Since its discovery, porcine epidemic diarrhea virus (PEDV) has significantly affected the agricultural economy worldwide. The available commercialized coronavirus vaccines cannot adequately control emerging strains. Therefore, investigating the correlation between viruses and antiviral host factors is necessary. In this study, we showed that zinc finger protein 219 (ZNF219) was upregulated by viral nonstructural protein 12 (nsp12) upon PEDV challenge. Moreover, ZNF219 inhibited the replication of PEDV through selective autophagic degradation of the PEDV S2 protein. ZNF219 recruited TRAF6, the ubiquitin E3 ligase, to ubiquitinate the PEDV S2 protein. After recognition, the ubiquitinated PEDV S2 protein was delivered to autolysosomes via the cargo receptor p62 for degradation by autophagy, thus inhibiting the proliferation of PEDV. To summarize, after sensing PEDV infection by recognizing the viral nsp12 protein, host cells upregulated the intracellular expression of ZNF219, which degraded the viral S2 protein by activating autophagy, thus suppressing viral replication. Our study revealed a novel antiviral mechanism involving ZNF219 and provided a novel target for preventing and treating PEDV.
• GlycoRNAs on host cell surfaces serve as adhesive molecules for viral internalization. • GlycoRNAs may interact with immunomodulatory receptors and facilitate immune recognition and responses. • Targeting glycoRNAs shows promise for antiviral therapy.
Human metapneumovirus (hMPV) is a prevalent respiratory virus in children with acute lower respiratory tract infections that is highly homologous with respiratory syncytial virus (RSV), the primary etiological agent of pediatric upper and lower respiratory tract infections. Although hMPV and RSV are the only human pathogens within the Pneumoviridae family and share similar clinical manifestations, the mechanisms underlying their divergent pathogenicity remain poorly understood. In this study, we performed transcriptomic analysis on clinical respiratory samples collected between 2017 and 2019 from 61 children: including hMPV-infected, RSV-infected and healthy controls. This analysis revealed a shared upregulation of antiviral response pathways, including neutrophil activation and signaling mediated by interferons and interleukins. Conversely, cilium organization and assembly pathways were commonly downregulated in both infections. hMPV infection uniquely upregulated pathways associated with extracellular component activity, ion channel complexes, and neuroactive ligand‒receptor interactions. In contrast, pathways related to membrane rafts and membrane microdomains were uniquely downregulated in hMPV-infected patients. Analysis of differentially expressed immune-related and interferon-stimulated genes revealed significant hMPV-specific increases in EGF and FCGR1A, alongside decreased EPAS1 expression. The genes that were uniquely upregulated during hMPV infection were enriched in cytokine production regulation, cytokine-cytokine receptor interactions, and PI3K/AKT signaling, whereas those that were uniquely downregulated involved the viral entry and endocytic vesicle pathways. Both hMPV infection and RSV infection significantly increased the proportions of M1 macrophages and neutrophils but decreased the proportions of M0 and M2 macrophages. Notably, hMPV infection resulted in a significant increase in monocytes and activated NK cells coupled with a decrease in resting memory CD4+ T cells, compared with RSV infection. The results also revealed a significantly greater relative abundance of Prevotella salivae in the hMPV infection group, whereas Streptococcus salivarius and Streptococcus mitis were enriched in the RSV group. These distinct immune and microbial signatures provide novel insights into the pathogenesis of pediatric hMPV and RSV infections.
