IntroductionHepatitis E virus (HEV) is a globally prevalent zoonotic pathogen posing major public health risks. Swine, a major meat source, carry HEV strains genetically similar to those in humans, highlighting the risk of zoonotic foodborne transmission. This study aimed to investigate the evolutionary history of HEV through phylogenetic and recombination analyses, further provide key reference bases for public health management, improve food safety standards, and offer support for developing effective strategies to prevent foodborne hepatitis E infections.MethodsWe analyzed 348 full-length genomes of HEV isolated from humans and pigs in Asia over the past three decades. Phylogenetic analysis was conducted using the neighbor-joining method in MEGA11. Recombination analysis was performed with seven methods in RDP4, and sequence similarity was visualized using Simplot.ResultsHEV-4 predominated in Asia, especially China, whereas HEV-3 was regionally endemic. Through genomic analysis, we identified 34 potential natural recombination events, predominantly occurring in the RNA-dependent RNA polymerase (RdRp) region; 14 events occurred between swine and human strains, supporting the hypothesis of cross-species transmission. Moreover, 20 recombination events occurred in China and mainly involved HEV-4 strains, suggesting that HEV has distinct evolutionary dynamics. The detection of five inter-genotypic recombination events may highlight ongoing genetic exchange within HEV populations in Asia, and the biological significance of these events remains to be determined.DiscussionThese findings highlight the importance of tracking the evolutionary dynamics of HEV through genomic surveillance, and further underscore the necessity of conducting ongoing HEV surveillance and research to inform prevention strategies.

Hepatitis E virus (HEV) is a major zoonotic foodborne pathogen causing human acute hepatitis, with genotype 4 endemic in China and swine as its main reservoir. Guangdong has a large intensive swine industry, yet stratified molecular epidemiological data on swine HEV are lacking. This study explored the prevalence, risk factors and genetic traits of swine HEV in Guangdong to support zoonotic disease control. A total of 1280 pig fecal samples from farms and abattoirs were tested for HEV RNA via RT-nPCR targeting ORF2. All positive samples underwent Sanger sequencing, and partial isolates were amplified for near-full-length genomes. Phylogenetic trees were built using the maximum likelihood method with 1000 bootstraps. Samples were grouped by region, season and sampling site; subgroup detection rates were compared via χ² and Fisher’s exact tests, and binary logistic regression identified independent risk factors (P < 0.05). The overall HEV positive detection rate was 2.66

Although commercial vaccines against porcine circovirus type 2 (PCV2) have been widely implemented globally, PCV2 remains endemic in swine populations, accompanied by ongoing genotype replacement. Meanwhile, the emergence of novel porcine circoviruses (PCVs), including PCV3 and PCV4, has further complicated the prevention and control of porcine circovirus-associated diseases (PCVAD). This study systematically characterized the epidemiological patterns and genetic diversity of PCVs circulating in Eastern China. A total of 739 clinical samples collected between 2010 and 2016 were screened for PCV2. Additionally, 653 samples obtained during 2023-2024 were analyzed using a triplex real-time quantitative PCR (qPCR) assay for the simultaneous detection of PCV2, PCV3, and PCV4. Full-genome amplification and sequencing were subsequently performed on all PCR-positive samples. Epidemiological analysis revealed an overall PCV2 positivity rate of 37.62% during 2010-2016. In the 2023-2024 cohort, the positivity rates for PCV2 and PCV3 were 35.99% and 16.39%, respectively, with a co-infection rate of 10.26%. Notably, no PCV4-positive samples were detected. Phylogenetic analysis demonstrated that PCV2d is the predominant genotype in Eastern China. Furthermore, PCV2g strains were identified in clinical samples for the first time in mainland China, while PCV3b were determined to be the dominant circulating subtype of PCV3. Multiple critical amino acid substitutions were identified within the neutralizing epitopes of the PCV2 Cap protein, and a recombination event involving a PCV2d vaccine strain and a PCV2c reference strain was detected. In contrast, the PCV3 genome exhibited a high degree of genetic conservation. Collectively, these findings expand the molecular epidemiological landscape of PCVs in Eastern China and elucidate the evolutionary dynamics of circulating PCV strains, providing important insights for the development of next-generation vaccines and region-specific PCVAD control strategies.

