Japanese encephalitis virus NS1 and NS1' protein disrupts the blood-brain barrier through macrophage migration inhibitory factor-mediated autophagy.

Flaviviruses in the Japanese encephalitis virus (JEV) serogroup, such as JEV, West Nile virus, and St. Louis encephalitis virus, can cause severe neurological diseases. The nonstructural protein 1 (NS1) is a multifunctional protein of flavivirus that can be secreted by infected cells and circulate in the host bloodstream. NS1' is an additional form of NS1 protein with 52 amino acids extension at its carboxy-terminal and is produced exclusively by flaviviruses in the JEV serogroup. In this study, we demonstrated that the secreted form of both NS1 and NS1' can disrupt the blood-brain barrier (BBB) of mice, with NS1' exhibiting a stronger effect. Using the in vitro BBB model, we found that treatment of soluble recombinant JEV NS1 or NS1' protein increases the permeability of human brain microvascular endothelial cells (hBMECs) and leads to the degradation of tight junction proteins through the autophagy-lysosomal pathway. Consistently, NS1' protein exhibited a more pronounced effect compared to NS1 in these cellular processes. Further research revealed that the increased expression of macrophage migration inhibitory factor (MIF) is responsible for triggering autophagy after NS1 or NS1' treatment in hBMECs. In addition, TLR4 and NF-κB signaling was found to be involved in the activation of MIF transcription. Moreover, administering the MIF inhibitor has been shown to decrease viral loads and mitigate inflammation in the brains of mice infected with JEV. This research offers a novel perspective on the pathogenesis of JEV. In addition, the stronger effect of NS1' on disrupting the BBB compared to NS1 enhances our understanding of the mechanism by which flaviviruses in the JEV serogroup exhibit neurotropism.IMPORTANCEJapanese encephalitis (JE) is a significant viral encephalitis worldwide, caused by the JE virus (JEV). In some patients, the virus cannot be cleared in time, leading to the breach of the blood-brain barrier (BBB) and invasion of the central nervous system. This invasion may result in cognitive impairment, behavioral disturbances, and even death in both humans and animals. However, the mechanism by which JEV crosses the BBB remains unclear. Previous studies have shown that the flavivirus NS1 protein plays an important role in causing endothelial dysfunction. The NS1' protein is an elongated form of NS1 protein that is particularly produced by flaviviruses in the JEV serogroup. This study revealed that both the secreted NS1 and NS1' of JEV can disrupt the BBB by breaking down tight junction proteins through the autophagy-lysosomal pathway, and NS1' is found to have a stronger effect compared to NS1 in this process. In addition, JEV NS1 and NS1' can stimulate the expression of MIF, which triggers autophagy via the ERK signaling pathway, leading to damage to BBB. Our findings reveal a new function of JEV NS1 and NS1' in the disruption of BBB, thereby providing the potential therapeutic target for JE.

Single-cell RNA sequencing reveals the immune features and viral tropism in the central nervous system of mice infected with Japanese encephalitis virus.

Japanese encephalitis virus (JEV) is a neurotropic pathogen that causes lethal encephalitis. The high susceptibility and massive proliferation of JEV in neurons lead to extensive neuronal damage and inflammation within the central nervous system. Despite extensive research on JEV pathogenesis, the effect of JEV on the cellular composition and viral tropism towards distinct neuronal subtypes in the brain is still not well comprehended. To address these issues, we performed single-cell RNA sequencing (scRNA-seq) on cells isolated from the JEV-highly infected regions of mouse brain. We obtained 88,000 single cells and identified 34 clusters representing 10 major cell types. The scRNA-seq results revealed an increasing amount of activated microglia cells and infiltrating immune cells, including monocytes & macrophages, T cells, and natural killer cells, which were associated with the severity of symptoms. Additionally, we observed enhanced communication between individual cells and significant ligand-receptor pairs related to tight junctions, chemokines and antigen-presenting molecules upon JEV infection, suggesting an upregulation of endothelial permeability, inflammation and antiviral response. Moreover, we identified that Baiap2-positive neurons were highly susceptible to JEV. Our findings provide valuable clues for understanding the mechanism of JEV induced neuro-damage and inflammation as well as developing therapies for Japanese encephalitis.

H3K27me3 of Rnf19a promotes neuroinflammatory response during Japanese encephalitis virus infection.