Hepatitis B virus (HBV) has been implicated in hepatocellular carcinoma (HCC) progression, partly through regulation of the tumor suppressor phosphatase and tensin homolog (PTEN). Although transcriptional regulation of PTEN by HBV is well characterized, its post-transcriptional regulation remains poorly understood. Because RNA 5-methylcytosine (m5C) modification influences post-transcriptional gene control and cancer development, we investigated whether HBV modulates PTEN through m5C. Methylated RNA immunoprecipitation (MeRIP)-quantitative polymerase chain reaction showed a marked reduction in m5C on PTEN mRNA in HBV-producing cells. MeRIP sequencing further identified decreased m5C within the PTEN coding sequence region (chr10:89717747-89717771) in HBV-producing HepAD38/tetracycline-off cells, with chr10:89717756 emerging as a critical site where HBV suppresses m5C enrichment and PTEN expression. Mechanistically, the m5C "writer" NOP2/Sun RNA methyltransferase 2 (NSUN2) and the "reader" Y-box binding protein 1 (YBX1) stabilized PTEN mRNA in an m5C-dependent manner. HBV disrupted this pathway, decreasing PTEN mRNA stability via NSUN2- and YBX1-mediated m5C. Overexpression of NSUN2 or YBX1 attenuated HBV-driven proliferation, migration, and invasion, and these effects were partially reversed by the PTEN inhibitor VO-Ohpic. The small hepatitis B surface antigen and hepatitis B X protein downregulated NSUN2 and YBX1, linking viral proteins to PTEN suppression. Further, HBV is associated with reduced NSUN2 expression in HBV transgenic (HBV-Tg) mice, HBV-infected primary human hepatocytes as well as HBV-positive clinical HCC specimens, supporting the physiological and clinical relevance of this finding. Together, these findings identify the NSUN2/YBX1/PTEN axis as a potential therapeutic target in HBV-associated HCC.
Swine enteric coronaviruses (SECoVs) cause severe watery diarrhea and high mortality in piglets, resulting in significant economic losses to the global pig industry. However, frequent mutations in SECoVs significantly compromise vaccine-induced immunity and limit cross-protection against emerging variants. Therefore, there is an urgent need to develop new broad-spectrum antiviral drugs to be the last line of defense to supplement vaccine immunity. In this study, we utilized molecular docking and molecular dynamics simulations to identify phloretin as a broad-spectrum SECoV inhibitor. Phloretin has demonstrated prophylactic and therapeutic efficacy in vitro and in vivo, improving the survival of SECoV-infected piglets. It was further found that phloretin exerts a broad-spectrum antiviral effect by acting on the conserved 3CLpro Cys144 site of three SECoVs. It is worth noting that derivative A12, designed on the basis of the structure-activity relationship (SAR) between phloretin and 3CLpro, showed a 15.7, 2.6, and 8.4-fold increase in antiviral effect against porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine delta coronavirus (PDCoV), respectively. This study reveals a 3CLpro Cys144 targeting broad-spectrum strategy for use against SECoVs, providing a candidate drug to bridge the vaccine immunity gap.
Herpes simplex virus type 1 (HSV-1) infects over 70% of the population and establishes lifelong latent infection with periodic reactivation in humans, resulting in various related diseases. However, the molecular and cellular events underlying the transition of HSV-1 from latency to reactivation remain poorly understood. In this study, we used bulk RNA sequencing and single-cell transcriptomic analyses to dissect the cellular and molecular events of HSV-1 latency-reactivation transition in infected trigeminal ganglia (TG) in both mouse and tree shrew infection models. We found that mice exhibited fluctuating host gene responses during the acute phase and relatively quiescent latency, whereas tree shrews displayed a relatively mild acute phase and active latency characteristics. Single-cell analysis revealed that HSV-1 infects TG neuronal subpopulations expressing growth hormone and pituitary hormones. Importantly, we observed that HSV-1 latency in tree shrew TGs exhibited inhibition of cellular autophagy function, while HSV-1 latency in mice was accompanied by the attenuation of monocyte-related immune surveillance. Given that infected cell protein 0 (ICP0) has autophagy inhibitory activity, we further investigated the role of this viral protein in tree shrew models using an ICP0-deficient HSV-1 strain. Notably, the mutant virus could not undergo spontaneous reactivation from latency. These findings support the hypothesis that ICP0 may be essential for spontaneous reactivation by inhibiting autophagy in vivo.
