Overcoming immunosuppressive tumor microenvironments remains a critical challenge in advanced vaccine development. Here, we evaluated Pygenic acid A (PA), an intracellular small-molecule inhibitor targeting the PD-1/SHP-2 axis, as a novel vaccine adjuvant. The adjuvant efficacy of PA was systematically assessed in two murine models: a therapeutic B16-F10 melanoma lung metastasis model and a prophylactic lethal H1N1 influenza virus challenge model. In the melanoma metastasis model, PA potentiated the anti-tumor effect of the mTRP2 vaccine, markedly inhibiting pulmonary metastatic lesions and prolonging the survival of tumor-bearing mice. Mechanistically, PA robustly boosted the intratumoral infiltration of functional T cells, thereby reversing local tumor immunosuppression. In the influenza vaccination model, consistent immunostimulatory effects were observed: the PA-adjuvanted hemagglutinin (HA) vaccine effectively elicited broad-spectrum cross-neutralizing antibody responses and provided complete protection against lethal heterologous influenza virus challenge. Further mechanistic investigations demonstrated that PA specifically promoted the differentiation of T follicular helper (Tfh) cells and the expansion of germinal center (GC) B cells in draining lymph nodes, while triggering a robust Th1-type cellular immune response dominated by IFN-γ secretion. Furthermore, in vivo safety assessments verified that PA intervention induced no obvious systemic inflammation, hematological abnormalities, or visceral organ injury, indicating a favorable safety profile. Collectively, these results demonstrate that PA serves as a potent and safe intracellular checkpoint-targeting adjuvant capable of potentiating both cellular immunity and cross-protective humoral immunity, holding great translational promise for the development of advanced cancer vaccines and broad-spectrum influenza vaccines.
Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus that causes a life-threatening infectious disease with high mortality. Currently, there are no approved vaccines or specific therapeutic drugs for clinical use against SFTSV infection. Here, we screened a lignan compound library to identify candidates with anti-SFTSV activity. Two type I arylnaphthalide lignan lactones, Pronaphthalide A and Procumbenoside I, were identified as potent inhibitors of SFTSV infection. Mechanistic investigations indicated that these lignans exert their inhibitory effects at the viral binding and internalization stages. Molecular docking analysis revealed that these lignans can bind to the conserved B domain of the SFTSV glycoprotein Gn, an interaction validated by subsequent experiments as the core mechanism underlying their antiviral activity. In a mouse model of lethal SFTSV infection, treatment with these compounds, particularly Procumbenoside I, significantly inhibited viral infection, attenuated histopathological abnormalities, and improved survival rates. The antiviral activity of these lignans was further expanded to other bunyaviruses, including Lymphocytic choriomeningitis virus (LCMV) and Wetland virus (WELV). These findings uncover the broad-spectrum antiviral activity of type I arylnaphthalide lignans and support their potential as candidate therapeutic agents for the clinical intervention of bunyavirus infections.
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• The H4N6/G030 strain, a novel H4N6 avian influenza virus (AIV), was isolated from a red-necked stint. • The H4N6/G030 is a novel cross-species reassortant derived from wild bird and poultry AIV lineages. • The H4N6/G030 binds avian and mammalian-type sialic acid receptors, replicates in their cells, and causes disease in animals. • High seroprevalence of H4N6 was observed on surveyed poultry farms nearby.
Monkeypox virus (MPXV), a pathogenic orthopoxvirus, has caused major outbreaks and emerged as a global public health threat. Although antivirals approved for smallpox are used therapeutically against monkeypox, their clinical utility is limited by drug availability and emerging resistance. The conserved strategy by which viruses remodel host nucleotide metabolism to secure biosynthetic precursors for replication and spread has emerged as a pivotal target for the development of broad-spectrum antiviral therapeutics. In this study, leveraging the high genetic and biological similarity between vaccinia virus (VACV) and MPXV, we employed VACV as a surrogate model to screen 10 FDA-approved inhibitors targeting nucleotide metabolism enzymes, aiming to identify potential novel inhibitors against MPXV. Mycophenolate mofetil (MMF), an inosine 5'-monophosphate dehydrogenase type II (IMPDH2) inhibitor, displayed potent inhibition effects against both VACV and MPXV. Subsequent downstream time-course studies revealed that MMF targets a post-entry stage of the viral replication cycle. Mechanistic studies suggest that MMF inhibits IMPDH2 activity by suppressing ubiquitin-specific protease 5 (USP5)-mediated deubiquitination of IMPDH2 and inducing rod-and-ring (R&R) assembly, leading to reducing dNTP pools and enhancing antiviral effects. In conclusion, our findings demonstrate that MMF is an effective antiviral drug against VACV and MPXV infection and establish a host-directed therapeutic strategy to combat future orthopoxvirus outbreaks.