Rabies virus (RABV) causes severe central nervous system damage, though the underlying mechanisms remain unclear. Circular RNAs (circRNAs) have been identified in various cells and tissues and are known to regulate gene expression in eukaryotes. Here, we investigated the expression patterns of circRNAs in brain tissues from mice infected with two strains of RABV (CVS-11 and SRV9) and compared them to brain tissues from uninfected mice. Differential expression analysis identified 1,306 circRNAs, primarily derived from coding exons, with functional enrichment implicating synaptic and nervous system pathways. Critically, we identified and validated a novel regulatory axis, mmu_circ_0012122/mmu-miR-1843-5p/Sertad2, where mmu_circ_0012122 acts as a sponge for mmu-miR-1843-5p, leading to Sertad2 upregulation. This axis differentially modulated murine nerve cell fate: mmu_circ_0012122 knockdown reduced necrotic and non-viable apoptotic cells, while mmu-miR-1843-5p overexpression suppressed viable apoptosis and necrosis. Comprehensive characterization further identified Sertad2 as a key mediator promoting both nerve cell apoptosis and neuroinflammatory responses during infection. These findings underscored circRNAs as critical regulators of neuronal survival during RABV infection and highlighted the mmu_circ_0012122-driven network as a potential therapeutic target.

AbstractDengue virus serotype 4 (DENV-4) has rarely been reported in the China-Myanmar-Laos (CML) border region since 2013, where serotypes 1, 2, and 3 have predominated. In 2024, after a nine-year absence of DENV-4 in this region, 17 DENV-4 cases were identified from 2,417 dengue patients through the surveillance network in Jinghong City, China. Over half of the patients presented with dengue fever with warning signs, accompanied by hepatic dysfunction and coagulopathy. Nine complete envelope gene sequences were obtained, and phylogenetic analysis classified the strains into DENV-4 genotype I lineages I_A.3 and I_A.3.2. Integration of epidemiological and phylogenetic data demonstrated that importation of I_A.3.2 from Myanmar initiated limited local transmission in China, whereas I_A.3 importation occurred later and was not associated with local spread. The 2024 I_A.3.2 strains formed a distinct subcluster characterized by the I351 V substitution in envelope protein domain III. Multiple nonsynonymous mutations were identified across the envelope protein, several of which differed from current tetravalent vaccine reference strains, highlighting potential implications for vaccine efficacy. This study documents the importation-initiated limited local transmission of DENV-4 genotype I in the CML border region in 2024 and reveals the accumulation of critical envelope protein mutations. The findings fill important gaps in understanding DENV-4 evolution in this region and underscore the urgency of sustained genomic surveillance in high-risk border areas to inform regional dengue prevention and control strategies.

Single-cell RNA sequencing (scRNA-seq) is a powerful technique for exploring cellular heterogeneity and host-pathogen interactions. This protocol details the Zika virus (ZIKV)-targeted scRNA-seq workflow for preparing high-quality single-cell suspensions from the whole brain tissues of neonatal mice, high-quality single-cell sorting, cDNA reverse transcription, amplification, ZIKV enrichment and host transcriptome library preparation, and sequencing dataset integration in downstream analysis to complete the quantification of ZIKV RNA in individual cells. Key features • Preparation of high-quality single-cell suspensions from the whole brain tissues of neonatal mice. • ZIKV-specific magnetic beads for using the ZIKV and host cell RNA capture. • ZIKV enrichment and host transcriptome library construction, providing a framework for quantifying viral load within individual cells. • Integration of viral enrichment and host transcriptomic datasets enables the visualization and quantification of ZIKV at single-cell resolution.