Hainan Island, located in the South China Sea, is known as an area with diseases related to Rickettsia spp. or spirochete infection; however, the potential threat there from infection with tick-borne viruses (TBVs) remains obscure. In the present study, the dominant tick species, including Rhipicephalus sanguineus and Rhipicephalus microplus, were collected in Hainan Island, and tick viromes were investigated by metagenomic sequencing. In total, 27 viral species were identified belonging to the families Orthomyxoviridae, Flaviviridae, Nairoviridae, Phenuiviridae, Totiviridae, Chuviridae, Rhabdoviridae, and Parvoviridae, amongst which one novel virus and 13 new strains were discovered. Subsequently, individual ticks were screened for seven TBVs, Huanggang Rhabd tick virus 1 (HRTV1), Lihan tick virus (LHTV), Mivirus (MIV), Guangdong tick quaranjavirus (GTQV), Wenchang Ephemerovirus (WEPMV), Jingmen tick virus (JMTV), and brown dog tick phlebovirus (BDPTV), resulting in high prevalence rates of 16.97%, 9.59%, 10.33%, 7.38%, 7.01%, 6.27%, and 3.69%, respectively. While co-infection with multiple viruses was more frequent in R. sanguineus, R. microplus ticks generally had higher viral loads. Four febrile patients showed antibody responses to three TBVs, one each to LHTV and JMTV, and two to GTQV; the patient with antibodies to JMTV also showed neutralizing activity against this virus. This study promoted our understanding of the diversity and complexity of the TBV community in Hainan Island. The results provide serological evidence that human exposure to TBVs like JMTV may have occurred in Hainan, raising concern about potential risks from TBVs and the need to perform further surveys of TBVs among ticks, animals and humans.
African swine fever (ASF), caused by the African swine fever virus (ASFV), is characterized by high mortality in infected pigs. ASFV infection triggers severe inflammatory response in the host, which is a crucial contributor to the high lethality of this disease. However, the underlying mechanism by which ASFV infection induces inflammatory response is still poorly understood. In this study, we found that UV-inactivated ASFV induces interleukin-1β (IL-1β) production, suggesting that certain structural proteins incorporated in the virion possess the ability to trigger inflammatory response. Further investigations demonstrated that deletion of the ASFV A137R gene significantly inhibited the ASFV-induced upregulation of the mRNA transcription of various proinflammatory genes and phosphorylation of p65 and IκBα. Furthermore, the purified pA137R protein promoted the mRNA transcription of these proinflammatory genes and phosphorylation of p65 and IκBα. Additionally, pA137R protein interacted with the NACHT and LRR domains of NLRP3 through its N terminal 1-99 amino acid (aa) domain, thereby promoting the oligomerization of NLRP3 and ASC and subsequently facilitating NLRP3 inflammasome assembly. Collectively, our findings identify ASFV pA137R protein as a key proinflammatory determinant of ASFV, which not only advances our understanding of the molecular mechanisms underlying ASFV-induced inflammatory response but also provides new insights into ASFV pathogenesis.
Viral hemorrhagic septicemia virus (VHSV) is a major pathogen affecting freshwater and marine fish species, posing a significant threat to global aquaculture. Reverse genetics systems are essential for studying viral replication, and host interactions, as well as developing vaccines and therapeutics. In this study, we developed a reverse genetics platform for VHSVLB2018 strain, a genetically distinct VHSV genotype IVa strain which exhibits low genomic identity with other Asian isolates, using a dual RNA polymerase I/II transcription vector. We successfully rescued recombinant VHSV in mammalian (B7GG) and fish (FHM and EPC) cell lines, and engineered recombinant VHSV strains expressing EGFP (rVHSV-EGFP) and cherry (rVHSV-Cherry) fluorescent proteins. Phenotypic analysis revealed that unmodified recombinant VHSV (rVHSV) exhibited growth kinetics and virulence similar to the wild-type VHSV, while fluorescent protein-expressing variants showed attenuated replication and virulence, with the rVHSV-EGFP strain displaying the greatest attenuation. Utilizing the rVHSV-EGFP strain, we conducted antiviral compound screening and identified three promising inhibitors-xanthohumol, octyl gallate, and rottlerin that effectively inhibit VHSV replication. Time-of-addition assays further revealed that xanthohumol and rottlerin targeted the viral replication stage, while octyl gallate interfered with viral internalization. This reverse genetics system provides a versatile platform for studying VHSV pathogenesis, developing live-attenuated vaccines, and screening antiviral compounds, enhancing our understanding of this pathogen and offering new tools for aquaculture disease management.
