysosome-associated proteins, alters the lysosomal pH, disrupts lysosomal homeostasis, and impedes autophagolysosomal degradation. Additionally, UGCG inhibition influences the conversion of light chain 3-II (LC3-II) and the formation of LC3-STING complexes, negatively regulates the autophagic degradation of STING, and ensures sustained activation of the PRV-induced STING signaling pathway, thereby achieving resistance against PRV infection. Finally, through in vivo evaluation, this study revealed that UGCG inhibitors, Eliglustat hemitartrate and Ibiglustat, hold promise as potential therapeutics for the treatment of PRV infection. In summary, this study preliminarily elucidates the impact of UGCG on PRV infection and its associated molecular mechanisms, suggesting UGCG could serve as a potential novel target for the prevention and treatment of viral diseases such as PRV.

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.

This study successfully isolated a novel bovine enterovirus strain from a bovine fecal sample, which was designated as Sichuan/SQ/20. The isolate showed typical enterovirus morphology under electron microscopy. Phylogenetic analysis showed that this strain exhibits the closest genetic relationship with the HeN-YR91 and JPN/TottoriU-31 strains, and all three belong to the BEV-F1 genosubtype. Subsequently, comprehensive investigations were conducted on the biological characteristics of this virus, both in vivo and in vitro. In vitro characterization revealed that viral replication commenced at 3 h post-infection (hpi) in Madin-Darby bovine kidney cells, reaching peak at 48 hpi with a virus titer of 1 × 108.73 TCID50/0.1 mL. Cytopathic effects initially appeared at 12 hpi. A 12-minute treatment at 55°C was sufficient to completely inactivate the virus. In vivo analysis revealed that significant pathological changes were specifically observed in the spleen, with no lesions observed in other organs. Immunofluorescence assay detected specific fluorescent signals in the liver, spleen, and small intestine, which were consistent with the PCR results. These findings provide a scientific foundation for vaccine design and antiviral drug screening, as well as for the development of effective prevention and control strategies.IMPORTANCEBovine enterovirus (BEV) is an important pathogen causing calf diarrhea and has been detected in the feces of calves with diarrhea, although its pathogenicity remains unclear. This study systematically established an isolation and identification protocol for BEV, characterized its physicochemical properties, and further investigated the pathogenicity and tissue tropism of the isolated strain in mice. These findings establish crucial baseline data for future vaccine development and therapeutic intervention strategies.

Porcine reproductive and respiratory syndrome virus (PRRSV), a highly contagious pathogen in swine, poses significant economic challenges to global pork production. This study elucidated the regulatory interplay between PRRSV infection and the pentose phosphate pathway (PPP), a critical metabolic axis for anabolism. Comparative metabolomic profiling of porcine alveolar macrophages (PAMs) pre- and post-PRRSV infection demonstrated marked upregulation of PPP activity, concomitant with elevated levels of nucleotide biosynthesis. This metabolic shift was driven by PRRSV-induced upregulation of glucose-6-phosphate dehydrogenase (G6PD), the PPP’s rate-limiting enzyme. Mechanistic investigations revealed that PRRSV infection stimulated hypoxia-inducible factor 1α (HIF-1α) expression, which transcriptionally activates G6PD. Genetic silencing of HIF-1α abolished PRRSV-mediated G6PD induction. Furthermore, reactive oxygen species (ROS) accumulation was identified as the upstream regulator of HIF-1α activation during PRRSV infection. Pharmacological ROS scavenging disrupted the ROS/HIF-1α/G6PD signaling cascade, diminished NADPH and reduced glutathione production, and consequently attenuated viral proliferation. These results established that PRRSV exploited the ROS/HIF-1α axis to reprogram host glucose metabolism through PPP potentiation, creating a biosynthetic environment conducive to viral propagation.

As obligate parasites, viruses exploit host cell organelles and molecular components to complete their life cycle. Among which, viruses firstly hijack the cytoskeleton of host cells to ensure their efficiently cell entry and replication. Although formin family members play a key role in both microfilament and microtubule cytoskeletal remodeling, few studies addressed the detailed function and mechanism of formins in the process of viral infection. Here, we showed that sus scrofa DIAPH1 was involved in the regulation of cytoskeletal dynamics during PRV replication. Firstly, we found that DIAPH1 showed significant changes in the expression level and intracellular localization during PRV infection of PK-15 cells. Next, inhibition of DIAPH1 by RNA interference or small molecular inhibitor SMIFH2 was found to diminish the outcome of PRV infection. Besides, DIAPH1 partially co-localized with actin and tubulin in PRV-infected cells. Cross-talk occurred between microfilaments and microfilaments, which also had an influence on the intracellular localization of DIAPH1. What's more, inhibition of DIAPH1 induced the reorganization of microfilament and the stability of microtubule. These results suggested that DIAPH1 regulated PRV infection by remodeling microfilament and microtubule cytoskeletal dynamics.