H9N2 avian influenza viruses (AIVs), which are enzootic in poultry and possess spillover potential to humans, represent a considerable global public health threat. Since 2021, we have observed an unprecedented decline in the ability of H9N2 viruses to agglutinate chicken red blood cells (CRBCs), a change that impairs effective detection in routine surveillance. Substituting CRBCs with turkey or guinea pig erythrocytes in hemagglutination assays effectively restored virus detectability. Through integrated bioinformatic and biological approaches, we identified that this phenotype is associated with specific amino acid substitutions at residues 131 and 132 of the 130-loop in the hemagglutinin (HA) protein. The HA motif 131/132-NT emerged in 2015 and rapidly increased in frequency, becoming the dominant pattern since 2021 in chickens and humans. Critically, the 131/132-NT motif attenuated viral binding affinity to short-chain α2,6-linked sialic acid receptors, explaining the loss of agglutination with CRBCs. Animal experiments further demonstrated that viruses carrying the HA 131/132-NT mutations maintained strong infectivity in chickens but altered tissue tropism in mice, showing reduced replication in the lungs. Collectively, our findings reveal a potential surveillance gap caused by reduced hemagglutination activity of H9N2 viruses, which may compromise the sensitivity of CRBC-based detection and potentially lead to under-detection of circulating viruses. Incorporating turkey or guinea pig red blood cells into surveillance protocols is therefore recommended to enhance detection sensitivity.
Mollusca, the second-largest animal phylum, includes many aquaculture species used as important food resources for humans. While DNA viruses that threaten molluscan aquaculture have received much attention, molluscan RNA viromes are still understudied. Here, using a multi-stage RdRP discovery pipeline combining six-frame translation, profile-based homology search, and phylogenetic validation, 203 RNA viruses spanning five viral phyla and fifteen viral orders were identified, based on 223 molluscan metatranscriptomes covering eight classes. Phylogenetic analysis, combined with structural modeling, revealed a significant increase in the number of Pisuviricota-related lineages. Extensive modular evolution in viral genomes was observed, including gene rearrangements and co-evolution of capsid and RdRP genes. Host prediction linked 76% of the RNA viruses to a range of eukaryotes. These findings expand the diversity of RNA viruses associated with molluscs and shed light on their phylogenetic relationships, highlighting that molluscs might serve as an important reservoir of diverse RNA viruses infecting a range of eukaryotic hosts.
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.
• Identification of a novel HIV-1 circulating recombinant form (CRF161_0107) in Chinese men who have sex with men. • CRF161_0107 emerged around 2016, exhibiting unique CRF01_AE-C5 and CRF07_BC-N parental lineages. • This is the first report of a CRF derived from CRF01_AE-C5 in southwestern China, suggesting viral migration. • The finding underscores the ongoing evolution of HIV-1 via recombination in key populations such as MSM.
Blood-tissue barriers are specialized interfaces that safeguard organ homeostasis by restricting pathogen dissemination. Zika virus (ZIKV), an emerging flavivirus of global concern, exhibits an exceptional ability to breach multiple barriers-including the blood-brain, blood-placental, blood-testis, and blood-retinal barriers-enabling neuroinvasion, vertical and sexual transmission, and ocular disease. ZIKV employs diverse strategies to cross these barriers: receptor-mediated entry, disruption of tight junctions, and hijacking immune cells or extracellular vesicles as viral carriers. Adaptive mutations further refine tissue tropism and enhance barrier traversal efficiency. Insights from cell culture, organoid, animal, and ex vivo tissue models reveal not only the conserved and tissue-specific mechanisms of barrier penetration but also the downstream pathological consequences in the affected organs. Understanding how ZIKV breaches these interfaces and induces organ-specific pathology deepens our knowledge of host-pathogen interactions and provides a framework for designing barrier-protective and disease-mitigating strategies against ZIKV and other pathogens that breach blood-tissue barriers.
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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) infection can induce herpes simplex encephalitis (HSE), a life-threatening neurological disorder characterized by active viral replication within the central nervous system accompanied by excessive neuroinflammatory responses. Cepharanthine hydrochloride (CH) is a natural bisbenzylisoquinoline alkaloid with diverse pharmacological activities. CH was evaluated for its antiviral and anti-inflammatory activities against HSV-1 infection. In microglia, CH markedly inhibited viral replication and suppressed STING-NF-κB signaling, thereby reducing HSV-1-associated neuroinflammation. During HSV-1 infection, CH binds to Nrf2, disrupting Keap1-Nrf2 interaction and subsequent ubiquitination. This hindered Nrf2 degradation and attenuated STING activation. In an HSE mouse model, CH significantly reduced viral loads in brain tissue, improved survival rates, prevented weight loss, and alleviated neurological symptoms. Furthermore, CH promoted the accumulation of Nrf2 and inhibited the STING-NF-κB signaling pathway in vivo. Collectively, these results indicate that CH represents a potential antiviral strategy for HSE.