The evolution of SARS-CoV-2 has been driven by successive globally circulating waves, including the Alpha and Delta lineages, early Omicron (BA.1-BA.5), XBB, and the recently dominant JN.1 lineages. Although the marked advantage in fitness of early Omicron over Delta lineages has been recognized, there is a lack of systematic evaluation of SARS-CoV-2 fitness across 2020 to 2025. Here, we analyzed 15.23 million SARS-CoV-2 genomes available through May 2025. The accumulation of mutations in the spike protein of the virus has continued to accelerate over time, whereas the trend slowed in the other viral proteins. Using a Bayesian genomic-epidemiological framework, we estimated that lineage fitness increased approximately linearly from 2021 to 2025. Notably, JN.1 lineages exhibited a significantly higher rate of fitness gain than their predecessor XBB and earlier Omicron lineages. We further analyzed characteristic mutations of JN.1 and found that those in the receptor-binding domain were associated with larger alterations in residue hydropathy, charge, and structural surface exposure relative to other lineages. These findings suggest JN.1 as a distinct evolutionary stage and underscore the importance of sustained genomic surveillance.
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Coxsackievirus B4 (CVB4) is a highly pathogenic enterovirus associated with severe neurological disease and mortality. To establish research models that recapitulate severe CVB4 infection, two clinical isolates with distinct neurovirulence-the high-virulence strain GZ-HFM01 and low-virulence strain GZ-R6-were used. An in vitro neurocytotoxicity model with human neuroblastoma SH-SY5Y cells showed that GZ-HFM01 produced significantly larger plaques than GZ-R6, reflecting its increased capacity to damage neuronal cells. Concurrently, an optimized in vivo severe infection model was established in 3-day-old ICR mice through intraperitoneal inoculation, which reproduced key clinical features of severe disease. Compared with GZ-R6, GZ-HFM01 infection resulted in significantly reduced survival, progressive neurological impairment, time-dependent viral accumulation in brain tissue, pronounced histopathological injury, and elevated serum pro-inflammatory cytokine levels. To map genomic determinants of neurovirulence, a chimeric virus panel was generated by replacing individual genome segments (5'untranslated region [UTR], P1, P2, P3, or 3'UTR) of GZ-HFM01 with the corresponding regions from GZ-R6. Evaluation with the established models demonstrated that replacement of the P2 region significantly attenuated both the cytopathic effect in SH-SY5Y cells and pathogenicity in 3-day-old ICR mice. Animal studies further indicated that substitution of the 5'UTR or P1 region also reduced virulence-a phenotype absent from the cell-based model-underscoring the multifactorial regulation of CVB4 pathogenesis. In conclusion, this study provides validated in vitro and in vivo models of severe CVB4 infection and identifies key genomic segments that contribute to neurovirulence, offering a foundation for mechanistic research and the development of targeted interventions against severe CVB4-induced disease.
Chikungunya virus (CHIKV) infection in humans is typically characterized by acute febrile illness, rash, and polyarthralgia, and often progresses to chronic arthralgia. However, existing small-animal models fail to capture both the acute and chronic phases of the disease. In this study, we compared Chinese tree shrews (Tupaia belangeri) and C57BL/6 mice in terms of infection dynamics, viral characteristics, histopathological changes, and immune responses. Following dual-site inoculation (subcutaneous injection into the abdomen and footpad), Tupaia belangeri exhibited typical symptoms that more closely resembled those of humans, including pronounced fever, foot swelling, and skin lesions, and were accompanied by higher and more sustained viral loads in blood and multiple tissues. Histopathological examination revealed marked inflammation and tissue damage in muscle and joint tissues, consistent with pathological changes observed in humans. Transcriptomic analysis further demonstrated significant upregulation of multiple key immune-related genes (CXCL10, ISG15, IFIT3, SERPING1, MCEMP1, IFI6), indicating a human-like immune response profile. Collectively, our results show that Tupaia belangeri faithfully mirrors the infection features, viral dynamics, and immune responses of human CHIKV infection, establishing it as a valuable model for dissecting disease mechanisms and testing vaccines or antiviral therapies.
