Screening of Neutralizing Antibodies against FaeG Protein of Enterotoxigenic Escherichia coli.

The misuse of antibiotics in veterinary medicine presents significant challenges, highlighting the need for alternative therapeutic approaches such as antibody drugs. Therefore, it is necessary to explore the application of antibody drugs in veterinary settings to reduce economic losses and health risks. This study focused on targeting the F4ac subtype of the FaeG protein, a key adhesion factor in enterotoxigenic Escherichia coli (ETEC) infections in piglets. By utilizing formaldehyde-inactivated ETEC and a soluble recombinant FaeG (rFaeG) protein, an antibody library against the FaeG protein was established. The integration of fluorescence-activated cell sorting (FACS) and a eukaryotic expression vector containing murine IgG Fc fragments facilitated the screening of anti-rFaeG IgG monoclonal antibodies (mAbs). The results demonstrate that the variable regions of the screened antibodies could inhibit K88-type ETEC adhesion to IPEC-J2 cells. Furthermore, in vivo neutralization assays in mice showed a significant increase in survival rates and a reduction in intestinal inflammation. This research underscores the potential of antibody-based interventions in veterinary medicine, emphasizing the importance of further exploration in this field to address antibiotic resistance and improve animal health outcomes.

Associating prenatal antibiotics exposure with attention deficit hyperactivity disorder symptoms in preschool children: The role of maternal vitamin D.

BACKGROUND: The associations between prenatal antibiotics exposure and attention-deficit/hyperactivity disorder (ADHD) in preschoolers, and the role of maternal vitamin D in these associations, remain to be explored. OBJECTIVES: To evaluate the relationships between multiple maternal urinary antibiotics levels and preschoolers' ADHD symptoms, and to identify the potential modifying effects of maternal vitamin D. METHODS: Based on a prospective birth cohort, the present study included 2033 motherchild pairs. Maternal urine and serum samples were collected during all three trimesters to measure the urinary concentrations of 43 antibiotics (including two metabolites) and the serum vitamin D levels. The ADHD symptoms of preschoolers were assessed using the Diagnostic and Statistical Manual-oriented ADHD problems scale in the Achenbach Child Behavior Checklist. Multiple informant models in the form of logistic regression were conducted to investigate the associations between prenatal antibiotics exposure and preschooler ADHD symptoms, and these associations were stratified by child sex and maternal vitamin D status. RESULTS: Compared with the lowest tertile concentrations, maternal exposure to the middle tertile concentrations of doxycycline and human antibiotics/preferred as human antibiotics (HAs/PHAs), and the highest tertile concentrations of doxycycline during the first trimester were associated with an increased risk of ADHD symptoms in children. An increased risk of ADHD symptoms was observed in girls exposed to the highest tertile levels of sulfamethazine during the second trimester. Furthermore, pregnant women with vitamin D deficiency have a greater risk of ADHD symptoms in their offspring after exposure to doxycycline in the first trimester. CONCLUSIONS: Maternal exposure to doxycycline and HAs/PHAs during the first trimester increases the risk of ADHD symptoms in preschoolers. Mid-pregnancy sulfamethazine exposure increases the risk of ADHD symptoms in girls. Maternal vitamin D deficiency during pregnancy may exacerbate the adverse effects of doxycycline exposure on ADHD symptoms.

Exosomal CTHRC1 from cancer-associated fibroblasts facilitates endometrial cancer progression via ITGB3/FAK signaling pathway.

The emerging tumor microenvironment (TME) is a complex and constantly evolving entity. Cancer-associated fibroblasts (CAFs) are a vital component of the TME with diverse functions. They interact closely with cancer cells through reciprocal signaling and play a crucial role in tumor progression. Exosomes, which contain diverse biological information, are identified as an important mediator of cell-cell communication. This study aimed to investigate how CAF-derived exosomes promote metastasis of endometrial cancer (EC). Our findings revealed that CAF-derived exosomes significantly enhanced EC cell proliferation and migration compared to normal fibroblast-derived exosomes. Quantitative proteomics analysis of CAF/NF-derived exosomes demonstrated differential expression of CTHRC1, a protein overexpressed in multiple tumors, promoting cancer progression through enhanced cell migration and invasion. Exosomal overload of CTHRC1 significantly contributes to EC cell migration. Mechanically, we determined that ITGB3 was immunoprecipitated by CTHRC1 and phosphorylated FAK on Tyr397, which was important for exosomal CTHRC1 mediated migratory ability of EC cells. Overexpression of CTHRC1 in secreted exosomes promotes the metastatic ability of EC cells in mouse models and may be eliminated by Defactinib, an inhibitor of FAK Tyr397 phosphorylation. Moreover, overexpression of CTHRC1 was increased in EC patients, elevating with cancer progression, and correlated with negative tumor prognosis. Our results revealed that CAF mediated endometrial cancer progression is related to high levels of CTHRC1 and exosomal CTHRC1 derived from CAF may be a promising therapeutic strategy for metastatic endometrial cancer.

