Macrophages are frontline effectors of innate immunity, acting as essential elements for clearing pathogens. However, viruses have continuously evolved sophisticated mechanisms to subvert these critical defenses. This review comprehensively decodes how viral pathogens systematically dismantle macrophage microbicidal capacities. It delineates the evasion of pattern recognition receptor surveillance, the hijacking of endocytic trafficking, and the active arrest of phagolysosomal maturation to secure intracellular replication niches. Furthermore, the review explores the paradoxical viral manipulation of the host oxidative burst, where pathogens weaponize reactive oxygen species or exploit antioxidant machineries, driving severe redox dysregulation. Profound metabolic reprogramming, including shifts toward aerobic glycolysis and the skewing of inflammatory polarization alongside cell death pathways, is also examined. Ultimately, these evasion strategies inflict functional paralysis on macrophages, heavily predisposing hosts to severe secondary bacterial coinfections. Mapping this virus-host crosstalk highlights critical vulnerabilities, providing a foundation for novel host-directed antiviral immunotherapies.

Getah virus (GETV) is an emerging mosquito-borne zoonotic alphavirus that poses a threat to multiple animal species, particularly pigs, and carries significant public health implications. Nevertheless, the molecular evolution and pathogenicity of circulating GETV strains in China remain incompletely defined. In this study, three GETV strains were isolated from diseased piglets during concurrent outbreaks on three geographically distinct commercial pig farms in China in August 2025. The isolates shared 99.67%-99.91% genomic nucleotide (nt) identity and were phylogenetically classified into subgroup GⅢ-c. Amino acid sequence alignment against 136 reference strains from the GenBank database identified four substitutions in Nsp1 and Nsp3 that were absent from all reference strains, along with six conserved residues largely concentrated in a distinct subclade formed by the three isolates and five related reference strains. Pathogenicity evaluation using a representative isolate (GETV-YY/2025) in 7-day-old piglets revealed that the infected animals developed severe clinical signs, including fever, diarrhea, ataxia, and hindlimb paralysis, with a high mortality rate of 77.8% (7/9). The strain exhibited broad tissue tropism, with high viral loads detected in pharyngeal and rectal swabs, serum, and multiple visceral organs as early as 1 day postinoculation, accompanied by obvious cerebral vascular congestion and pulmonary hemorrhage. Notably, efficient contact transmission to sentinel piglets was observed, resulting in one fatality among the three contact animals (33.3%). Collectively, these findings characterize the emergence of a highly virulent GETV subgroup GⅢ-c circulating in Chinese pig populations, highlighting its capacity for efficient contact transmission and severe pathogenesis in neonatal pigs. This study provides critical insights into the evolutionary dynamics of GETV and informs the urgent development of prevention and control strategies against this re-emerging veterinary pathogen.

Pseudorabies virus (PRV), a member of the Alphaherpesvirinae subfamily, primarily infects pigs and poses a significant threat to the swine industry. In recent years, emerging PRV variants have been reported to infect humans, predominantly causing encephalitis. Ferroptosis is a recently identified form of programmed cell death, characterized by iron-dependent lipid peroxidation. It has been implicated in the pathogenesis of various diseases, including neurodegenerative disorders and viral infections. However, whether PRV induces ferroptosis in the central nervous system and the underlying mechanisms remain to be fully elucidated. This study demonstrates that PRV infection induces ferroptosis in N2a cells, mouse primary neurons, and murine brains. Specifically, PRV infection triggers ferritinophagy in N2a cells, mouse primary neurons, and brain, increasing intracellular free iron levels and subsequent lipid peroxidation, ultimately driving ferroptosis. Furthermore, PRV-induced ferroptosis is closely associated with neuroinflammation. Mechanistically, ferroptosis upregulates prostaglandin-endoperoxide synthase 2 (PTGS2), thereby enhancing prostaglandin E2 (PGE2) synthesis and exacerbating inflammatory responses. Integrated transcriptomic and metabolomic analyses further confirm that PRV-induced ferroptosis drives neuroinflammation through the PTGS2/PGE2 pathway. Notably, treatment with the ferroptosis inhibitor deferoxamine effectively mitigates PRV-induced ferroptosis, reduces viral titers in mouse brains, and alleviates viral encephalitis. In conclusion, our study reveals the critical role of ferroptosis in PRV-induced viral encephalitis, providing therapeutic insights for treating PRV-associated neurological diseases.