Proventricular dilatation disease (PDD) is a severe neurological and gastrointestinal disorder of psittacine birds, and parrot bornavirus (PaBV) has been identified as an important virus associated with PDD. Parrots are widely kept as companion animals and are frequently maintained in rescue centers, zoos, breeding farms, and pet trade settings, where close contact among birds and frequent movement of captive parrots may facilitate viral transmission. Despite its recognized importance to psittacine health, the epidemiology of PaBV in captive parrot populations in China, including its prevalence, genetic diversity, and genotype distribution, remains poorly characterized. Therefore, we combined viral metagenomic analysis of selected pooled specimens with individual-level RT-PCR screening to characterize the parrot virome, assess PaBV positivity, and investigate the genetic diversity of the detected PaBV strains in captive parrots in Guangdong Province, China. Among the 1,829,113 viral reads (0.40

Parrot bornavirus (PaBV) is a neurotropic virus that causes chronic infection in parrots, affecting their nervous and gastrointestinal systems and often resulting in high mortality in captive populations. It is a major threat to the parrot breeding industry and the ornamental bird trade. We used Enzyme-Mediated Dual Exponential Amplification (EmDEA) rapid nucleic acid detection technology to create a novel, simple, and highly sensitive method for detecting parrot bornavirus type 4 (PaBV-4). Primers and probes specific to the M gene of PaBV-4 were designed. After two rounds of screening and optimization, the optimal primer pair was identified as F4R7RNA1. The assay was tested for specificity, sensitivity, and clinical usefulness. The test showed no cross-reactivity with H5N2, H7N9, H9N2, NDV, IBV, or IBDV. It had a detection limit of 5 copies/µL and a repeatability coefficient of variation of less than 5

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease (LRTD) in infants and older adults worldwide. Although vaccines based on the fusion (F) protein have shown progress, their efficacy remains limited by antigenic instability and viral evolution. The metastable transition of the F protein between prefusion (preF) and postF conformations critically determines the exposure of neutralizing epitopes, with most potent antibodies targeting preF–specific sites. In addition, the glycosylated G protein contributes to immune evasion through glycan shielding and CX3C-mediated immunomodulation. Recent advances in structural biology and computational protein design have improved the stabilization of preF conformations; however, these approaches do not fully address antigenic variability. Emerging methods, including protein language models (PLMs) and structure prediction frameworks, enable antigen design to be guided by sequence–structure relationships, allowing researchers to prioritize candidate antigens with favorable stability profiles. Here, we propose the term “Rational Design 2.0” to describe this emerging framework. By integrating structural information with evolutionary and sequence-level constraints, Rational Design 2.0 extends RSV vaccine design beyond static structural optimization and provides a conceptual framework for future vaccine-development strategies.

Flaviviruses intricately rewire host metabolic networks to establish a replication-permissive environment; however, the role of glucose transporter-mediated uptake, particularly via glucose transporter 4 (GLUT4), remains insufficiently defined. Japanese encephalitis virus (JEV) infection induces extensive remodeling of glucose metabolism, exemplified by the coordinated upregulation of critical metabolic effectors. Pharmacological blockade of glucose metabolic pathways markedly attenuates JEV replication, whereas exogenous glucose supplementation enhances viral propagation in a concentration-dependent manner. A targeted screen of 111 metabolism-oriented compounds identified selective GLUT4 inhibitors with potent antiviral efficacy. Notably, GLUT4 expression is consistently upregulated during JEV infection across multiple cell types, albeit to varying degrees, and is similarly induced by duck Tembusu virus (DTMUV), suggesting a potentially conserved mechanism shared by these two flaviviruses. However, broader validation across additional members of the Flavivirus genus remains warranted. Mechanistically, the viral nonstructural protein 3 (NS3) engages insulin receptor substrate 1 (IRS1), thereby activating the IRS1-PI3K-Akt-mTORC1-SREBP-1c signaling axis to transcriptionally drive GLUT4 expression. Concurrently, JEV infection induces PI3K-Akt-dependent phosphorylation of AS160, promoting GLUT4 vesicular trafficking via the coordinated action of Rab8 and Rab10. Collectively, these findings delineate a previously unrecognized mechanism whereby JEV commandeers host insulin signaling to orchestrate GLUT4 biosynthesis and membrane translocation, thereby ensuring continuous metabolic substrate availability to sustain replication. This GLUT4-centric metabolic circuitry represents a mechanistically tractable target for host-directed antiviral strategies against Flavivirus.