Histone methylation is an important epigenetic modification that affects various biological processes, including the inflammatory response. In this study, we found that infection with Japanese encephalitis virus (JEV) leads to an increase in H3K27me3 in BV2 microglial cell line, primary mouse microglia and mouse brain. Inhibition of H3K27me3 modification through EZH2 knockdown and treatment with EZH2 inhibitor significantly reduces the production of pro-inflammatory cytokines during JEV infection, which suggests that H3K27me3 modification plays a crucial role in the neuroinflammatory response caused by JEV infection. The chromatin immunoprecipitation-sequencing (ChIP-sequencing) assay revealed an increase in H3K27me3 modification of E3 ubiquitin ligases Rnf19a following JEV infection, which leads to downregulation of Rnf19a expression. Furthermore, the results showed that Rnf19a negatively regulates the neuroinflammatory response induced by JEV. This is achieved through the degradation of RIG-I by mediating its ubiquitination. In conclusion, our findings reveal a novel mechanism by which JEV triggers extensive neuroinflammation from an epigenetic perspective.

A novel small-molecule inhibitor of hepatitis C virus replication acts by suppressing signal transducer and activator of transcription 3.

OBJECTIVES: Hepatitis C virus (HCV) infects hepatocytes and causes liver damage. The aim of this study was to identify new classes of host-targeting anti-HCV compounds that may provide novel approaches for antiviral treatment regimens. METHODS: Cell culture-derived HCV (HCVcc), replicons and pseudoparticles were used in combination with high-throughput screening, reporter gene assays and cytotoxicity and signalling pathway analyses. RESULTS: A small-molecule inhibitor of HCV, N-(cyclopropyl(phenyl)methyl)thieno[2,3-d]pyrimidin-4-amine, designated IB-32, was identified by screening a compound library with a Jc1-luc HCVcc assay. By using various virus models, HCV replication was identified as the predominant step of IB-32's action. IB-32 inhibited HCVcc (genotype 2a) and HCV replicons (genotype 1b) at low nanomolar ranges (with IC50s of 40 ±â€Š8 and 100 ±â€Š15 nM, respectively). IB-32 was found to be non-toxic when tested against a panel of human cell lines in vitro at the effective antiviral dose. Mechanistically, IB-32 strongly inhibited STAT3 (Tyr705) phosphorylation, a necessary cellular factor for HCV replication and a pivotal therapeutic target for multiple cancers. Furthermore, the inhibition of HCV replication by IB-32 was augmented in cells with STAT3 knockdown. In contrast, the inhibitory effect of IB-32 was attenuated in cells overexpressing a constitutively active form of STAT3. CONCLUSION: The results presented here identify a promising STAT3-targeting anti-HCV therapeutic candidate. This novel small molecule could be further optimized and developed for use as both an antiviral and an anti-cancer drug.

High-throughput profiling of alpha interferon- and interleukin-28B-regulated microRNAs and identification of let-7s with anti-hepatitis C virus activity by targeting IGF2BP1.

Hepatitis C virus (HCV) infection is a major cause of severe liver disease. Interferon (IFN)/ribavirin treatment remains the standard therapeutic regimen for HCV infection in most countries. IFN-stimulated genes are believed to contribute to antiviral effects. However, emerging evidence suggests that microRNAs (miRNAs), a class of noncoding small RNAs, are involved in the control of viral infection. Here, we systematically profiled the hepatocyte expression of a set of 750 miRNAs in response to alpha interferon (IFN-α) and interleukin-28B (IL-28B) treatments. The anti-HCV activity of differentially expressed miRNAs was evaluated using cell culture-derived HCV in vitro. The results demonstrate that let-7b had a significant anti-HCV effect by inhibiting HCV replication and viral protein translation in human hepatoma cells. In particular, we show that the inhibition of let-7b attenuated the anti-HCV effects of IFN-α and IL-28B. Furthermore, we show that the host factor insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is a target of let-7b. IGF2BP1 was required for HCV replication, and its expression was downregulated by IFN-α and IL-28B. Deletion of the wild-type seed region of let-7b abolished its antiviral activity. Finally, we demonstrate that other let-7 family miRNAs were able to inhibit HCV and to suppress IGF2BP1 expression. In conclusion, we provide an example of a host miRNA regulated by type I and type III IFNs that inhibits HCV replication and infectivity by targeting host targets. These results highlight the important role of miRNAs in the host antiviral immune response and provide a novel candidate for anti-HCV therapy.