Porcine deltacoronavirus (PDCoV) is an enteropathogenic virus that causes severe diarrhea in pigs, particularly suckling piglets, and exhibits cross-species transmission with zoonotic potential. The S1 subunit of the viral spike (S) protein mediates cell entry and elicits neutralizing antibodies, making it an ideal target for diagnostics, prophylaxis, and therapeutics. Here, we produced two monoclonal antibodies (mAbs), B8F10 and G10C2, by immunizing BALB/c mice with a recombinant PDCoV S1 protein fused to a human IgG Fc fragment, expressed in an insect baculovirus system and purified using Protein A/G magnetic beads. Both mAbs can specifically recognize native PDCoV S protein in indirect immunofluorescence assays and western blot analyses under denaturing conditions, indicating their binding to linear epitopes. Isotyping classified both as IgG1/κ, and sequence analysis revealed distinct heavy-chain complementarity-determining regions (CDRs) but identical light-chain CDRs. Using truncated S1 proteins coupled with Pepscan ELISA, the B8F10 and G10C2 epitopes were precisely mapped to 342LETNFMCT349 and 491VINNTVVG498, respectively. These epitopes can be recognized by both mAbs and swine PDCoV antiserum, confirming their immunodominance. While global PDCoV strain alignments revealed high conservation of these epitopes, a V491A mutation within the G10C2-binding site abolished mAb binding. Structural analysis confirmed both epitopes are surface-exposed on S1. These findings demonstrate the diagnostic potential of these mAbs and the epitopes’ suitability as vaccine targets. Their high conservation suggests broad applicability, whereas the V491A mutation may represent an immune escape mechanism. These epitopes could serve as diagnostic markers, immunotherapeutics, or the foundation for epitope-based vaccines to induce protective immunity.

Recombination is a primary driver of porcine reproductive and respiratory syndrome virus (PRRSV) evolution. Currently, lineage 1 NADC30-like virus (L1C) and lineage 8 HP-PRRSV (L8E) recombinants dominate in China, whereas recombinants harboring the lineage five backbone are infrequently detected. To elucidate the mechanisms underlying this disparity, we investigated the interplay between recombination frequency, viral fitness, and strain-specific phenotypes using co-infection models. Next-generation sequencing (NGS) revealed that the lineage 5 replicase is prone to template switching, generating high frequencies of recombination junctions comparable to NADC30-like strains when viral RNA abundance is high. However, plaque purification demonstrated a disconnection between the generation of recombination events and the survival of viable progeny. While NADC30-like strains acted as a highly recombinogenic backbone, yielding up to 100% recombinant progeny, lineage 5-based recombinants were largely eliminated due to competitive exclusion. Furthermore, although a mutation known to reduce recombination frequency (K541R) successfully reduced errors at the nucleic acid level, it failed to prevent the emergence of recombinants under strong selective pressure. Our findings suggest that PRRSV recombination is not merely a stochastic event but a complex outcome dictated by strain-specific phenotypic profiles. The scarcity of lineage 5 recombinants is driven by a phenotypic deficit, which is a combination of replication efficiency and susceptibility to host clearance. This study highlights the dominant role of NADC30-like strains in driving viral diversity and underscores that vaccine safety depends on the holistic phenotypic fitness of the virus.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) recombination poses a significant threat to the farm as it accelerates viral evolution and leads to changes in pathogenicity and immune protection. Understanding why certain strains recombine frequently while others do not is critical for reducing this risk. This study unravels the complexity of PRRSV recombination, showing that it is determined by the specific phenotypic profile of the virus strains involved. We discovered that current epidemic strains (NADC30-like) act as highly recombinogenic backbones due to their biological traits. Surprisingly, classical vaccine strains (lineage 5) infrequently form viable recombinants not because they are genetically accurate, but because their distinct phenotypic weaknesses lead to their elimination by the host or competitors. This finding shifts the paradigm of vaccine safety, emphasizing that preventing recombination requires a holistic approach that considers the complex interplay of viral fitness and host adaptation, thereby providing critical guidance for the industry.

