Jing An

Type I interferon (IFN) signaling is a central antiviral defense, with STAT2 driving the expression of IFN-stimulated genes (ISGs) to restrict viral infection. Zika virus (ZIKV) and dengue virus evade this pathway through nonstructural protein 5 (NS5)-mediated ubiquitination and proteasomal degradation of STAT2, yet the contribution of this immune-evasion strategy to viral fitness and pathogenesis remains incompletely defined. Here, using a multistep mutational scanning strategy that jointly assessed STAT2 degradation and viral RNA replication, we identified a single NS5 residue, L162, as uniquely permissive to substitution that abolishes STAT2 degradation without compromising intrinsic replication functions. Substitution of L162 with alanine or glycine (L162A or L162G) selectively disrupted NS5 recruitment of the ZSWIM8-CUL3 E3 ubiquitin ligase, thereby preserving STAT2 stability and restoring IFN signaling. Recombinant ZIKV carrying these mutations exhibited reduced replication and enhanced ISG induction in human cells, defects fully rescued by STAT2 knockout. In A129 mice (type I IFN receptor deficient), mutant and WT viruses replicated comparably, but in human STAT2 knock-in mice, ZIKV-NS5 ^L162G exhibited markedly reduced viral loads and disease. In pregnant human STAT2 knock-in mice, the mutant also reduced placental and fetal viral burdens and adverse pregnancy outcomes. Notably, infection with ZIKV-NS5 ^L162G elicited robust neutralizing antibody and T cell responses that conferred protection against WT challenge. Together, these findings establish NS5-mediated STAT2 degradation as a central determinant of ZIKV immune-evasion, viral fitness, and pathogenesis and highlight disruption of NS5–STAT2 antagonism as a promising strategy for antiviral intervention and rational attenuation.

Adverse pregnancy outcomes associated with dengue virus (DENV) infection are a severe manifestation of dengue fever; however, we still poorly understand the underlying mechanisms. Trophoblast dysfunction in the placenta contributes centrally to various pregnancy complications. As the functional unit of the placenta, the syncytiotrophoblast (STB) requires continuous fusion of precursor trophoblasts and adequate energy supply. In this study, we established a model of adverse pregnancy by infecting Ifnar 1 ^−/− pregnant mice with DENV-2. We did not detect viral replication within the placental trophoblast layer but observed substantial structural damage to the STB. RNA-seq analysis demonstrated that precursor trophoblasts exhibit disrupted glucose metabolism, and placental tissue upregulates pro-fusion genes—yet fails to complete syncytialization. Metabolic assays revealed reduced ATP levels and elevated lactate concentrations, indicating that an inadequate energy supply impedes trophoblast differentiation. Treatment with the glycolytic inhibitor 2-DG restored oxidative phosphorylation (OXPHOS) activity in the placenta and markedly improved pathological outcomes. Further mechanistic studies established that DENV infection induces oxidative stress, which activates hypoxia-inducible factor Hif1α, leading to subsequent upregulation of lactate dehydrogenase (LDHA) expression. We identify this cascade as a fundamental mechanism underlying metabolic reprogramming in trophoblasts. Our findings not only clarify the key pathological processes driving DENV-induced adverse pregnancy outcomes but also propose novel therapeutic strategies for clinical intervention. IMPORTANCE: Dengue virus infection during pregnancy is linked to adverse fetal outcomes, but the underlying mechanisms remain unclear. In Ifnar 1 ^−/− pregnant mice, we show that fetal growth restriction is driven by disruption of the maternal environment that impairs placental function. We identify syncytiotrophoblast damage and defective trophoblast fusion despite compensatory upregulation of pro-fusion genes. Placental metabolic analysis reveals a shift from oxidative phosphorylation to glycolysis, accompanied by reduced ATP production and impaired syncytialization. Inhibition of glycolysis in vivo partially restores placental structure and improves fetal outcomes, supporting a key role for metabolic reprogramming. Mechanistically, ROS activates HIF-1α and upregulates its downstream target LDHA, thereby linking metabolic reprogramming to placental dysfunction and highlighting this pathway as a potential therapeutic target. These findings reveal a mechanism underlying DENV-associated pregnancy complications and highlight metabolic pathways as potential therapeutic targets.