Semi-synthetic chondroitin sulfate CS-semi5 upregulates miR-122-5p, conferring a therapeutic effect on osteoarthritis via the p38/MMP13 pathway.

Osteoarthritis (OA) is an aging-associated disease characterized by joint stiffness pain and destroyed articular cartilage. Traditional treatments for OA are limited to alleviating various OA symptoms. There is a lack of drugs available in clinical practice that can truly repair cartilage damage. Here, we developed the chondroitin sulfate analog CS-semi5, semi-synthesized from chondroitin sulfate A. In vivo, CS-semi5 alleviated inflammation, provided analgesic effects, and protected cartilage in the modified Hulth OA rat model and papain-induced OA rat model. A bioinformatics analysis was performed on samples from OA patients and an exosome analysis on papain-induced OA rats, revealing miR-122-5p as the key regulator associated with CS-semi5 in OA treatment. Binding prediction revealed that miR-122-5p acted on the 3'-untranslated region of p38 mitogen-activated protein kinase, which was related to MMP13 regulation. Subsequent in vitro experiments revealed that CS-semi5 effectively reduced cartilage degeneration and maintained matrix homeostasis by inhibiting matrix breakdown through the miR-122-5p/p38/MMP13 axis, which was further validated in the articular cartilage of OA rats. This is the first study to investigate the semi-synthesized chondroitin sulfate CS-semi5, revealing its cartilage-protecting, anti-inflammatory, and analgesic properties that show promising therapeutic effects in OA via the miR-122-5p/p38/MMP13 pathway.

Enzymatic characterization and dominant sites of foot-and-mouth disease virus 2C protein.

Foot-and-mouth disease virus (FMDV) 2C protein is a conserved non-structural protein and crucial for replication of the virus. In this study, FMDV 2C protein was prepared and the enzymatic activities were investigated in detail. The protein could digest ssDNA or ssRNA into a small fragment at about 10 nt, indicating that the protein has nuclease activity. But it did not show digestion to blunt-end dsDNA or dsRNA. The nuclease activity of 2C protein could be inhibited in 2 mM Zn2+ or Ca2+ while enhanced by Mg2+ or Mn2+. FMDV 2C protein exhibited unwinding activity to all the three kinds of dsDNA and dsRNA (5' protruded, 3' protruded, and blunt-end). The unwinding velocity to 5' protruded dsRNA was higher than to the blunt-end dsRNA. 2C protein only showed unwinding activity in high concentration of Mg2+, but no unwinding activity in physiological concentrations of Mg2+ and Ca2+, as well as in cell lysate. The 2C protein could catalyze two structured ssRNA to form double strand, thus it was proved to have RNA chaperone activity. The Mg2+ and ATP in different concentrations did not show promotion to the RNA chaperone activity. Finally, six mutant proteins (K116A, D160A, D170A, N207A, R226A, and F316A) were constructed and the enzymatic activities were analyzed. All the six mutations reduced the ATPase activity, D170A and F361A could inactivate the nuclease activity, while the N207A and F316A could inactivate the helicase activity. Our study provides a comprehensive understanding of the enzymatic activities of FMDV 2C protein.

Knowledge graph-derived feed efficiency analysis via pig gut microbiota.