African swine fever virus (ASFV) employs sophisticated regulatory strategies to manipulate host cell apoptosis, a process critical for its pathogenesis and immune evasion; however, the mechanisms underlying this process remain incompletely understood. Here, we report a novel mechanism by which the ASFV-encoded envelope protein CD2v suppresses apoptosis by activating the TPL2 (tumor progression locus 2)-MEK (mitogen-activated protein kinase kinase)-ERK (extracellular signal-regulated kinase) signaling axis, leading to proteasomal degradation of the pro-apoptotic protein BimEL in primary porcine alveolar macrophages and wild boar lung (WSL) cells. We further demonstrated that ASFV infection triggers ERK1/2-dependent phosphorylation and degradation of BimEL, a process independent of viral replication and mediated by viral structural components. A targeted screen identified CD2v as the key viral protein driving this pathway. Both the purified extracellular domain of CD2v (Asp17-Tyr206) and virion-associated CD2v activated TPL2-MEK-ERK signaling without requiring internalization into cells, resulting in BimEL downregulation and subsequent suppression of apoptosis. Crucially, CRISPR-Cas9-mediated knockout of CD2v abolished ASFV-induced ERK1/2 activation and consequential BimEL degradation. Furthermore, we discovered that soluble CD2v released from ASFV-infected cells can activate this signaling axis in uninfected bystander cells, thereby inhibiting apoptosis distantly. This paracrine function, alongside its intrinsic role in directly infected cells, enables CD2v to establish a pro-survival microenvironment conducive to viral propagation. Our findings uncover a multifaceted anti-apoptotic mechanism employed by ASFV, expanding the functional repertoire of CD2v and providing new insights into ASFV pathogenesis with potential therapeutic implications.IMPORTANCEThis study elucidates a distinct mechanism of apoptosis inhibition by African swine fever virus (ASFV), a pathogen that causes a devastating disease in swine. We identify the ASFV CD2v protein as a key suppressor of cell death that operates by hijacking the host TPL2-MEK-ERK signaling pathway to degrade the pro-apoptotic protein BimEL. Importantly, CD2v mediates this effect not only within infected cells but also, in a soluble form, on surrounding uninfected bystander cells. This dual action helps create a protective, pro-survival cellular environment that facilitates viral spread and persistence. Understanding this novel apoptotic suppression mechanism advances our knowledge of ASFV-host interactions and highlights potential new avenues for therapeutic intervention.

Identifying cellular proteins and processes crucial for viral infection is vital for comprehending virus-induced disease mechanisms and developing host-targeted therapies. PRRSV has been shown to take advantage of host metabolic reprogramming and immunosuppression to promote virus production, but the host factors that coordinate these processes have not been fully elucidated. Here, we showed that NUDT7 expression was significantly increased during PRRSV infection by ETS1 targeting its promoter. We also found NUDT7 enhance PRRSV replication by reprograms viral infection-induced intracellular lipid droplets (LDs) synthesis. Mechanistically, NUDT7 interacts with and targets the ubiquitin-ribosomal fusion protein UBA52 for proteasomal degradation. NUDT7 enhances lipid droplet formation and stabilizes the lipogenic transcription factor SREBF1 by blocking UBA52-mediated K11/K27/K48 polyubiquitination. NUDT7-UBA52-SREBF1 axis drives lipid metabolic reprogramming, creating a favorable environment for PRRSV replication. Additionally, NUDT7 inhibits type I interferon signaling and the expression of interferon-stimulated genes, facilitating viral immune evasion. These findings suggest that NUDT7 could be a therapeutic target for combating PRRSV infection, offering a novel perspective and theoretical foundation for developing targeted metabolic-immune antiviral strategies.