Introduction:Porcine Reproductive and Respiratory Syndrome (PRRS) is a highly contagious disease that causes reproductive disorders in sows and respiratory problems in pigs of different ages. It first appeared in the late 20th century in the United States and Europe before spreading globally, leading to significant economic losses in the swine industry. Porcine Reproductive and Respiratory Syndrome virus (PRRSV) has a high rate of genetic recombination, resulting in considerable genetic diversity within the virus. The lack of cross-protection between different lineages often leads to unsuccessful vaccination attempts. Methods:To accurately distinguish PRRSV lineages and develop effective vaccination strategies for pigs, we have developed a fluorescence quantitative PCR (qPCR) method by designing specific primers and SYBR green dye. This method allows for the simultaneous identification of different PRRSV genotypes. Results:Our experimental results show that these methods have good specificity and do not react with other common viral pathogens in pigs. This method also demonstrates good sensitivity, with the ability to detect low levels of the virus. The detection limits of these assay were 102 copies/μL for PRRSV-1 (European-type PRRS) and 101 copies/μL for PRRSV-2 (American-type PRRSV), HP-PRRSV (Highly Pathogenic PRRSV), and NL-PRRSV (NADC30-like PRRSV), respectively. Furthermore, the reproducibility of this method is commendable, with intra- and inter-assay coefficients of variation remaining below 3%. In the subsequent study, a total of 316 clinical samples of porcine with respiratory and reproductive failure symptoms were collected from 14 cities in Guangdong. The results showed that among these samples, 22.78% (72 out of 316) tested positive for PRRSV-2, 15.51% (49 out of 316) tested positive for HP-PRRSV, and 0.95% (3 out of 316) tested positive for NL-PRRSV. However, PRRSV-1 was not detected in any of the samples. Discussion:Our method provides a quick way to identify PRRSV genotypes in pig herds in Guangdong, which has certain significance for developing effective vaccination strategies against PRRS.

Interferon-induced transmembrane proteins (IFITMs) are essential components of the innate immune system, demonstrating potent resistance to various enveloped viruses (such as influenza, West Nile, and dengue viruses) both in laboratory settings and in living organisms. Newcastle disease (ND), resulting from Newcastle disease virus (NDV), is a severe avian viral ailment with notable economic impact due to its significant mortality and morbidity rates. On the basis of the efficient antiviral effects of IFITMs, an in-depth study of the role and mechanism of NDV inhibition by chicken IFITMs (chIFITMs) is highly important for the prevention and control of this disease. In this study, we found that transient overexpression of chIFITMs effectively inhibited NDV (NDV Lasota, NDV Na) infection in DF-1 cells, with the highest inhibition rates of up to 89% and 99%, respectively, and that there was no significant difference in the antiviral effects of chIFITM1/2/3, which were not significantly different. Virus‒cell binding-entry assays revealed that chIFITMs restrict the entry process of NDV. Deleting endogenous chIFITMs enhances viral replication (more than 1.27-fold) and diminishes chIFNL3-mediated antiviral effects. Concurrently, overexpressing chIFITMs influences the expression level of the W protein; and co-immunoprecipitation experiments confirmed interaction between them. These findings suggest that the W protein could represent a novel target for the inhibition of NDV by chIFITMs. In summary, our results provide the initial comprehensive analysis of the antiviral effects of chIFITMs against NDV. This observation suggests that IFITMs are important barriers against zoonotic infections and important targets against viral invasion.

Unlike preceding MERS-related coronaviruses, the recently identified MjHKU4r-CoV-1 strain can directly infect human cells. Nonetheless, its potential pathogenic attributes and underlying molecular mechanisms remain unclear. We find that MjHKU4r-CoV-1 induces significant inflammation, including interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α), and exhibits pronounced fusogenicity mediated by its spike (S) protein, leading to extensive syncytium formation. This suggests the possibility that MjHKU4r-CoV-1 possesses strong pathogenic potential in humans. Further, we successfully reveal the molecular mechanism of MjHKU4r-S-driven membrane fusion by crystallizing the six-helix bundle (6-HB) structure, a fusion apparatus composed of HR1 and HR2 domains. Concurrently, we develop a series of peptide-based fusion inhibitors that target the viral HR1 domain to impede the formation of viral 6-HB. Among these fusion inhibitors, a stapled peptide, MjHKU4r-HR2P10, shows the most potent inhibitory activity against MjHKU4r-CoV-1, MERS-CoV, SARS-CoV-2, and HCoV-OC43 infections at nanomolar level and thus holds considerable promise for further development as effective antiviral agents in clinic.