Feed efficiency (FE) is essential for pig production, has been reported to be partially explained by gut microbiota. Despite an extensive body of research literature to this topic, studies regarding the regulation of feed efficiency by gut microbiota remain fragmented and mostly confined to disorganized or semi-structured unrestricted texts. Meanwhile, structured databases for microbiota analysis are available, yet they often lack a comprehensive understanding of the associated biological processes. Therefore, we have devised an approach to construct a comprehensive knowledge graph by combining unstructured textual intelligence with structured database information and applied it to investigate the relationship between pig gut microbes and FE. Firstly, we created the pgmReading knowledge base and the domain ontology of pig gut microbiota by annotating, extracting, and integrating semantic information from 157 scientific publications. Secondly, we created the pgmPubtator by utilizing PubTator to expand the semantic information related to microbiota. Thirdly, we created the pgmDatabase by mapping and combining the ADDAGMA, gutMGene, and KEGG databases based on the ontology. These three knowledge bases were integrated to form the Pig Gut Microbial Knowledge Graph (PGMKG). Additionally, we created five biological query cases to validate the performance of PGMKG. These cases not only allow us to identify microbes with the most significant impact on FE but also provide insights into the metabolites produced by these microbes and the associated metabolic pathways. This study introduces PGMKG, mapping key microbes in pig feed efficiency and guiding microbiota-targeted optimization.

Oral administration of PEDV-dissolved Alg-CS gel induces high and sustained mucosal immunity in mice.

Porcine epidemic diarrhea (PED) is a serious disease in piglets that leads to high mortality. An effective measure that provides higher IgA levels in the intestine and milk is required to decrease losses. Porcine epidemic diarrhea virus (PEDV) was dissolved in calcium alginate (Alg) and combined with chitosan (CS) via electrostatic interactions between cationic chitosan and anionic alginate to create a porous gel (Alg-CS+PEDV). The gel was used to immunize mice orally or in combination with subcutaneous injections of inactivated PEDV vaccine. At 12 and 24 days after immunization, levels of IgA and IgG in Alg-CS+PEDV were higher than with normal PEDV oral administration. At 24 days after immunization, the concentration of IFN-γ in Alg-CS+PEDV was higher than with normal PEDV oral administration. Furthermore, oral administration combining subcutaneous immunization induced higher levels of IgG and IgA than oral administration alone. Our study provides a new method for the preparation and administration of oral vaccines to achieve enhanced mucosal immunity against PEDV.

Single-Cell Transcriptome Analysis of Small Cell Neuroendocrine Carcinoma of the Endometrium Reveals ISL1 as a Potential Biomarker for Diagnosis and Treatment.

BACKGROUND: As a dedifferentiated tumor, small cell endometrial neuroendocrine tumors (NETs) are rare and frequently diagnosed at an advanced stage with a poor prognosis. Current treatment recommendations are often extrapolated from histologically similar tumors in other sites or based on retrospective studies. The exploration for diagnostic and therapeutic markers in small cell NETs is of great significance. METHODS: In this study, we conducted single-cell RNA sequencing on a specimen obtained from a patient diagnosed with small cell endometrial neuroendocrine carcinoma (SCNEC) based on pathology. We revealed the cell map and intratumoral heterogeneity of the cancer cells through data analysis. Further, we validated the function of ISL LIM Homeobox 1 (ISL1) in vitro in an established neuroendocrine cell line. Finally, we examined the association between ISL1 and tumor staging in small cell lung cancer (SCLC) patient samples. RESULTS: We observed the significant upregulation of ISL1 expression in tumor cells that showed high expression of the neuroepithelial markers. Additionally, in vitro cell function experiments demonstrated that the high ISL1 expression group exhibited markedly higher cell proliferation and migration abilities compared to the low expression group. Finally, we showed that the expression level of ISL1 was correlated with SCLC stages. CONCLUSIONS: ISL1 protein in NETs shows promise as a potential biomarker for diagnosis and treatment.

Crystal structure of the ATPase domain of porcine circovirus type 2 Rep protein.