Porcine reproductive and respiratory syndrome virus (PRRSV) remains a major threat to the swine industry. Although redox imbalance and oxidative stress are known to favor PRRSV infection, the host determinants that preserve redox homeostasis during PRRSV replication remain insufficiently defined. Here, we identify phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme of the de novo serine synthesis pathway, as a novel restriction factor for PRRSV. We found that PRRSV infection downregulated PHGDH at both mRNA and protein levels. PHGDH knockdown significantly enhanced PRRSV replication in MARC-145 cells and primary porcine alveolar macrophages, whereas ectopic PHGDH expression suppressed viral replication. Specifically, PHGDH depletion promoted PRRSV replication and release, but not attachment or internalization. Catalytically inactive PHGDH mutants retained PRRSV-restriction activity comparable to wild type, indicating a non-canonical, catalysis-independent function. Mechanistically, PHGDH depletion coordinately reduced antioxidant defense genes, oxidized cellular reducing pools, and increased intracellular ROS and mitochondrial superoxide. Importantly, antioxidant treatment with N-acetylcysteine, reduced glutathione, or Mito-TEMPO abolished the proviral effect of PHGDH knockdown, establishing oxidative stress as an essential mediator of enhanced PRRSV replication. PHGDH depletion also caused mitochondrial structural and functional abnormalities and promoted glycolytic features; however, antioxidants failed to restore mitochondrial integrity, and glycolysis inhibition did not reverse the elevated viral replication in PHGDH-depleted cells, indicating that oxidative stress-rather than glycolysis or secondary mitochondrial injury-drives the proviral phenotype. Collectively, our data uncover a PHGDH-redox axis that limits PRRSV replication and highlight redox homeostasis as a tractable host process for antiviral intervention.IMPORTANCEPorcine reproductive and respiratory syndrome virus is a leading cause of respiratory disease and reproductive failure in pigs, creating major economic losses worldwide. Effective control remains challenging, so identifying host processes that naturally limit infection can reveal new intervention opportunities. Our study shows that a host metabolic enzyme, phosphoglycerate dehydrogenase, helps cells maintain antioxidant capacity and redox balance, and this cellular "buffer" restricts porcine reproductive and respiratory syndrome virus replication. When this protection is weakened, oxidative stress rises and the virus benefits; restoring redox balance counters this effect. These findings highlight redox homeostasis as an important, druggable host pathway and suggest that strengthening the cell's own stress defenses may complement existing strategies to reduce virus burden and improve swine health.

Porcine deltacoronavirus (PDCoV) infects a wide range of hosts and can spread across species. Moreover, it is capable of infecting pAPN-knockout cell lines and pigs, suggesting that other important host factors facilitate PDCoV infection. To identify these important host factors, in this study, we employed co-immunoprecipitation (Co-IP) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify 19 significantly upregulated host membrane proteins that interact with the PDCoV S1 protein. Among these, glucose-regulated protein 78 (GRP78) was found to positively regulate PDCoV attachment and internalization, as validated by knockdown, blocking, and overexpression assays. Affinity assays confirmed a strong interaction between GRP78 protein and PDCoV S1 protein. This regulatory function of GRP78 operates in a pAPN-independent way. Mechanistically, we demonstrated that GRP78 interacts with the PDCoV S1 protein via its substrate-binding domain (SBD) and facilitates viral entry through clathrin-mediated endocytosis. Notably, the role of GRP78 in promoting viral entry and replication has also been applied by other coronaviruses, underscoring its potential as a conserved host factor in coronavirus infection. Together, these findings reveal a novel mechanism of PDCoV-host interaction that centers on GRP78-mediated viral attachment and internalization via clathrin-dependent endocytosis.IMPORTANCEPorcine deltacoronavirus (PDCoV) represents a significant zoonotic threat with pandemic potential, exhibiting a broad tissue tropism that underscores its capacity for cross-species spread. Current understanding of PDCoV entry mechanisms, however, remains largely limited to the porcine aminopeptidase N (pAPN), whose knockout fails to fully block infection-highlighting the critical need to identify alternative host entry factors. In this study, we identified the cell membrane protein GRP78 as a novel host factor that binds the C-terminal domain (CTD) of the PDCoV S1 protein via its substrate-binding domain (SBD), thereby mediating viral adsorption and internalization. Furthermore, GRP78 engages clathrin to facilitate viral internalization through endocytosis. Notably, GRP78-mediated entry operates independently of pAPN and demonstrates a degree of broad-spectrum activity relevant to other coronaviruses. Collectively, these findings provide new insights into the early entry mechanisms of PDCoV and identify GRP78 as a potential broad-spectrum target for antiviral intervention.