Screening and rational design of hepatitis C virus entry inhibitory peptides derived from GB virus A NS5A.

Chronic infection by hepatitis C virus (HCV) is a cause of the global burden of liver diseases. HCV entry into hepatocytes is a complicated and multistep process that represents a promising target for antiviral intervention. The recently reported amphipathic α-helical virucidal peptide (C5A) from the HCV NS5A protein suggests a new category of antiviral drug candidates. In this study, to identify C5A-like HCV inhibitors, synthetic peptides derived from the C5A-corresponding NS5 protein region of selected Flaviviridae viruses were evaluated for their anti-HCV activities. A peptide from GB virus A (GBV-A), but not other flaviviruses, demonstrated an inhibitory effect on HCV infection. Through a series of sequence optimizations and modifications of the peptide helicity and hydrophobicity, we obtained a peptide designated GBVA10-9 with highly potent anti-HCV activity. GBVA10-9 suppressed infection with both cell culture-derived and pseudotyped HCV in vitro, and the 50% cell culture inhibitory concentration ranged from 20 nM to 160 nM, depending on the genotypic origin of the envelope proteins. GBVA10-9 had no detectable effects on either HCV attachment to Huh7.5.1 cells or viral RNA replication. No virucidal activity was found with GBVA10-9, suggesting an action mechanism distinct from that of C5A. The inhibitory effect of GBVA10-9 appeared to occur at the postbinding step during viral entry. Taken together, the results with GBVA10-9 demonstrated a potent activity for blocking HCV entry that might be used in combination with other antivirals directly targeting virus-encoded enzymes. Furthermore, GBVA10-9 also provides a novel tool to dissect the detailed mechanisms of HCV entry.

Ferroptosis contributes to JEV-induced neuronal damage and neuroinflammation.

Ferroptosis is a newly discovered prototype of programmed cell death (PCD) driven by iron-dependent phospholipid peroxidation â€‹accumulation, and it has been linked to numerous organ injuries and degenerative pathologies. Although studies have shown that a variety of cell death processes contribute to JEV-induced neuroinflammation and neuronal injury, there is currently limited research on the specific involvement of ferroptosis. In this study, we explored the neuronal ferroptosis induced by JEV infection in vitro and in vivo. Our results indicated that JEV infection induces neuronal ferroptosis through inhibiting the function of the antioxidant system mediated by glutathione (GSH)/glutathione peroxidase 4 (GPX4), as well as by promoting lipid peroxidation mediated by yes-associated protein 1 (YAP1)/long-chain acyl-CoA synthetase 4 (ACSL4). Further analyses revealed that JEV E and prM proteins function as agonists, inducing ferroptosis. Moreover, we found that treatment with a ferroptosis inhibitor in JEV-infected mice reduces the viral titers and inflammation in the mouse brains, ultimately improving the survival rate of infected mice. In conclusion, our study unveils a critical role of ferroptosis in the pathogenesis of JEV, providing new ideas for the prevention and treatment of viral encephalitis.

A human claudin-1-derived peptide inhibits hepatitis C virus entry.

UNLABELLED: Hepatitis C virus (HCV) entry is a complicated process that requires multiple host factors, such as CD81, scavenger receptor BI, claudin-1 (CLDN1), and occludin. The interaction of virus and cellular entry factors represents a promising target for novel anti-HCV drug development. In this study, we sought to identify peptide inhibitors for HCV entry by screening a library of overlapping peptides covering the four above-mentioned entry factors. An 18-amino acid peptide (designated as CL58) that was derived from the CLDN1 intracellular and first transmembrane region inhibited both de novo and established HCV infection in vitro. Unlike previously reported peptides corresponding to CLDN1 extracellular loops, CL58 did not alter the normal distribution of CLDN1 and was not cytotoxic in vitro at concentrations nearly 100-fold higher than the effective antiviral dose. The inhibitory effect of CL58 appeared to occur at a late step during viral entry, presumably after initial binding. Finally, overexpressed CL58 was able to interact with HCV envelope proteins. CONCLUSION: We identified a novel CLDN1-derived peptide that inhibits HCV entry at a postbinding step. The findings expand our knowledge of the roles that CLDN1 play in HCV entry and highlight the potential for developing a new class of inhibitors targeting the viral entry process.

Development and evaluation of neutralizing antibodies for cross-protection against West Nile virus and Japanese encephalitis virus.