Viruses are dependent on the host factors for their replication and survival. Therefore, identification of host factors that druggable for antiviral development is crucial. The actin cytoskeleton plays an important role in the virus infection. The dynamics change of actin and its function are regulated by multiple actin-associated proteins (AAPs). However, the role and mechanism of various AAPs in the life cycle of virus are still enigmatic. In this study, we analyzed the roles of actin and AAPs in the replication of pseudorabies virus (PRV). Using a library of compounds targeting AAPs, our data found that multiple AAPs, such as Rho-GTPases, Rock, Myosin and Formin were involved in PRV infection. Besides, our result demonstrated that the actin-binding protein Drebrin was also participated in PRV infection. Further studies are necessary to elucidate the molecular mechanism of AAPs in the virus life cycle, in the hope of mining host factors for antiviral developments.

The vitamin D receptor (VDR) is a nuclear steroid receptor that regulates the expression of genes across various biological functions. However, the role of VDR in pseudorabies virus (PRV) infection has not yet been explored. We discovered that VDR positively influenced PRV proliferation because knockdown of VDR impaired PRV proliferation, whereas its overexpression promoted it. Additionally, we observed that PRV infection upregulated VDR transcription alongside 1,25-dihydroxyvitamin D3 (VD3) synthesis, contingent on p53 activation. Furthermore, VDR knockdown hindered PRV-induced lipid synthesis, implicating VDR's involvement in this process. To decipher the mechanism behind VDR's stimulation of lipid synthesis during PRV infection, we conducted RNA sequencing (RNA-seq) and found significant enrichment of genes in the Ca2+ signaling pathway. Measurements of Ca2+ indicated that VDR facilitated Ca2+ absorption. Moreover, the PI3K/AKT/mTORC1 and AMPK/mTORC1 pathways were also enriched in our RNA-seq data. Interfering with VDR expression, or chelating Ca2+ using BAPTA-AM, markedly impacted the activation of PI3K/AKT/mTORC1 and AMPK/mTORC1 pathways, lipid synthesis, and PRV proliferation. In summary, our study demonstrates that PRV infection promotes VDR expression, thereby enhancing Ca2+ absorption and activating PI3K/AKT/mTORC1- and AMPK/mTORC1-mediated lipid synthesis. Our findings offer new insights into strategies for PRV prevention.IMPORTANCEVitamin D, beyond its well-known benefits for bone health and immune function, also plays a pivotal role in regulating gene expression through its receptor, the vitamin D receptor (VDR). Although VDR's influence spans multiple biological processes, its relationship with viral infections, particularly pseudorabies virus (PRV), remains underexplored. Our research illustrates a complex interplay where PRV infection boosts VDR expression, which in turn enhances Ca2+ absorption, leading to the activation of critical lipid synthesis pathways, PI3K/AKT/mTORC1 and AMPK/mTORC1. These findings not only deepen our understanding of the intricate dynamics between host molecular mechanisms and viral proliferation but also open avenues for exploring new strategies aimed at preventing PRV infection. By targeting components of the VDR-related signaling pathways, we can potentially develop novel therapeutic interventions against PRV and possibly other similar viral infections.

ABSTRACT Pseudorabies virus (PRV) is the causative agent of Aujeszky’s disease in pigs. The low-density lipoprotein receptor (LDLR) is a transcriptional target of the sterol-regulatory element-binding proteins (SREBPs) and participates in the uptake of LDL-derived cholesterol. However, the involvement of LDLR in PRV infection has not been well characterized. We observed an increased expression level of LDLR mRNA in PRV-infected 3D4/21, PK-15, HeLa, RAW264.7, and L929 cells. The LDLR protein level was also upregulated by PRV infection in PK-15 cells and in murine lung and brain. The treatment of cells with the SREBP inhibitor, fatostatin, or with SREBP2-specific small interfering RNA prevented the PRV-induced upregulation of LDLR expression as well as viral protein expression and progeny virus production. This suggested that PRV activated SREBPs to induce LDLR expression. Furthermore, interference in LDLR expression affected PRV proliferation, while LDLR overexpression promoted it. This indicated that LDLR was involved in PRV infection. The study also demonstrated that LDLR participated in PRV invasions. The overexpression of LDLR or inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), which binds to LDLR and targets it for lysosomal degradation, significantly enhanced PRV attachment and entry. Mechanistically, LDLR interacted with PRV on the plasma membrane, and pretreatment of cells with LDLR antibodies was able to neutralize viral entry. An in vivo study indicated that the treatment of mice with the PCSK9 inhibitor SBC-115076 promoted PRV proliferation. The data from the study indicate that PRV hijacks LDLR for viral entry through the activation of SREBPs. IMPORTANCE Pseudorabies virus (PRV) is a herpesvirus that primarily manifests as fever, pruritus, and encephalomyelitis in various domestic and wild animals. Owing to its lifelong latent infection characteristics, PRV outbreaks have led to significant financial setbacks in the global pig industry. There is evidence that PRV variant strains can infect humans, thereby crossing the species barrier. Therefore, gaining deeper insights into PRV pathogenesis and developing updated strategies to contain its spread are critical. This study posits that the low-density lipoprotein receptor (LDLR) could be a co-receptor for PRV infection. Hence, strategies targeting LDLR may provide a promising avenue for the development of effective PRV vaccines and therapeutic interventions.