Recruitment of viral RNA polymerase to replicase membrane proteins is a critical step for assembly of the replication and transcription complex (RTC) during the replication of positive-stranded RNA viruses. In this study, we report that the efficient recruitment of porcine reproductive and respiratory syndrome virus (PRRSV) RNA polymerase RdRp domain requires conformational rearrangement of replicase membrane proteins nsp2 and nsp3 via formation of heterodimeric complex (nsp2/3). Specifically, nsp2 or nsp3 interacts poorly with the nsp9 RdRp core domain (aa.500-685) in pairwise transfection, but the interaction became efficient when the two membrane proteins were co-expressed. Further analysis mapped the nsp9 RdRp-binding region to the cytoplasmic tails of both nsp2 and nsp3 and revealed that reciprocal co-expression of the transmembrane domain (TM) was sufficient to activate the binding of nsp2 or nsp3 to the nsp9 RdRp domain, whereas specific amino acid mutations within the cytoplasmic tails (nsp2 P1073A/G, N1074A/G, N1081A/G, and nsp3 R210E) could disable this interaction. The significance of nsp2/3-nsp9 RdRp interactions was revealed in studies of viral mutants, which showed that PRRSV mutants carrying the above corresponding mutations were either non-viable or severely crippled in replication and exhibited a defect particularly in viral subgenomic RNA (sgRNA) synthesis. Together, our findings highlight the importance of structure change-induced engagement of PRRSV membrane proteins (nsp2 and nsp3) with viral polymerase core domain and provide insight into the orchestrated RTC assembly in PRRSV RNA synthesis.IMPORTANCEPRRSV represents a major threat to the global pork production, but there are no effective vaccines or antiviral drugs yet available. This report concerns the viral RTC assembly. We show that PRRSV replicase membrane proteins nsp2/3 heterodimerization induces a conformational rearrangement of their cytoplasmic tails to allow efficient interaction with nsp9 RdRp core domain. Mutations within the cytoplasmic tails that block the interactions lead to a defect in viral sgRNA synthesis. These findings add insights into the mechanisms of orderly assembly of PRRSV RTC and regulation of viral sgRNA synthesis and provide potential vulnerable targets for drug interventions.

Getah virus (GETV) is an emerging arthropod-borne zoonotic alphavirus that poses a growing threat to animal and public health, yet its virulence determinants remain poorly understood. Here, we report the first identification of a natural GETV variant isolated from the brain of diseased piglets. This variant harbors a 9-nucleotide deletion in the E1 glycoprotein stem region, resulting in the deletion of glutamine 397 (Q397) and valine 398 (V398), along with an asparagine‑to‑isoleucine substitution at position 396 (N396I) and the loss of phenylalanine 399 (F399). It also carries a threonine-to-methionine substitution at position 49 (T49M) in nonstructural protein 2 (NSP2). The NQVF motif (residues 396-399) in E1 protein is highly conserved among GETV strains and related alphaviruses, suggesting a shared functional role. Using reverse genetics, we demonstrated that the E1 deletion-but not the NSP2 mutation-drastically attenuates viral replication in vitro and completely abrogates lethality in neonatal mice by restricting systemic dissemination. Stepwise mutagenesis further revealed that the four‑residue motif functions synergistically as a virulence switch: single deletions partially reduce pathogenicity, while the quadruple deletion confers full attenuation, limiting tissue tropism and histopathology. Although both wild‑type and mutant viruses crossed the placental barrier in pregnant mice, the mutant exhibited reduced vertical transmission and maternal tissue replication. Our findings identify a highly conserved E1 stem motif as a critical regulator of GETV virulence, offering a strategic target for developing attenuated vaccines against GETV and other alphaviruses.