Background: West Nile virus is a severe zoonotic pathogen that can cause severe central nervous system symptoms in humans and horses, and is fatal for birds, chickens and other poultry. With no specific drugs or vaccines available, antibody-based therapy is a promising treatment. This study aims to develop neutralizing antibodies against West Nile virus and assess their cross-protective potential against Japanese encephalitis virus. Methods: Monoclonal antibodies against WNV and JEV were isolated by hybridoma technology. The therapeutic efficacy of these antibodies was evaluated using a mouse model, and a humanized version of the monoclonal antibody was generated for potential human application. Results: In this study, we generated eight monoclonal antibodies that exhibit neutralizing activity against WNV. Their therapeutic effects against WNV were validated both in vivo and in vitro. Among these antibodies, C9-G11-F3 also exhibited cross-protective activity against JEV. We also humanized the antibody to ensure that it could be used for WNV infection treatment in humans. Conclusion: This study highlights the importance of neutralizing antibodies as a promising approach for protection against West Nile virus infection and suggests their potential utility in the development of therapeutic interventions.

A novel triptolide analog downregulates NF-κB and induces mitochondrial apoptosis pathways in human pancreatic cancer.

Pancreatic cancer is the seventh leading cause of cancer-related death worldwide, and despite advancements in disease management, the 5 -year survival rate stands at only 12%. Triptolides have potent anti-tumor activity against different types of cancers, including pancreatic cancer, however poor solubility and toxicity limit their translation into clinical use. We synthesized a novel pro-drug of triptolide, (E)-19-[(1'-benzoyloxy-1'-phenyl)-methylidene]-Triptolide (CK21), which was formulated into an emulsion for in vitro and in vivo testing in rats and mice, and used human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids. A time-course transcriptomic profiling of tumor organoids treated with CK21 in vitro was conducted to define its mechanism of action, as well as transcriptomic profiling at a single time point post-CK21 administration in vivo. Intravenous administration of emulsified CK21 resulted in the stable release of triptolide, and potent anti-proliferative effects on human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids in vitro, and with minimal toxicity in vivo. Time course transcriptomic profiling of tumor organoids treated with CK21 in vitro revealed <10 differentially expressed genes (DEGs) at 3 hr and ~8,000 DEGs at 12 hr. Overall inhibition of general RNA transcription was observed, and Ingenuity pathway analysis together with functional cellular assays confirmed inhibition of the NF-κB pathway, increased oxidative phosphorylation and mitochondrial dysfunction, leading ultimately to increased reactive oxygen species (ROS) production, reduced B-cell-lymphoma protein 2 (BCL2) expression, and mitochondrial-mediated tumor cell apoptosis. Thus, CK21 is a novel pro-drug of triptolide that exerts potent anti-proliferative effects on human pancreatic tumors by inhibiting the NF-κB pathway, leading ultimately to mitochondrial-mediated tumor cell apoptosis.

Pancreatic cancer is a major cause of cancer-related deaths worldwide, with only 12% of patients surviving for five years after diagnosis. Individuals generally experience few symptoms of the disease in the early stages and are often diagnosed once the cancer has already spread to other parts of the body. By this point, options for treatment are limited. A molecule known as triptolide has been shown to kill breast, lung, pancreatic and other types of cancer cells. However, triptolide is toxic to humans and other animals, making it unsuitable for use in patients. One way to make drugs safer without compromising their beneficial effects is to modify their molecular structure. By formulating triptolide into an emulsion ­ a mixture of liquids allowing it to dissolve ­ Tian, Zhang et al. synthesized a new analogue called CK21. Experiments showed that CK21 inhibited the growth of human pancreatic cancer cells grown in a laboratory including cells grown in artificial organs similar to the pancreas, known as pancreatic tumor organoids. Furthermore, CK21 killed large tumors in mice pancreases with very few side effects, suggesting the structural modification of triptolide increased safety of the drug. To better understand how CK21 works, Tian, Zhang et al. examined the genes that were induced in the pancreatic tumor organoids at various time points after treatment with the drug. This revealed that CK21 switched off genes involved in the NF-κB cell signaling pathway, which regulates how cells grow and respond to stress. In turn, it triggered programmed cell death, killing the tumor cells in a controlled manner. The findings suggest that CK21 could be a promising candidate for treating pancreatic cancer. In the future, clinical trials will be required to establish whether CK21 is a safe and effective therapy for humans.