Extensive clinical cases and epidemiological analyses have indicated that symptomatic dengue virus (DENV) infection may lead to adverse outcomes during pregnancy; however, the precise mechanisms remain elusive. In our previous work, we demonstrated that DENV-2 infection induces intrauterine growth restriction by triggering neutrophil-mediated destruction of the placental vascular system in mice. However, the specific mechanisms driving neutrophil infiltration into the placenta remain unclear, especially given that the placenta constitutes a unique immune-privileged niche dedicated to maintaining maternal–fetal tolerance. In this study, we generated a time-resolved single-cell atlas of placentas from interferon-alpha/beta receptor-deficient mice infected with DENV-2. Integrated analysis of single-cell transcriptomes and single-nucleotide polymorphism sequencing revealed that infection primarily activated fetal-origin tissue-resident PF4 + macrophages. Activated fetal macrophages mediate preferential recruitment and the subsequent aberrant accumulation of neutrophils in the labyrinth zone through the secretion of CXCL2, which binds to neutrophil CXCR2. Notably, the recruited neutrophils further exhibited an enhanced self-recruitment phenotype. Blocking CXCL2-CXCR2 signaling effectively inhibited neutrophil recruitment, restored placental microvascular density, and alleviated fetal intrauterine growth restriction. This study revealed that the activation of placental tissue-resident macrophages is responsible for the aberrant neutrophil infiltration in the placenta following DENV-2 infection. Mechanistically, macrophages initiate the recruitment of self-amplifying neutrophils, and the CXCL2-CXCR2 signaling pathway plays a crucial role in this process. These results provide important information for the clinical development of therapeutic strategies against DENV-2-induced adverse pregnancy outcomes.

Sexual transmission of Zika virus (ZIKV) presents a distinct public health challenge, yet its kinetics and mechanisms remain poorly defined due to the lack of suitable immunocompetent animal models. Here, we established a mouse model of ZIKV sexual transmission using human STAT2 knock-in (hSTAT2 KI) mice. We show that infected males efficiently transmit ZIKV to females via mating. Beyond sperm, non-sperm components in semen substantially contributed to transmission. ZIKV sexual transmission was time-dependent, peaking early at 5 days post-infection (dpi) and declining rapidly thereafter, with transmission virtually ceasing by 40 dpi—a short transmission window that did not align with the prolonged presence of ZIKV RNA in sperm, suggesting clearance of infectious virus or rising antibody responses. Both African and Asian ZIKV strains displayed similar transmission capacities, indicating evolutionary conservation of this trait. In females, infection resulted in viremia and viral RNA detection in multiple organs of the reproductive tract, with predominant localization in the vagina, where it triggered localized lipid metabolic disturbances with minimal impact on upper tract tissues. Our model recapitulates key aspects of human ZIKV sexual transmission, offering a robust platform for mechanistic study and intervention development.

Recent studies have shown that dengue virus (DENV) infection during pregnancy is linked to adverse pregnancy outcomes, presenting an emerging clinical challenge with poorly understood pathogenic mechanisms. Uterine vascular remodeling, a critical determinant of successful pregnancy, is predominantly regulated by uterine natural killer (uNK) cells. In this study, we explored the effect of uNK cells on uterine injury in a DENV-induced adverse pregnancy mouse model. Transcriptomic analysis revealed significant dysregulation of uterine vascular remodeling and immune response-related gene expression after DENV-2 infection. Histopathological analysis revealed vascular remodeling disorders characterized by the preservation of vascular smooth muscle and a high level of tight junction protein in the vascular wall, accompanied by abnormal dilation of the lumen. These structural abnormalities coincided with a marked increase in uNK cell infiltration in the uterus. Moreover, the depletion of NK cells with anti-Asialo-GM1 mitigated uterine vascular damage and improved pregnancy outcomes. Single-cell RNA sequencing revealed a subpopulation of uNK cells (uNK2) that is characterized by upregulated expression of cytotoxicity-related genes. We further revealed that monocytes/macrophages (Mon_Macro) serve as intermediates in uNK2-mediated neutrophil (NE) recruitment. The SPP1–CD44 signaling axis is the dominant pathway governing uNK2-Mon_Macro crosstalk, and activated Mon_Macro2 can secrete chemokines such as CXCL2 to recruit NE. These findings provide valuable insights into the pathogenesis of DENV-induced uterine injury and facilitate the development of targeted therapeutic interventions for DENV-associated gestational disorders. IMPORTANCE: Dengue virus (DENV), a globally prevalent mosquito-borne virus, increasingly threatens maternal health because its infection is linked to adverse pregnancy outcomes. However, the mechanisms underlying DENV-induced damage to the uterus remain elusive. This study revealed impaired uterine vascular remodeling and abnormal vascular dilation followin g DENV-2 infection. Notably, uterine natural killer (uNK) cells are the critical pathological determinants of vascular injury in the uterus. Mechanistically, uNK cells may influence monocytes/macrophages (Mon_Macro) through SPP1–CD44 signaling to drive neutrophil infiltration, leading to uterine damage and adverse pregnancy outcomes. Successful depletion of NK cells reduces vascular damage and improves pregnancy outcomes. These findings provide critical mechanistic insights into DENV-2-induced uterine pathological lesions, fundamentally advancing our understanding of mosquito-borne virus-associated gestational diseases. The identified molecular cascades not only reveal the etiological landscape of pregnancy complications but also establish therapeutic measures for intervention strategies in mosquito-borne viral disorders.