Porcine reproductive and respiratory syndrome virus(PRRSV) is an economically important pathogen for global pork industry. As a positive-strand RNA virus, lacking exonuclease-mediated proofreading, its RNA-dependent RNA polymerase (RdRP) domain within the nonstructural protein 9(nsp9) plays a vital role in maintaining replication accuracy. To identify the residues of PRRSV that regulates replication fidelity, its RdRP structure was predicted by using Alpha Fold 2 and aligned with the solved structure of coxsackievirus B3 (CVB3) RdRP. This comparison identified conserved residues in PRRSV RdRP that are potentially involved in fidelity. Using site-directed mutagenesis, nucleoside analog sensitivity tests, and next-generation sequencing(NGS), it was found that the nsp9 K541R mutation enhances fidelity, as increasing viral resistance to mutagens like ribavirin, 5-Fluorouracil(5-FU), and 5-Azacytidine(5-AZC), as well as generating lower rate of non-contiguous junctions. In contrast, mutations at other positions, including A394G, L396S, and R401A, reduced fidelity and elevated frequency of recombination and mutation accumulation. Structural modeling revealed that the highly conserved residue K336 is spatially adjacent to the key fidelity site K541 but situated on the opposite side of the RNA channel. We found that K336R exhibits a dissociated “resistance-high recombination” phenotype. The findings reveal the importance of specific residues in PRRSV RdRP for replication fidelity and provide insights into the potential for improving the stability and safety of live attenuated vaccines through targeted modifications. Furthermore, the study emphasizes the structural conservation of fidelity determinants across RNA viruses, despite low sequence similarity, which can offer a framework for identifying fidelity key sites in other viral RdRPs.

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.

Viruses frequently hijack host metabolic enzymes to fuel replication. However, the mechanisms underlying this hijacking and utilization of metabolic enzymes remain poorly understood. In this study, we report a sophisticated mechanism by which porcine reproductive and respiratory syndrome virus (PRRSV) exploits a non-canonical enzymatic function of PHGDH (phosphoglycerate dehydrogenase) to modulate macroautophagy/autophagy. We demonstrate that PRRSV infection recruits the transcription factor ZNF143 (zinc finger protein 143) to transcriptionally repress PHGDH expression. Importantly, the antiviral restriction activity of PHGDH is entirely uncoupled from its canonical enzymatic role in serine biosynthesis. Mechanistically, PHGDH depletion triggers the initiation of autophagy via the AMP-activated protein kinase (AMPK)-ULK1 (unc-51 like autophagy activating kinase 1) signaling axis; however, it paradoxically arrests autophagic flux at the autophagosome-lysosome fusion stage. PHGDH is identified as a critical scaffold that facilitates the assembly of the autophagic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex; its downregulation disrupts the interaction between STX17 (syntaxin 17) and SNAP29 (synaptosome associated protein 29), thereby blocking autophagosome-lysosome fusion. This induction of incomplete autophagy creates a favorable cytosolic niche for viral replication. Furthermore, the antiviral effect of PHGDH is also observed in two other swine pathogens, porcine epidemic diarrhea virus (PEDV) and pseudorabies virus (PRV). Collectively, these findings revealed that viruses weaponized the moonlighting function of a metabolic enzyme to dismantle autophagic flux, highlighting PHGDH as a broad-spectrum antiviral target that bridged metabolism and membrane trafficking.Abbreviation: AMPK: AMP-activated protein kinase; BECN1: beclin 1; CQ: chloroquine; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; PEDV: porcine epidemic diarrhea virus; PHGDH: phosphoglycerate dehydrogenase; PRV: pseudorabies virus; PRRSV: porcine reproductive and respiratory syndrome virus; SGOC: serine-glycine-one-carbon; siRNA: small interfering RNA; SNAP29: synaptosome associated protein 29; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; SSP: serine synthesis pathway; STX17: syntaxin 17; ULK1: unc-51 like autophagy activating kinase 1; VAMP8: vesicle associated membrane protein 8; ZNF143: zinc finger protein 143.