Lung cDC1 and cDC2 dendritic cells priming naive CD8+ T cells in situ prior to migration to draining lymph nodes.

The current paradigm indicates that naive T cells are primed in secondary lymphoid organs. Here, we present evidence that intranasal administration of peptide antigens appended to nanofibers primes naive CD8+ T cells in the lung independently and prior to priming in the draining mediastinal lymph node (MLN). Notably, comparable accumulation and transcriptomic responses of CD8+ T cells in lung and MLN are observed in both Batf3KO and wild-type (WT) mice, indicating that, while cDC1 dendritic cells (DCs) are the major subset for cross-presentation, cDC2 DCs alone are capable of cross-priming CD8+ T cells both in the lung and draining MLN. Transcription analyses reveal distinct transcriptional responses in lung cDC1 and cDC2 to intranasal nanofiber immunization. However, both DC subsets acquire shared transcriptional responses upon migration into the lymph node, thus uncovering a stepwise activation process of cDC1 and cDC2 toward their ability to cross-prime effector and functional memory CD8+ T cell responses.

Recombinant human interleukin 28B: anti-HCV potency, receptor usage and restricted cell-type responsiveness.

OBJECTIVES: Interleukin 28B (IL28B) genetic variation has been recently reported as a potent predictor of hepatitis C virus (HCV) response to interferon (IFN) therapy. The aim of this study was to produce recombinant human IL28B (rhIL28B) in yeast and explore the action mechanisms of rhIL28B as a novel anti-HCV agent. METHODS: A simple and efficient protocol for producing rhIL28B in the methylotrophic yeast Pichia pastoris was developed. The anti-HCV activity, induction of IFN-stimulated genes (ISGs), receptor usage and cellular responsiveness of rhIL28B were characterized. RESULTS: The yield of secreted rhIL28B was optimized to 200 mg/L, and soluble rhIL28B that was approximately 95% pure was achieved using a one-step ion-exchange purification procedure. rhIL28B inhibited HCV propagation in Huh7.5.1 cells with an IC(50) of 0.15 × 10(-3) mg/L. Treatment of hepatoma cells with rhIL28B resulted in the phosphorylation of STAT1 within 1 h and expression of ISGs. The HCV inhibitory effects of rhIL28B were antagonized by the antibody neutralization of receptors IL10R2 and IL28R1. The combination of rhIL28B and ribavirin synergistically inhibited HCV production in cell culture. Importantly, compared with the broad-spectrum activity of IFN-α, we demonstrated restricted cell-type responsiveness of rhIL28B in liver, lung and prostate cells. CONCLUSIONS: This study established an easy and highly efficient approach for the production of rhIL28B with potent in vitro antiviral activity and restricted cell tropism, and thus provides a novel antiviral candidate for improving the treatment of HCV-infected patients.

Protective Immune Responses Induced by an mRNA-LNP Vaccine Encoding prM-E Proteins against Japanese Encephalitis Virus Infection.

Japanese encephalitis virus (JEV) is an important zoonotic pathogen, which causes central nervous system symptoms in humans and reproductive disorders in swine. It has led to severe impacts on human health and the swine industry; however, there is no medicine available for treating yet. Therefore, vaccination is the best preventive measure for this disease. In the study, a modified mRNA vaccine expressing the prM and E proteins of the JEV P3 strain was manufactured, and a mouse model was used to assess its efficacy. The mRNA encoding prM and E proteins showed a high level of protein expression in vitro and were encapsulated into a lipid nanoparticle (LNP). Effective neutralizing antibodies and CD8+ T-lymphocytes-mediated immune responses were observed in vaccinated mice. Furthermore, the modified mRNA can protect mice from a lethal challenge with JEV and reduce neuroinflammation caused by JEV. This study provides a new option for the JE vaccine and lays a foundation for the subsequent development of a more efficient and safer JEV mRNA vaccine.

Rapid Detection of Genotype II African Swine Fever Virus Using CRISPR Cas13a-Based Lateral Flow Strip.