Identification of a conserved G-quadruplex in E165R of ASFVAfrican swine fever virus (ASFV) is a double-stranded DNA arbovirus with high transmissibility and mortality rates. It has caused immense economic losses to the global pig industry. Currently, no effective vaccines or medications are to combat ASFV infection. G-quadruplex (G4) structures have attracted increasing interest because of their regulatory role in vital biological processes. In this study, we identified a conserved G-rich sequence within the E165R gene of ASFV. Subsequently, using various methods, we verified that this sequence could fold into a parallel G4. In addition, the G4-stabilizers pyridostatin and 5,10,15,20-tetrakis-(N-methyl-4-pyridyl) porphin (TMPyP4) can bind and stabilize this G4 structure, thereby inhibiting E165R gene expression, and the inhibitory effect is associated with G4 formation. Moreover, the G4 ligand pyridostatin substantially impeded ASFV proliferation in Vero cells by reducing gene copy number and viral protein expression. These compelling findings suggest that G4 structures may represent a promising and novel antiviral target against ASFV.

The porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious pathogen in pigs. This study aimed to investigate the impact of PRRSV infection on cellular metabolism, particularly focusing on lipid metabolism to understand its role in promoting viral replication. We conducted a metabolic analysis on MARC-145 cells before and after PRRSV infection. Our results demonstrated that the most significant alterations in cellular metabolism, accounting for 40.8 % of total changes, were related to lipid metabolism. These changes were primarily driven by the activation of sterol regulatory-element binding proteins (SREBPs), critical regulators of lipid biosynthesis. To understand the mechanisms behind SREBPs activation by PRRSV, we investigated the involvement of upstream effectors, specifically protein kinase B (AKT) and phosphoenolpyruvate carboxykinase 1 (PCK1). Our findings indicated that PRRSV infection triggered AKT activation, leading to the subsequent activation of PCK1. Activated PCK1 then phosphorylated insulin-induced genes (INSIGs), resulting in their degradation. This degradation facilitated the translocation of SREBPs from the endoplasmic reticulum to the nucleus. Additionally, we observed that PRRSV infection stimulated the production of reactive oxygen species (ROS), which played a critical role in activating AKT. Collectively, our findings demonstrate that PRRSV enhances lipid synthesis through a ROS-dependent AKT/PCK1/INSIG/SREBPs signaling axis, which provides new insights into the metabolic strategies employed by PRRSV.

RAB GTPases (RABs) control intracellular membrane trafficking with high precision. In the present study, we carried out a short hairpin RNA (shRNA) screen focused on a library of 62 RABs during infection with porcine reproductive and respiratory syndrome virus 2 (PRRSV-2), a member of the family Arteriviridae. We found that 13 RABs negatively affect the yield of PRRSV-2 progeny virus, whereas 29 RABs have a positive impact on the yield of PRRSV-2 progeny virus. Further analysis revealed that PRRSV-2 infection transcriptionally regulated RAB18 through RIG-I/MAVS-mediated canonical NF-κB activation. Disrupting RAB18 expression led to the accumulation of lipid droplets (LDs), impaired LDs catabolism, and flawed viral replication and assembly. We also discovered that PRRSV-2 co-opts chaperone-mediated autophagy (CMA) for lipolysis via RAB18, as indicated by the enhanced associations between RAB18 and perlipin 2 (PLIN2), CMA-specific lysosomal associated membrane protein 2A (LAMP2A), and heat shock protein family A (Hsp70) member 8 (HSPA8/HSC70) during PRRSV-2 infection. Knockdown of HSPA8 and LAMP2A impacted on the yield of PRRSV-2 progeny virus, implying that the virus utilizes RAB18 to promote CMA-mediated lipolysis. Importantly, we determined that the C-terminal domain (CTD) of HSPA8 could bind to the switch II domain of RAB18, and the CTD of PLIN2 was capable of associating with HSPA8, suggesting that HSPA8 facilitates the interaction between RAB18 and PLIN2 in the CMA process. In summary, our findings elucidate how PRRSV-2 hijacks CMA-mediated lipid metabolism through innate immune activation to enhance the yield of progeny virus, offering novel insights for the development of anti-PRRSV-2 treatments.

Viral infection is a significant risk factor for fertility issues. Here, we demonstrated that infection by neurotropic alphaherpesviruses, such as pseudorabies virus (PRV), could impair female fertility by disrupting the hypothalamus-pituitary-ovary axis (HPOA), reducing progesterone (P4) levels, and consequently lowering pregnancy rates. Our study revealed that PRV exploited the transient receptor potential mucolipin 1 (TRPML1) and its lipid activator, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), to facilitate viral entry through lysosomal cholesterol and Ca2+. P4 antagonized this process by inducing lysosomal storage disorders and promoting the proteasomal degradation of TRPML1 via murine double minute 2 (MDM2)-mediated polyubiquitination. Overall, the study identifies a novel mechanism by which PRV hijacks the lysosomal pathway to evade P4-mediated antiviral defense and impair female fertility. This mechanism may be common among alphaherpesviruses and could contribute significantly to their impact on female reproductive health, providing new insights for the development of antiviral therapies.