Porcine reproductive and respiratory syndrome virus (PRRSV) remains a major threat to global swine industry, yet the immune mechanisms underlying protective vaccination are incompletely understood. Here, we applied integrated single-cell RNA sequencing and T cell receptor (TCR) profiling to characterize immune responses in a PRRSV vaccination-challenge model spanning complete, partial, and non-protection outcomes. We identified distinct CD8+ T cell subsets that were selectively enriched in protected animals vaccinated with modified live vaccine (MLV) and marked by clonal expansion, strong cytotoxic transcriptional programs, and enhanced functional activity, which correlated with rapid control of viremia after challenge. In contrast, non-protected animals accumulated dysfunctional CD8+ T cells expressing exhaustion-associated markers such as CTLA4 despite partial cytotoxic signatures. Mechanistically, the protection-associated responses were primarily driven by viral structural proteins (SP). Replacing the SP-coding region of a heterologous strain reshaped the CD8+ T cell landscape from a mixed cytotoxic/exhausted profile toward a protective program, accompanied by improved clinical outcomes. Further, optimal CD8+ T cell activation required macrophages/monocytes-derived innate signaling, including TLR4 and TLR8 pathways, and was enhanced by CD4+ T cell help. Together, our findings define protection-associated CD8+ T cell states linked to viral control and provide insights for rational PRRSV vaccine design.

Senecavirus A (SVA) is a newly emerging picornavirus threatening the global swine industry, causing vesicular disease and neonatal mortality in pigs. The non-structural protein 2C of SVA is a multifunctional virulence factor. To provide robust tools for a comprehensive study of this protein's function, we successfully generated two monoclonal antibodies (mAbs; 1F9 and 6B4) by immunizing BALB/c mice with the prokaryotically expressed 2C protein as the immunogen. Indirect immunofluorescence assays confirmed that these mAbs specifically recognized the native 2C protein. Western blot analysis further substantiated their reactivity, revealing that the recognized epitopes are linear. Both 1F9 and 6B4 were characterized as IgG1/κ isotypes. Sequence analysis of the heavy and light chain variable regions showed that the framework and complementarity-determining region (CDR) sequences were entirely distinct between the two mAbs. The antigenic epitopes recognized by 1F9 and 6B4 were precisely mapped to amino acids 162DGYKGQF168 and 34LQAWINKE41, respectively, through the expression of a series of truncated forms of 2C protein. Amino acid sequence alignment of the 2C protein from global SVA strains in the GenBank database indicated that these epitopes are highly conserved. Molecular docking revealed that mAbs 1F9 and 6B4 bind to SVA 2C via hydrophobic interactions, hydrogen bonds, and salt bridges involving specific residues in their heavy and light chain CDRs. The successful development of these mAbs provides a powerful tool for the functional investigation of SVA 2C protein.

As a member of nucleocytoplasmic large DNA viruses, African swine fever virus (ASFV) has been proposed to include an intriguing nuclear stage for DNA replication. In this report, we revisited this early event by using the ASFV type II strain HN09 as a model organism. Our studies began with dissecting the early stages of the ASFV life cycle via analyses by viral growth kinetics, electron microscopy, and quantitative PCR (qPCR). We showed that the single replication cycle lasted about 9 h, with the initial early mRNA transcription taking place around 0.5 h upon virus incubation with host cells, a timing that is much earlier than previously found; the viral DNA replication occurred around 4-5 hours post-infection (hpi), coinciding with the formation of early viral factory (VF), followed by virion morphogenesis at 6-9 hpi. The time-course tracking of viral genomic and newly synthesized DNA via EdU (5-ethynyl-2-deoxyuridine) labeling and DNAscope in situ hybridization combined with 3D reconstruction revealed that the viral DNA remained all the time in the cytoplasm. Further transcriptional studies by RNAscope targeting three early mRNAs (CP204L, F334L, and H359L) and EU labeling of nascent viral RNAs revealed similar results regarding their localization. In line with these observations, disruption of the nucleocytoplasmic shuttling with either small interfering RNA (siRNA) or chemical inhibitor did not affect ASFV replication. In contrast, the replication of porcine pseudorabies virus (PRV) was significantly reduced. Thus, we provide strong evidence to suggest that ASFV DNA replication does not involve a nuclear stage. Our findings provide great insight into the replication biology of ASFV.IMPORTANCEAfrican swine fever virus (ASFV) represents a devastating threat to the global swine industry. This virus has a large genomic size of 170 to 200 kb with a complex virion structure, but how this virus coordinates transcription/replication cascades has remained poorly defined. By using modern techniques, including EdU (5-ethynyl-2-deoxyuridine) and EU labeling, DNAscope and RNAscope, 3D reconstruction, and RNA interference (RNAi), we provide compelling evidence to show that the ASFV life cycle does not involve a nuclear stage, with both viral transcription and DNA replication confined to the cell cytoplasm. Our findings provide important insight into ASFV replication biology and into seeking targets for antiviral drug development.