The African swine fever virus (ASFV) is a dsDNA virus that can cause serious, highly infectious, and fatal diseases in wild boars and domestic pigs. The ASFV has brought enormous economic loss to many countries, and no effective vaccine or treatment for the ASFV is currently available. Therefore, the on-site rapid and accurate detection of the ASFV is key to the timely implementation of control. The RNA-guided, RNA-targeting CRISPR effector CRISPR-associated 13 (Cas13a; previously known as C2c2) exhibits a "collateral effect" of promiscuous RNase activity upon the target recognition. The collateral cleavage activity of LwCas13a is activated to degrade the non-targeted RNA, when the crRNA of LwCas13a binds to the target RNA. In this study, we developed a rapid and sensitive ASFV detection method based on the collateral cleavage activity of LwCas13a, which combines recombinase-aided amplification (RAA) and a lateral flow strip (named CRISPR/Cas13a-LFD). The method was an isothermal detection at 37 °C, and the detection can be used for visual readout. The detection limit of the CRISPR/Cas13a-LFD was 101 copies/µL of p72 gene per reaction, and the detection process can be completed within an hour. The assay showed no cross-reactivity to eight other swine viruses, including classical swine fever virus (CSFV), and has a 100% coincidence rate with real-time PCR detection of the ASFV in 83 clinical samples. Overall, this method is sensitive, specific, and practicable onsite for the ASFV detection, showing a great application potential for monitoring the ASFV in the field.

Tissue specificity drives protective immunity against Staphylococcus aureus infection.

Infections caused by Staphylococcus aureus range from mild to severe and frequently recur. Emerging evidence suggests that the site and severity of infection drive the potency of elicited immune responses and susceptibility to recurrent infection. In this study, we used tractable mouse models of S. aureus skin infection (SSTI) and pneumonia to determine the relative magnitude of elicited protective immunity. Surprisingly, despite both SSTI and pneumonia eliciting antibody and local effector T cell responses, only SSTI elicited protective antibody and memory T cell responses and subsequent protection against secondary SSTI and pneumonia. The failure of pneumonia to elicit protective immunity was attributed to an inability of S. aureus pneumonia to elicit toxin-specific antibodies that confer protection during secondary infection and was associated with a failure to expand antigen-specific memory T cells. Taken together, these findings emphasize the importance of understanding protective immunity in the context of the tissue-specificity.

Development of efficient, sensitive, and specific detection method for Encephalomyocarditis virus based on CRISPR/Cas13a.

The Encephalomyocarditis virus (EMCV) is one of the major zoonosis pathogens, and it can cause acute myocarditis in young pigs or reproductive failure in sows. EMCV has been recognized as a pathogen infecting many species and causes substantial economic losses worldwide. Therefore, the development of a rapid, sensitive, and accurate detection of this virus is essential for the diagnosis and control of the EMCV-induced disease. The RNA-guiding, RNA-targeting CRISPR effector CRISPR/Cas13a (Cas13a, previously known as C2c2) exhibits a "collateral effect" of promiscuous RNase activity upon the target recognition. When the crRNA of LwCas13a binds to the target RNA, the collateral cleavage activity of LwCas13a is activated to degrade the non-targeted RNA. In this study, we developed an efficient, sensitive, and specific EMCV detection method based on the collateral cleavage activity of LwCas13a by combining recombinase-aided amplification (RAA) and a lateral flow strip. This method was an isothermal detection at 37 °C, which allowed visual observation by the naked eyes. We also optimized the reaction conditions of this method, and the detection result could be obtained within 60 min. The sensitivity of our method reached up to 101 copies/µL. Furthermore, no cross-reactions with other 8 major swine viruses were observed, indicating the excellent specificity of this method. At the same time, the assay had a 100 % coincidence rate with qPCR detection of the EMCV in 37 clinical samples. In addition, our developed method requires only 2-step operations and basic equipment, and thus it is simple and inexpensive. Overall, CRISPR/Cas13a-based detection has a great application potential for the EMCV detection.

Increased Cleavage of Japanese Encephalitis Virus prM Protein Promotes Viral Replication but Attenuates Virulence.

