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.
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 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.
Group A rotaviruses (RVs) continue to be one of the most important pathogens causing severe acute gastroenteritis in infants and young animals worldwide. Recently, the prevalence of porcine RV (PoRV) from pig farms has strikingly increased, adversely affecting the swine industry, particularly with the G9 genotype of PoRV VP7 emerging as the predominant genotype spreading in China. Current vaccines against PoRV fail to provide sufficient protective immunity, necessitating urgent development of effective vaccines and antiviral drugs against PoRV. Here, we successfully established and improved the entirely plasmid-based reverse genetics (RG) system to rescue a G9 genotype of recombinant PoRV AHFY2022 strain (G9P[23]). Using the improved RG system, we rescued recombinant AHFY2022 harboring the fluorescent UnaG protein or nano-luciferase (NLuc) reporter within gene segment 7 that encodes non-structural protein 3 (NSP3). Furthermore, we adopted the UnaG reporter virus to screen anti-PoRV drugs and identified two promising antiviral drugs, C8 and C9. Moreover, we generated the recombinant PoRV (rAHFY2022-G5-VP7) containing G5 genotype of VP7 from PoRV-positive samples in the backbone of AHFY2022 strain. The reassortant strain exhibited efficient replication and genetic stability. Mouse models were utilized to evaluate the immune responses elicited by rAHFY2022-G5-VP7 strain in vivo, revealing similar neutralizing antibodies and cellular immune response compared to the parental AHFY2022 strain in mice. Together, this study provides an important tool for screening potential anti-PoRV drugs and developing novel vaccines against prevalent PoRV strains.
Mitochondrial homeostasis is intricately linked to the pathogenesis of many viral infections. The maintenance of mitochondrial homeostasis and normal cellular functions relies heavily on the delicate balance of mitochondrial dynamics. However, the precise impact of porcine epidemic diarrhea virus (PEDV) infection on mitochondrial dynamics and subsequent pathological processes is largely unexplored. In this study, through both in vivo and in vitro infections, we have demonstrated that PEDV infection induces mitochondrial fission and leakage of mtDNA by upregulating Drp1 expression, and identified Smad3 as a crucial transcription factor responsible for regulating Drp1 expression. By establishing an inflammatory model using PEDV-infected cells, we have identified Drp1-mediated mtDNA release as an upstream event triggering TLR9/NF-κB pathway activation during PEDV infection. These findings provide novel insights into the relationship between PEDV infection and host inflammatory responses from the perspective of mitochondrial dynamics.
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.
• 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.
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.
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.
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.
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.
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.
The Zaire Ebola virus (EBOV) and Bundibugyo virus (BDBV) cause severe hemorrhagic fever with high mortality, highlighting the urgent need for broad-spectrum antiviral therapies. Neutralizing nanobodies, with their small size, structural stability, and ability to access sterically restricted epitopes, represent a promising antiviral modality. Here, we identified a high-affinity nanobody, BDBV-Nb02, from a fully synthetic phage display library targeting the glycan cap of BDBV glycoprotein (GP1). BDBV-Nb02 demonstrated strong binding kinetics (KD ≈ 1 nM) and potent neutralizing activity against both BDBV and EBOV pseudoviruses, with half-maximal inhibitory concentration (IC50) values in the nanomolar range. Engineering a bivalent format significantly enhanced neutralization potency, achieving up to a 56-fold reduction in the 90% inhibitory concentration (IC90) compared with the monovalent form. Epitope competition assays and molecular docking revealed that BDBV-Nb02 targets a conserved glycan cleft, with residues F248 and NP278/279 identified as critical neutralization sites. In contrast, Fc-fusion constructs impaired the nanobody's efficacy, highlighting the importance of preserving the structural features that enable access to glycan-shielded epitopes. Our findings demonstrate that BDBV-Nb02 is a promising candidate for broad-spectrum orthoebolavirus therapy and may serve as a valuable component in future antiviral cocktail formulations.
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.
Vaccination stands as the single most effective and cost-efficient public health intervention in human history, serving as a cornerstone of modern medicine that profoundly transforms global health outcomes. Beyond preventing disease, it acts as a catalyst for equitable socioeconomic development. In recent decades, recurrent seasonal viral outbreaks and sporadic yet catastrophic pandemics have continued to pose challenges to global public health systems. Traditional vaccine technologies, however, not only often fall short in protection efficacy, but also fail to keep pace with the evolving demands of next-generation vaccine development. These scientific gaps have directed cutting-edge research to prioritize critical objectives in terms of enhancing antigen effectiveness, achieving stable pan-protection against diverse variant strains, and strengthening production robustness. The advent of genomics spurred the emergence of reverse vaccinology 1.0, leading to breakthroughs like the MenB vaccine. Today, the advanced reverse vaccinology 2.0 paradigm thoroughly redefines vaccine design process by organically integrating human immunology with state-of-the-art computational protein structure analysis tools. This review explores the transformative shifts in influenza and respiratory syncytial virus vaccine development, along with specific case studies, to deepen understanding of the evolving principles and methodologies in novel vaccine designs and offer strategic insights for addressing emerging infectious pathogens.