The urokinase-type plasminogen activator system (uPAs) consists of the urokinase-type plasminogen activator (uPA), its receptor (uPA receptor, uPAR) and the plasminogen activator inhibitor (PAI). Recent studies have revealed that, beyond its well-established roles in promoting fibrinolysis and degrading extracellular matrix (ECM) proteins-thereby regulating tissue repair and tumor metastasis-this system also plays a significant role in viral infections. Specifically, the uPAs modulates viral infection processes by regulating the expression and activity of uPA and uPAR, which are involved in inflammatory response modulation, immune cell migration, and the infiltration of inflammatory cells during tissue repair in the context of viral infections. In this review, we summarize the roles of uPAs in various viral infections, aiming to deepen our understanding of the contributions of each uPA component and provide insights into potential strategies for inhibiting viral infection processes.

In this study, we identified a new chicken-specific protein, named chicken interferon-related antiviral protein (chIRAP) after sequence analysis and comparison, which inhibited the proliferation of various viruses including influenza A virus (IAV) and Newcastle Disease Virus (NDV) in vitro, and chicken embryos with high expression of chIRAP reduced IAV infection. Mass spectrometry analysis of chIRAP interacting proteins and screening of interacting proteins affecting the function of chIRAP revealed that the deletion of endogenous chicken peroxiredoxin 1 (chPRDX1) significantly reduced the antiviral effect of chIRAP. In order to clarify the functional site of chPRDX1 affecting the antiviral effect of chIRAP, we constructed the point mutants of chPRDX1 based on the results of molecular docking (D79A, T90A, K93A, Q94A, R110A, R123A), and screened the sites affecting the antiviral effects of chIRAP by knockdown of endogenous chPRDX1 combined with the overexpression mutant strategy, the results showed that the mutations in the sites affected the antiviral effects of chIRAP to different degrees, with D79A being the most significant, and the D79A mutation of chPRDX1 reduces the ability of chPRDX1 to regulate reactive oxygen species (ROS). chIRAP may exert antiviral effects by regulating the intracellular ROS balance at the D79 site of chPRDX1. In conclusion, we identified a novel chicken-derived antiviral protein, clarified its antiviral effects and preliminarily explored its mechanism of action, which provides a new tool and option for the prevention and treatment of avian-origin viral diseases, especially avian-origin related zoonotic diseases.

IntroductionHepatitis E virus (HEV) is a major cause of acute hepatitis in humans and recognized as a zoonotic pathogen, with swine serving as a primary reservoir. Despite substantial research, comprehensive analysis encompassing regional variations and pig growth stages within China, as well as the influence of recent biosecurity measures on HEV prevalence, remains limited. In this study, we aim to assess the prevalence and risk factors associated with swine HEV in China.MethodsA thorough review of HEV infection studies was conducted using six databases: China National Knowledge Infrastructure, Wanfang, Wipro, Centre for Agriculture and Biosciences International, PubMed, and ScienceDirect, covering publications from January 1, 2004 to December 31, 2023. Eighty-seven studies investigating the seroprevalence of swine HEV IgG antibodies and HEV RNA detection rates were included. A rigorous meta-analysis and quality assessment followed.ResultsThe combined seroprevalence of swine HEV IgG antibodies was 58.0% (95% confidence interval [CI]: 52.0–65.0). The seroprevalence from 2019 to 2023 was lower (27.4, 95% CI: 26.3–28.2) than that in other years. The seroprevalence was higher in sows (67.2, 95% CI: 55.8–78.7) than in suckling, nursery, and fattening pigs. The detection rate of HEV RNA was 13.0% (95% CI: 11.0–15.0), with fattening pigs showing a significantly higher positivity rate (16.9, 95% CI: 13.2–20.7) than sows and suckling pigs. HEV RNA detection was significantly lower in bile (8.3, 95% CI: 6.3–10.3) than in feces and liver.DiscussionThis study highlights the widespread presence of HEV in pig farms across China, with prevalence strongly linked to pig growth stage, study year, and sample type. The findings underscore the importance of pig growth stage, sample type, and recent biosecurity measures in controlling HEV prevalence, providing actionable insights for improving biosecurity practices in pig farms.

