Xinna Ge

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. IMPORTANCE: Porcine 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.

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. IMPORTANCE: Porcine 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.

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. IMPORTANCE: Porcine 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.

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. IMPORTANCE: PRRSV 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.

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.

Porcine deltacoronavirus (PDCoV) is an emerging pathogen that causes severe, often fatal, diarrhea in suckling piglets and has zoonotic potential. Its nonstructural protein 12 (Nsp12), functioning as the RNA-dependent RNA polymerase (RdRp), is a central component of the viral replication–transcription complex and a critical target for host antiviral mechanisms. Here, we identified eukaryotic elongation factor 1 gamma (eEF1G) as a host interactor of PDCoV Nsp12 by immunoprecipitation-coupled mass spectrometry in IPEC-J2 cells. This interaction was confirmed by co-immunoprecipitation, pull-down assays, and confocal microscopy. Functional analyses involving siRNA knockdown and overexpression of eEF1G, combined with viral titration, strand-specific real-time quantitative PCR, and RNA immunoprecipitation assays, demonstrated that eEF1G directly binds to Nsp12. Knockdown of eEF1G significantly enhanced viral replication and increased negative-stranded RNA synthesis, whereas overexpression did not affect viral proliferation. Furthermore, eEF1G was found to bind PDCoV genomic RNA and competitively disrupt the interaction between Nsp12 and viral RNA, thereby impairing RdRp activity. Our results indicate that eEF1G acts as a novel host restriction factor that inhibits PDCoV replication by competing with Nsp12 for genomic RNA binding, ultimately blocking negative-stranded RNA synthesis. This study unveils a new antiviral mechanism and highlights a potential target for developing interventions against PDCoV.

The autophagy–lysosome axis is an evolutionarily conserved intracellular degradation pathway which constitutes an important component of host innate immunity against microbial infections. Here, we show that African swine fever virus (ASFV), one of most devastating pathogens to the worldwide swine industry, can reshape the autophagy–lysosome axis by recruiting the critical lysosome membrane proteins (LAMP1 and LAMP2) to viral factories while inhibiting autophagic induction in macrophages. The screening of viral membrane proteins led to the identification of several ASFV membrane proteins, exemplified by viral protein pEP153R, that could significantly alter the subcellular localization of LAMP1/2 when expressed alone in transfected cells. Further analysis showed that pEP153R was also a component of viral factories and could induce endoplasmic reticulum (ER) retention of LAMP1/2, leading to the inhibition of the fusion of autophagosomes with lysosomes. Interestingly, the ASFV mutant lacking EP153R could still actively recruit LAMP into viral factories (VFs) and inhibit autophagic flux, indicating the existence of a functional redundancy of other viral proteins in the absence of pEP153R and highlighting the complexity of ASFV replication biology. Taken together, our results reveal novel information about the interplay of ASFV with the autophagy–lysosome axis and a previously unrecognized function of ASFV protein pEP153R in regulating the cellular autophagic process.

The porcine reproductive and respiratory syndrome virus (PRRSV) has significantly impacted the global pork industry for over three decades. Its high mutation rates and frequent recombination greatly intensifies its epidemic and threat. To explore the fidelity characterization of Chinese highly pathogenic PRRSV JXwn06 and the NADC30-like strain CHsx1401, self-recombination and mutation in PAMs, MARC-145 cells, and pigs were assessed. In vitro, CHsx1401 displayed a higher frequency of recombination junctions and a greater diversity of junction types than JXwn06. In vivo, CHsx1401 exhibited fewer junction types yet maintained a higher junction frequency. Notably, JXwn06 showed more accumulation of mutations. To pinpoint the genomic regions influencing their fidelity, chimeric viruses were constructed, with the exchanged nsp9-10 regions between JXwn06 and CHsx1401. The SJn9n10 strain, which incorporates JXwn06’s nsp9-10 into the CHsx1401 genome, demonstrated reduced sensitivity to nucleotide analogs compared to CHsx1401. Conversely, compared with JXwn06, the JSn9n10 strain showed increased sensitivity to these inhibitors. The swapped nsp9-10 also influences the junction frequency and accumulated mutations as their donor strains. The results indicate a propensity for different types of genetic variations between these two strains and further highlight the nsp9-10 region as a critical determinant of their fidelity.

Porcine circovirus 3 (PCV3) is an emerging virus, causing substantial economic losses in pig populations, that was first detected in 2016. Furthermore, the virus has already been reported in Europe, the Americas, and Asia, including China, indicating that the virus has spread worldwide. However, the molecular epidemiology of PCV3 still needs further study. To investigate PCV3 epidemiological characteristics in China, 2707 serum samples of pigs were randomly collected from 17 provinces in China between September 2018 and March 2022 and analyzed via PCR assays. The study showed that PCV3 infection was prevalent in the overall population with 31.07% (841/2707) and 100.0% (17/17) at sample and province levels, respectively, though the positivity rate of PCV3 varied from 7.41 to 70.0% in different provinces, suggesting that PCV3 infection has a widespread distribution in China. We selected 22 serum samples from different regions that had high levels of viral DNA for amplification and sequenced their ORF2 (Cap) gene. According to the phylogenetic analysis, all isolates in the current study could be grouped into two separate subclades, with 15 strains belonging to clade 3a and 7 strains belonging to clade 3b, indicating that PCV3a and PCV3b were the predominant subtypes in the regions of China studied. Meanwhile, additional analysis revealed that the capsid gene sequences identified in this study displayed 97.46~99.8% nucleotide (nt) and 97.06~100% amino acid (aa) sequence similarity with other PCV3 available reference strains, respectively. In general, our studies provide important insights for understanding the prevalence and evolution of PCV3 in China and will guide future efforts to develop measures for preventing and controlling the disease.

As an emergent picornavirus pathogenic to pigs, Senecavirus A (SVA) can replicate in pig kidneys and proliferates well in porcine kidney epithelial PK-15 cells. Here, tandem mass tags (TMT) labeling coupled with liquid chromatography–tandem mass spectrometry (LC-MS/MS) was used to analyze the proteome dynamic changes in PK-15 cells during SVA infection. In total, 314, 697 and 426 upregulated differentially expressed proteins (DEPs) and 131, 263 and 342 downregulated DEPs were identified at 12, 24 and 36 hpi, respectively. After ensuring reliability of the proteomic data by quantitative PCR and Western blot testing of five randomly selected DEPs, Mx1, eIF4E, G6PD, TOP1 and PGAM1, all the DEPs were subjected to multiple bioinformatics analyses, including GO, COG, KEGG and STRING. The results reveal that the DEPs were mainly involved in host innate and adaptive immune responses in the early and middle stages of SVA infection, while the DEPs mainly participated in various metabolic processes in the late stage of infection. Finally, we demonstrated that Mx1 protein exerts antiviral activity against SVA by interacting with VP1 and VP2 proteins dependent on its GTPase, oligomerization and interaction activities, while Mx1 interacts with VP3 only depending on its oligomerization activity. Collectively, our study provides valuable clues for further investigation of SVA pathogenesis.