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Coxsackievirus A10 (CVA10) is a major causative agent of hand, foot and mouth disease and utilizes KREMEN1 (KRM1) as its cellular receptor. While our previous work identifies VP2 residue K140 as a universal anchor for KRM1 binding among KRM1-utilizing enteroviruses, the functional significance of other receptor-interface residues remains poorly characterized. Here, through structure-guided mutagenesis, we demonstrate that VP3-T234, a completely conserved residue at the C-terminus of VP3, is essential for CVA10 infectivity. The T234A mutation does not affect virion assembly but abolishes both KRM1 binding and cellular attachment. Interestingly, this requirement shows remarkable virus specificity: the homologous residue is critical for CVA8, but is not required for other KRM1-utilizing enteroviruses including CVA2-CVA6 and CVA12. The T234A mutation significantly attenuates the pathogenesis of both CVA10 and CVA8 in neonatal mice. Moreover, the CVA8-T234A mutant provides complete protection as an attenuated vaccine against lethal CVA8 challenge. Our findings establish a model wherein KRM1 engagement relies on the conserved VP2-K140 anchor complemented by virus-specific secondary residues, with VP3-T234 representing a key determinant for CVA10 and CVA8. These insights advance our understanding of enterovirus-receptor interactions and provide new directions for vaccine development.
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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.
CD39 exerts an inhibitory effect on tumour progression by impairing the cytotoxic capacity of natural killer (NK) cells against cancer cells. However, the impact of CD39 expression on the non-cytolytic functions of NK cells in treatment-naïve human immunodeficiency virus type 1 (HIV-1)-infected individuals remains poorly understood. In this study, thirty-four individuals with acute HIV-1 infection (AHI), thirty-eight with chronic HIV-1 infection (CHI), and twenty-four HIV-1-negative healthy controls (HC) were enrolled to explore the role of CD39 expression on NK cells in HIV-1 suppression at different infection stages. Flow cytometry was employed to analyze the immune phenotype and functional characteristics of NK cells. We found that CD39 expression on NK cells was significantly upregulated following HIV-1 infection, and its positive rate was positively associated with HIV-1 viral load in both AHI and CHI individuals. Compared with CD39- NK cells, CD39+ NK cells exhibited reduced activation; in AHI individuals, the activation level of CD39+ NK cells was positively associated with HIV-1 viral load but inversely correlated with CD4+ T-cell counts. In CHI individuals, the interleukin-10 (IL-10)-producing capacity of total NK cells, CD39+ NK cells, and CD39- NK cells was enhanced and positively correlated with HIV-1 viral load. Additionally, across the AHI and CHI groups, the overall IL-10-secreting ability of NK cells was positively correlated with the frequency of CD39+ NK cells. In both AHI and CHI individuals, CD39+ NK cells showed lower T-cell immunoglobulin and ITIM domain (TIGIT) expression than CD39- NK cells, while the CD39+TIGIT+ NK cell subset displayed significantly stronger IL-10-secreting capacity. POM-1, an inhibitor of CD39 ectonucleotidase activity, could enhance IL-10 secretion by NK cells in both HIV-1-infected individuals and the majority of healthy controls, but attenuate interferon-γ (IFN-γ) secretion by NK cells in HIV-1-infected individuals. In contrast, the CD39-blocking antibody A1 reduced IFN-γ secretion without affecting IL-10 secretion by NK cells in both HIV-1-infected individuals and healthy controls. Our findings reveal a novel CD39+ NK cell-associated mechanism that contributes to ineffective HIV-1 control, and suggest that CD39, alone or combined with TIGIT, may serve as a promising target to restore antiviral NK cell function in treatment-naïve individuals living with HIV-1.
Tick-borne viruses (TBVs) pose significant emerging threats to public and veterinary health worldwide. In Pakistan, the potential threats posed by TBVs extend far beyond Crimean-Congo hemorrhagic fever virus (CCHFV), which causes outbreaks and severe hemorrhaging with a high fatality rate among humans each year. However, the full extent of the tick-borne virome remains largely unexplored. This study presents the metagenomic profiling of viruses in livestock-associated ticks from Pakistan. Eighty-seven ticks belonging to the genera Ixodes, Rhipicephalus, Haemaphysalis, and Hyalomma species from livestock in Punjab. These ticks were subsequently grouped into 11 pools for RNA sequencing. Our analysis revealed extensive viral diversity, identifying sequences related to 31 viruses spanning at least 11 families. New strains of Jingmen tick virus (JMTV), Brown dog tick phlebovirus 2 (BDTPV-2), and Liman tick virus (LMTV) were characterized, confirming their presence in the region. Serological surveys performed among 319 livestock, 253 humans, and 214 rats detected antibodies against these viruses, indicating host exposure. Notably, the presence of JMTV-neutralizing antibodies was confirmed in two livestock animals, one human, and one rat, providing evidence of productive infection. Our findings significantly expand the known diversity and distribution of TBVs in Pakistan, establish the preliminary baseline of the tick virome in the country, and provide serological evidence of cross-species exposure to emerging TBVs. This study highlights the underestimated risk of tick-borne viral zoonoses in Pakistan and underscores the urgent need for enhanced surveillance and risk assessment.
• A quail siadenovirus (QAdV-1) was identified to be associated with ulcerative enteritis-like disease. • QAdV-1 is a novel member of the genus Siadenovirus in the family Adenoviridae. • QAdV-1 shows the highest identity to Turkey hemorrhagic enteritis virus. • Animal studies evidenced that QAdV-1 is pathogenic to quails and can transmit cross-species to chickens.