Merbecoviruses comprise four viral species with remarkable genetic diversity: MERS-related coronavirus, Tylonycteris bat coronavirus HKU4, Pipistrellus bat coronavirus HKU5, and Hedgehog coronavirus 1. However, the potential human spillover risk of animal merbecoviruses remains to be investigated. Here, we reported the discovery of HKU5-CoV lineage 2 (HKU5-CoV-2) in bats that efficiently utilize human angiotensin-converting enzyme 2 (ACE2) as a functional receptor and exhibits a broad host tropism. Cryo-EM analysis of HKU5-CoV-2 receptor-binding domain (RBD) and human ACE2 complex revealed an entirely distinct binding mode compared with other ACE2-utilizing merbecoviruses with RBD footprint largely shared with ACE2-using sarbecoviruses and NL63. Structural and functional analyses indicate that HKU5-CoV-2 has a better adaptation to human ACE2 than lineage 1 HKU5-CoV. Authentic HKU5-CoV-2 infected human ACE2-expressing cell lines and human respiratory and enteric organoids. This study reveals a distinct lineage of HKU5-CoVs in bats that efficiently use human ACE2 and underscores their potential zoonotic risk.

Enterovirus 71 (EV71) is a major cause of hand, foot, and mouth disease, particularly affecting pediatric populations worldwide. The role of ZYG11B, a CUL2-complex-associated E3 ubiquitin ligase from the Zyg-11 family, in antiviral defense against EV71 remains unclear. To our knowledge, this study is the first to reveal that ZYG11B targets EV71 VP1 for proteasomal degradation via the ubiquitin-proteasome pathway, with CRL2ZYG11B complex activity specifically driving K33-linked ubiquitination. Mass spectrometry and immunoprecipitation analyses confirmed the interaction between ZYG11B and VP1 and identified key domains required for binding both VP1 and CUL2. Comparative analyses showed that VP1 ubiquitination sites are highly conserved across related enteroviruses, including CA6, CA16, and EVD68. Functional assays further demonstrated that ZYG11B restricts these viruses, highlighting its potential as a broad-spectrum antiviral target. These findings establish ZYG11B as a critical effector in host antiviral responses and support its therapeutic potential for managing enterovirus infections. E3 ubiquitin ligases and deubiquitinases have become important topics of competition between viruses and hosts. Here, we identified CRL2ZYG11B as an E3 ubiquitin ligase complex capable of degrading structural protein VP1 of enteroviruses, making ZYG11B a broad-spectrum antiviral factor. We first proposed the inhibitory effect of ZYG11B on viruses and identified the structural domains of ZYG11B connecting substrates and CUL2, providing new targets for the design of antiviral drugs.

The angiotensin-converting enzyme 2 (ACE2) receptor is shared by various coronaviruses with distinct receptor-binding domain (RBD) architectures, yet our understanding of these convergent acquisition events remains elusive. Here, we report that two bat MERS-related coronaviruses (MERSr-CoVs) infecting Pipistrellus nathusii (P.nat)-MOW15-22 and PnNL2018B-use ACE2 as their receptor, with narrow ortholog specificity. Cryoelectron microscopy structures of the MOW15-22/PnNL2018B RBD-ACE2 complexes unveil an unexpected and entirely distinct binding mode, mapping >45 Å away from that of any other known ACE2-using coronaviruses. Functional profiling of ACE2 orthologs from 105 mammalian species led to the identification of host tropism determinants, including an ACE2 N432-glycosylation restricting viral recognition, and the design of a soluble P.nat ACE2 mutant with potent viral neutralizing activity. Our findings reveal convergent acquisition of ACE2 usage for merbecoviruses found in European bats, underscoring the extraordinary diversity of ACE2 recognition modes among coronaviruses and the promiscuity of this receptor.