Respiratory syncytial virus (RSV) bronchiolitis is the leading cause of hospitalization in infancy and exhibits pronounced age-dependent clinical heterogeneity. Fever becomes increasingly prevalent with age, yet whether febrile representation reflects a uniform inflammatory and immune phenotype across infancy remains unclear. In this prospective cohort of infants hospitalized with RSV bronchiolitis, we performed an integrated analysis of clinical features, pharyngeal microbiome composition, host transcriptomic profiles, and host-microbe interaction networks, with particular attention to age-related variation in fever-associated patterns. Clinically, fever prevalence exhibited a strong age-dependent increase across infancy. Correspondingly, canonical correspondence analysis identified age and fever as dominant gradients related to variation in both pharyngeal microbiome composition and host gene expression. Although no significant age-dependent correlations were observed at the global microbial and host transcriptomic levels in the fever-age interaction model, distinct patterns of microbial and host responses related to fever were observed across different age groups. Specifically, ranked gene set enrichment analysis indicated that febrile infants in early infancy showed relative attenuation of host defense-related programs, whereas older infants showed stronger enrichment of antiviral and inflammatory effector pathways, with more selective regulatory and signaling-associated patterns in late infancy. Integrated host-microbe network analysis further delineated a coherent developmental trajectory of fever-associated interaction architectures, evolving from densely interconnected regulatory networks in early infancy to modular, selectively coupled, host-centered configurations with advancing age. Together, febrile responses in RSV bronchiolitis should not be interpreted as a uniform biological phenotype across infancy and support age-aware interpretation of fever in pediatric RSV infection.
• An FCV-VP1 mRNA-LNP vaccine was constructed, utilizing a safe, flexible non-viral platform. • The vaccine elicits effective, durable neutralizing antibodies, protecting cats from FCV challenge. • This mRNA vaccine provided complete protection in cats, offering a novel effective strategy against FCV infection.
Klebsiella pneumoniae is an important opportunistic pathogen in both humans and animals. Controlling it has become increasingly difficult due to the rapid spread of antimicrobial resistance. In this study, we isolated and characterized a novel lytic bacteriophage, vB_Kp_Z1, and evaluated its therapeutic efficacy against K1-serotype K. pneumoniae. Host range analysis showed that vB_Kp_Z1 was strictly specific to K1 strains, as confirmed across multiple prevalent capsular types. The in vivo efficacy of vB_Kp_Z1 was assessed using intraperitoneal infection models in mice. Two hypervirulent K1 strains were used: a pigeon-derived strain (KP1897) and a human clinical strain (KP177). Phage treatment significantly improved survival compared with phosphate-buffered saline-treated controls. It provided complete protection in KP1897-infected mice and achieved an 87.5% survival rate in KP177-infected mice. In addition, phage administration markedly reduced bacterial loads in the blood, liver, and lungs, indicating effective control of systemic dissemination. These findings demonstrate that vB_Kp_Z1 is a K1-specific bacteriophage with therapeutic potential against hypervirulent K. pneumoniae, including strains from different host species.