IntroductionPorcine reproductive and respiratory syndrome virus (PRRSV), an economically significant threat to the world pork production, is notoriously known for its heterogeneity, and therefore the current vaccines often fail to provide efficient cross-protection against diverse PRRSV strains.MethodsBy making chimeric viruses using HP-PRRSV-2 lineage 8 (JXwn06) and lineage 1 NADC30-like strains (CHsx1401) as model organisms, the recently results have shown that the viral structural protein-coding region is critical for induction of homologous immunity. In this study, the chimeric viruses were further constructed by exchanging the region coding for the minor (GP2/3/4) or major (GP5/M) structural proteins of JXwn06 on the backbone of CHsx1401 to generate two mutants CHsx1401-GP234JX and CHsx1401-GP5MJX.ResultsThe subsequent animal experiment showed that all three chimeras could confer good protective immunity against the lethal challenge by HP-PRRSV strain JXwn06, and the survived pigs had much lower lung lesions, faster viremia clearance, and lower viral tissue load. However, the exchange of SP region as a whole performed better than either GP2/3/4 or GP5/M region alone, as the pigs in the latter groups showed transient fever following challenge and higher viral load in certain tissues, highlighting a synergistic role. Interestingly, as compared to the group CHsx1401-GP234JX, the group CHsx1401-GP5MJX showed excellent viremia clearance, comparable to the SP group.DiscussionOur results in this report revealed the important role of ORFs2-4 and ORFs5-6 regions in induction of protective immunity and have important implications in understanding viral pathogenesis and further vaccine development.

In recent years, porcine circovirus type 2 (PCV2) has become a significant pathogenic virus in the swine industry, causing huge economic losses globally. However, the impact of PCV2 on the differentiation of CD4+ T cells (Th1/Th2/Th17/Treg) remains unclear. In this study, we noticed that PCV2-infected piglets exhibited several clinical symptoms, including slow weight gain, diarrhea, and lethargy. In addition, we observed significant changes in the levels of IFN-γ, IL-4, and IL-17A during the early stages of infection, while the inhibitory cytokine IL-10 exhibited a significant increase in the later stages. Furthermore, the overall count of CD4+ T helper cells was significantly reduced, and the Th1/Th2/Th17/Treg immune balance was disrupted, with a shift towards Treg cells. What is more, we revealed that TGF-β, a cytokine that induces Treg cell differentiation, was highly expressed after PCV2 infection. This cytokine recruited and phosphorylated Smad3, which subsequently translocated into the nucleus to facilitate Foxp3 transcription. Besides, we also investigated the association between the changes in the intestinal microbiota caused by PCV2 infection and the immune balance of T cells. Overall, our findings enriched the mechanism of PCV2 promoting Treg cell differentiation and provided valuable insights for the prevention and treatment of immunosuppressive diseases. IMPORTANCE:Porcine circovirus type 2 (PCV2) infection can cause immunosuppression-related diseases in pigs. Currently, it is still recognized as an important infectious pathogen of the swine industry in the world. In this study, we discovered that PCV2 infection disrupted the Th1/Th2/Th17/Treg immune equilibrium, and the differentiation capacity of Treg cells increased significantly. Briefly, PCV2 infection promoted the secretion of cytokine TGF-β, recruited Smad3, and phosphorylated it. Subsequently, the phosphorylated Smad3 transmitted the signal from the cell membrane to the nucleus and bound to the enhancer of Foxp3, thereby enhancing the transcription level of Foxp3 and facilitating the differentiation of Treg cells. This study enriches the pathogenic mechanism of PCV2 persistent infection and provided a theoretical basis for the prevention and control of immunosuppressive diseases.