In flavivirus, the furin-mediated cleavage of prM is mandatory to produce infectious particles, and the immature particles containing uncleaved prM cannot undergo membrane fusion and release to the extracellular environment. However, the detailed relationship between viral replication or pathogenicity and furin in Japanese encephalitis virus (JEV) hasn't been clarified. Here, JEV with the mutations in furin cleavage sites and its nearby were constructed. Compared with WT virus, the mutant virus showed enhanced cleavage efficiency of prM protein and increased replication ability. Furthermore, we found that the mutations mainly promote genomic replication and assembly of JEV. However, the mutant formed smaller plaques than WT virus in plaque forming assay, indicating the lower cytopathogenicity of mutant virus. To assess the virulence of JEV mutant, an in vivo assay was performed using a mouse model. A higher survival rate and attenuated neuroinflammation were observed in JEV mutant-infected mice than those of WT-infected mice, suggesting the cleavage of prM by furin was closely related to viral virulence. These findings will provide new understanding on JEV pathogenesis and contribute to the development of novel JEV vaccines. IMPORTANCE Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis epidemics in Southeast Asia, affecting mostly children, with high morbidity and mortality. During the viral maturation process, prM is cleaved into M by the cellular endoprotease furin in the acidic secretory system. After cleavage of the prM protein, mature virions are exocytosed. Here, the mutant in furin cleavage sites and its nearby was constructed, and the results showed that the mutant virus with enhanced replication mainly occurred in the process of genomic replication and assembly. Meanwhile, the mutant showed an attenuated virulence than WT virus in vivo. Our study contributes to understanding the function of prM and M proteins and provides new clues for live vaccine designation for JEV.

Zika virus causes placental pyroptosis and associated adverse fetal outcomes by activating GSDME.

Zika virus (ZIKV) can be transmitted from mother to fetus during pregnancy, causing adverse fetal outcomes. Several studies have indicated that ZIKV can damage the fetal brain directly; however, whether the ZIKV-induced maternal placental injury contributes to adverse fetal outcomes is sparsely defined. Here, we demonstrated that ZIKV causes the pyroptosis of placental cells by activating the executor gasdermin E (GSDME) in vitro and in vivo. Mechanistically, TNF-α release is induced upon the recognition of viral genomic RNA by RIG-I, followed by activation of caspase-8 and caspase-3 to ultimately escalate the GSDME cleavage. Further analyses revealed that the ablation of GSDME or treatment with TNF-α receptor antagonist in ZIKV-infected pregnant mice attenuates placental pyroptosis, which consequently confers protection against adverse fetal outcomes. In conclusion, our study unveils a novel mechanism of ZIKV-induced adverse fetal outcomes via causing placental cell pyroptosis, which provides new clues for developing therapies for ZIKV-associated diseases.

Testosterone protects mice against zika virus infection and suppresses the inflammatory response in the brain.

Testosterone is essential to human growth and development as well as immune regulation. Zika virus (ZIKV), an emerging arbovirus associated with neurological complications including neuroinflammation, can also cause testicular damage and decrease testosterone secretion. However, whether the dysregulation of testosterone plays a role in the process of neuroinflammation during ZIKV pathogenesis is still unclear. In this study, we found that ZIKV infection caused testicular damage and decreased testosterone secretion in male mice, and testosterone supplementation after ZIKV infection reduced their mortality and attenuated the pathological symptoms. Further investigation revealed that testosterone treatment after ZIKV infection alleviated inflammation and nerve injury in the mouse brain. Additionally, reduced CD8+ T cell infiltration and interferon-gamma production were observed in brains of testosterone-treated mice. Overall, our results demonstrated that testosterone plays a protective role in ZIKV-infected mice, and thus it can be developed as a potential therapeutic drug against ZIKV infection.

Epidemiology and Comparative Analyses of the S Gene on Feline Coronavirus in Central China.

Feline coronavirus (FCoV) infections present as one of two forms: a mild or symptom-less enteric infection (FEC) and a fatal systemic disease termed feline infectious peritonitis (FIP). The lack of epidemiology of FCoV in central China and the reason why different symptoms are caused by viruses of the same serotype have motivated this investigation. Clinical data of 81 suspected FIP cases, 116 diarrhea cases and 174 healthy cases were collected from veterinary hospitals using body cavity effusion or fecal samples. Risk factors, sequence comparison and phylogenetic studies were performed. The results indicated that FIPV was distinguished from FECV in the average hydrophobicity of amino acids among the cleavage sites of furin, as well as the mutation sites 23,531 and 23,537. FIPV included a higher minimal R-X-X-R recognition motif of furin (41.94%) than did FECV (9.1%). The serotype of FCoV was insignificantly correlated with FIP, and the clade 1 and clade 2 strains that appeared were unique to central China. Thus, it is hypothesized that this, along with the latent variables of an antigenic epitope at positions 1058 and 1060, as well as mutations at the S1/S2 sites, are important factors affecting FCoV transmission and pathogenicity.