Human adenoviruses (HAdVs), particularly subgroup B serotypes HAdV-3 and HAdV-55, are associated with severe respiratory disease and currently lack targeted therapies. While neutralizing monoclonal antibodies (nMAbs) offer promising therapeutic potential, the specific nMAbs targeting these serotypes remain poorly characterized. Therefore, this study aimed to generate and evaluate the efficacy of serotype-specific nMAbs against HAdV-3 and HAdV-55. Mice were immunized with HAdV-3 virions, HAdV-55 virions, or recombinant fiber knob proteins (HAdV-55/-7) for the generation of serotype-specific nMAbs, and their efficacy was systematically evaluated using in vitro assays and an in vivo tree shrew model. Through comprehensive screening, eleven MAbs were identified with specificity against HAdV-3 (six clones) or HAdV-55/-7 fiber knob (five clones). Four nMAbs exhibited potent neutralizing activity: 13F12 (half-maximal inhibitory concentration, IC50: 3.8 μg/mL), 8D2 (IC50: 15.1 μg/mL), 3A3 (IC50: 14.9 μg/mL) against HAdV-3 virions, and 8F2 with neutralizing efficacy against HAdV-55 virions (IC50: 30.4 μg/mL). Western blot analysis revealed that MAbs 13F12 and 8F2 targeted the fiber protein, whereas 8D2 and 3A3 bound to the hexon protein. Furthermore, in vivo evaluations demonstrated that 13F12 significantly reduced viral loads in nasal turbinates and attenuated lung pathology in HAdV-3-infected tree shrews. Mechanistically, all tested anti-HAdV-3 nMAbs inhibited infection by blocking viral attachment (P < 0.01 vs. controls). In conclusion, this study underscores the therapeutic potential of targeting viral entry and highlights 13F12 as a promising candidate for HAdV prophylaxis.
Herpes simplex virus type 1 (HSV-1) causes lifelong latent infection and is associated with severe diseases, including herpes simplex encephalitis, neonatal herpes, and no licensed vaccine is currently available for this pathogen. Here, we systematically evaluated an attenuated HSV-1 platform with deletions in ICP34.5 and ICP47 genes (HSV-1 Δ34.5Δ47) for application as a dual-function vaccine. This construct, generated by BAC-galK recombination, showed attenuated replication in vitro. Notably, it elicited robust humoral and cellular immune responses in mice, and provided complete protection against lethal challenge with virulent HSV-1 McKrae strain through both corneal and genital tract infection routes. To assess its utility for heterologous antigen delivery, we engineered a recombinant HSV-1 Δ34.5Δ47-N, which expresses the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein. This recombinant virus retained the protective efficacy against HSV-1 and induced robust N-specific immune responses. Passive serum transfer and in vivo T-cell depletion studies demonstrated that humoral immunity was sufficient to mediate protective immunity against HSV-1 challenge. Safety assessment revealed no detectable viral mRNA or pathological lesions in the brains of immunized animals. These findings support HSV-1 Δ34.5Δ47 as a safe and versatile platform for both HSV-1 prophylaxis and heterologous antigen delivery.
Despite widespread use of multiple PCR, a substantial proportion of pediatric acute respiratory tract infections (ARTIs) lack identifiable pathogens and are classified as unknown etiology. The microbial characteristics and clinical relevance of these cases remain unclear. In this study, we compared the airway microbiomes of PCR-positive and PCR-negative ARTIs and examined their relationships with sampling site and disease severity. A total of 514 hospitalized children with ARTIs were enrolled. Nasopharyngeal swabs (NS) and bronchoalveolar lavage fluid (BALF) samples were tested using a 22-target multiplex PCR panel and subsequently stratified by pathogen status for pooled metatranscriptomic sequencing to profile active microbial communities, viral genotypes, and antibiotic resistance genes. PCR identified common respiratory pathogens in 77.0% of NS and 54.1% of BALF samples. Metatranscriptomic analysis showed that PCR-negative pools displayed markedly lower viral activity and comparatively higher bacterial transcript abundance, with notable enrichment of Pseudomonas. Microbial signatures differed between upper and lower airway samples and across clinical severity, with severe cases demonstrating increased bacterial burden and Pseudomonas enrichment, whereas mild infections exhibited relatively stronger viral signals. Under current thresholds, antibiotic resistance genes were detected in patient pools but not in healthy controls. Overall, PCR-negative pediatric ARTIs exhibited distinct, bacteria-enriched microbial profiles. Integrating metatranscriptomics with PCR enhances pathogen characterization and reveals site- and severity-related microbial patterns that may support diagnostic evaluation and clinical management.