H9N2 avian influenza virus (AIV) poses a persistent threat as inactivated vaccines (InV) often fail to prevent viral shedding. To address this, we developed a recombinant turkey herpesvirus (HVT-BNT) expressing conserved B and T cell epitopes from H9N2 AIV to enhance both humoral and cellular immunity. HVT-BNT exhibited genetic stability over 15 serial passages and growth kinetics comparable to the parental strain in vitro. We evaluated immunization strategies of HVT-BNT combined with InV in 1-day-old chicks and 18-day-old embryos via subcutaneous (HVT-BNT + InV) or in ovo (HVT-BNT-ovo + InV) routes, respectively. Compared to InV alone, HVT-BNT + InV elicited significantly higher HI and neutralizing antibody titers, elevated IgG and IgM levels, and increased proportions of CD8+ T cells. Similarly, the HVT-BNT-ovo + InV group exhibited a trend of higher values in these indicators. Notably, while the InV group displayed no significant differences in key immune cytokines compared to the control group, the combined immunization groups exhibited significant upregulation of IFN-α, IFN-β, IFN-γ, IL-2, IL-5, IL-6, IL-10, and IL-13. Furthermore, ELISPOT assays confirmed enhanced IFN-γ secretion in response to conserved AIV peptides (NP380-393, NP455-463, and NS198-106) in the combined immunization groups. Following heterologous H9N2 AIV challenge, oropharyngeal positivity rates in the combined immunization groups were lower than those in the InV group at 5 DPI. Similarly, cloacal positivity rates were more reduced in the combined groups compared to the InV group at 3 and 5 DPI. By 7 DPI, viral shedding was completely cleared in both combined immunization groups, whereas the InV group continued to shed virus via the oropharyngeal route. These findings demonstrate that the HVT-BNT-based vaccination strategy effectively enhances both humoral and cellular immune responses, providing superior early protection. While the in ovo strategy provides a viable hatchery intervention, the subcutaneous route exhibits the superior immune activation and protection compared with conventional InV alone.
• 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.
The recent global outbreak of mpox virus (MPXV) infections underscores the urgent need for antiviral therapies against orthopoxviruses. In this study, using a high-content screening (HCS) platform based on a modified vaccinia virus Tiantan strain with GFP insertion (MVTT-GFP) under BSL-2 conditions, we screened 1513 kinase inhibitors for antiviral activity. Among these, Bruton's tyrosine kinase inhibitor BTKi-2 emerged as a potent candidate, exhibiting IC50 of 0.535 μM against vaccinia virus (VACV) and 0.260 μM against MPXV in vitro, while maintaining low cytotoxicity. In a murine model of VACV-induced pneumonia, BTKi-2 treatment reduced lung viral loads by 90% and a significantly improved survival compared to vehicle-treated controls. Notably, mechanistic studies indicate that BTKi-2's antiviral effects cannot be completely attributed to the inhibition of BTK or EGFR/ErbB2 signaling. These findings highlight BTKi-2 as a promising antiviral agent in vitro and in vivo, suggesting that BTKi-2 may offer a potential avenue for future therapeutic development against orthopoxvirus infections.
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Enterovirus (EV) infections represent a significant global and national health concern in children, leading to various complications. Although most children with EV infections generally have a favorable prognosis, a small proportion may still develop severe complications. This study aimed to analyze the epidemiological characteristics, disease spectrum, and disease burden of EV infections in China. A total of 163,714 hospitalized children with EV infections from 37 member hospitals of the Futang Research Center of Pediatric Development were identified between Jan 1st, 2016 and Dec 31st, 2023, accounting for 1.49% of all pediatric hospitalizations. Most cases occurred in infants aged 28 days to ≤ 1 year (42.29%) and toddlers aged 1 to ≤ 3 years (39.93%), with a male predominance (male-to-female ratio, 1.62 : 1). Severe cases of EV infections can be life-threatening and increase the burden of disease. The median LOS for EV infections were 5 days, with an average hospitalization cost of $587.37. EV infections can affect multiple organs, including the heart, brain, and respiratory system and were associated with severe complications, including myocarditis, encephalitis, and meningitis. These findings underscore the substantial disease burden of EV infections and highlight the need for targeted prevention strategies in young children.