Dengue 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.

Antibody-dependent enhancement (ADE) remains a major obstacle to dengue virus (DENV) vaccine development and continues to make safe, broadly protective vaccination a global health priority (Aggarwal et al., 2024; Halstead et al., 2014; Martinez et al., 2021; Paz-Bailey et al., 2024; Shepard et al., 2016). This study generated two DNA-based DENV vaccine constructs encoding premembrane (prM) and envelope (E) proteins: a secretion-deficient mutant (DNSP) and a full-length wild-type (WT) prM-E protein. Both vaccines elicited cell-mediated immune responses and protected mice against lethal DENV1 challenge. DNSP immunization induced minimal anti-prM-E antibody production but conferred durable protection, as evidenced by sustained T cell responses and challenge experiments performed one year after vaccination. In vitro DENV infection assays further showed that DNSP reduced K562 cell infection across all four DENV serotypes. In vivo DENV2 challenge also demonstrated that DNSP conferred effective protection against DENV2 infection. These findings identify secretion-deficient prM-E DNA vaccination as a promising strategy for strengthening cell-mediated antiviral immunity while limiting antibody induction. DNSP may therefore serve as a booster immunogen with potential to reduce ADE-associated risk during DENV vaccination.

Zika virus (ZIKV), transmitted by mosquitoes, poses a serious public health threat. Currently, precise quantitative diagnostic methods are lacking. Existing single-cell RNA-sequencing (scRNA-seq) techniques are challenged to detect ZIKV RNA due to its absence of a poly(A) tail, hindering the identification of infected cells. In this study, we developed a novel ZIKV-targeted scRNA-seq method that enables precise quantification of ZIKV RNA in individual cells. Immunocompetent suckling mice were intracerebrally infected with ZIKV to establish persistent infection in the brain. Samples were collected at 10-day post infection for ZIKV-targeted and 10× Genomics scRNA-seq analysis. Comparative analysis identified 17 distinct cell types in both ZIKV-infected and control suckling mouse brains, with significant changes in cell type distribution and proportion post-infection. The ZIKV-targeted scRNA-seq method suggested higher efficiency in capturing exogenous cells compared to the 10× Genomics scRNA-seq method. Both methods identified multiple endogenous and exogenous cell types susceptible to ZIKV, with peripheral blood-derived monocytes/macrophages (PBDMMs), neurons, and T cells as primary cell types expressing ZIKV RNA. IFA validated scRNA-seq findings, revealing that neurons and microglia could be infected by ZIKV, with significant reductions in their numbers post-infection. This study presents a novel ZIKV-targeted scRNA-seq that enables accurate quantification of ZIKV RNA within individual cells, identifies key susceptible cell types, and offers advantages in detecting exogenous cells, making it a scalable solution for providing valuable insights into therapeutic and vaccine development. IMPORTANCE: This study marks the first use of a scRNA-seq method tailored for ZIKV, allowing accurate measurement of ZIKV RNA in individual cells and identification of critical susceptible cell types. A comparative analysis with the 10× Genomics scRNA-seq method highlighted the advantages of ZIKV-targeted scRNA-seq in terms of accuracy and practicality, particularly its superior ability to capture exogenous cells. Beyond ZIKV, this method also helps establish precise quantification of viral RNA at the single-cell level for other viruses by designing target-specific beads based on conserved regions of the viral genome. This advancement is set to greatly enhance studying pathogenesis of ZIKV infection and then significantly contribute to improve prevention and research in therapeutics and vaccines.