PCV2 belongs to the genus Circovirus in the family Circoviridae, whose genome is replicated by rolling circle replication (RCR). PCV2 Rep is a multifunctional enzyme that performs essential functions at multiple stages of viral replication. Rep is responsible for nicking and ligating single-stranded DNA and unwinding double-stranded DNA (dsDNA). However, the structure and function of the Rep are still poorly understood, which significantly impedes viral replication research. This study successfully resolved the structure of the PCV2 Rep ATPase domain (PRAD) using X-ray crystallography. Homologous structure search revealed that Rep belonged to the superfamily 3 (SF3) helicase, and multiple conserved residues were identified during sequence alignment with SF3 family members. Simultaneously, a hexameric PRAD model was generated for analysing characteristic structures and sites. Mutation of the conserved site and measurement of its activity showed that the hallmark motifs of the SF3 family influenced helicase activity by affecting ATPase activity and ß-hairpin just caused the loss of helicase activity. The structural and functional analyses of the PRAD provide valuable insights for future research on PCV2 replication and antiviral strategies.

Japanese encephalitis virus inhibits superinfection of Zika virus in cells by the NS2B protein.

Superinfection exclusion (SIE) is a phenomenon in which a preexisting infection prevents a secondary infection. SIE has been described for several flaviviruses, such as West Nile virus vs Nhumirim virus and Dengue virus vs yellow fever virus. Zika virus (ZIKV) is an emerging flavivirus posing threats to human health. The SIE between ZIKV and Japanese encephalitis virus (JEV) is investigated in this study. Our results demonstrate for the first time that JEV inhibits ZIKV infection in both mammalian and mosquito cells, whether co-infects or subsequently infects after ZIKV. The exclusion effect happens at the stage of ZIKV RNA replication. Further studies show that the expression of JEV NS2B protein is sufficient to inhibit the replication of ZIKV, and the outer membrane region of NS2B (46-103 aa) is responsible for this SIE. JEV infection and NS2B expression also inhibit the infection of the vesicular stomatitis virus. In summary, our study characterized a SIE caused by JEV NS2B. This may have potential applications in the prevention and treatment of ZIKV or other RNA viruses.IMPORTANCEThe reemerged Zika virus (ZIKV) has caused severe symptoms in humans and poses a continuous threat to public health. New vaccines or antiviral agents need to be developed to cope with possible future pandemics. In this study, we found that infection of Japanese encephalitis virus (JEV) or expression of NS2B protein well inhibited the replication of ZIKV. It is worth noting that both the P3 strain and vaccine strain SA14-14-2 of JEV exhibited significant inhibitory effects on ZIKV. Additionally, the JEV NS2B protein also had an inhibitory effect on vesicular stomatitis virus infection, suggesting that it may be a broad-spectrum antiviral factor. These findings provide a new way of thinking about the prevention and treatment of ZIKV.

Cellular nuclear-localized U2AF2 protein is hijacked by the flavivirus 3'UTR for viral replication complex formation and RNA synthesis.

Japanese encephalitis virus (JEV) is a zoonotic pathogen belonging to the Flavivirus genus, causing viral encephalitis in humans and reproductive failure in swine. The 3' untranslated region (3'UTR) of JEV contains highly conservative secondary structures required for viral translation, RNA synthesis, and pathogenicity. Identification of host factors interacting with JEV 3'UTR is crucial for elucidating the underlying mechanism of flavivirus replication and pathogenesis. In this study, U2 snRNP auxiliary factor 2 (U2AF2) was identified as a novel cellular protein that interacts with the JEV genomic 3'UTR (the SL-I, SL-II, SL-III, and DB region) via its 1 to 148 amino acids. JEV infection or JEV 3' UTR on its own triggered the nuclear-localized U2AF2 redistributed to the cytoplasm and colocalized with viral replication complex. U2AF2 also interacts with JEV NS3 and NS5 protein, the downregulation of U2AF2 nearly abolished the formation of flavivirus replication vesicles. The production of JEV protein, RNA, and viral titers were all increased by U2AF2 overexpression and decreased by knockdown. U2AF2 also functioned as a pro-viral factor for Zika virus (ZIKV) and West Nile virus (WNV), but not for vesicular stomatitis virus (VSV). Mechanically, U2AF2 facilitated the synthesis of both positive- and negative-strand flavivirus RNA without affecting viral attachment, internalization or release process. Collectively, our work paves the way for developing U2AF2 as a potential flavivirus therapeutic target.

A review on the recycling of spent lithium iron phosphate batteries.

Lithium iron phosphate (LFP) batteries have gained widespread recognition for their exceptional thermal stability, remarkable cycling performance, non-toxic attributes, and cost-effectiveness. However, the increased adoption of LFP batteries has led to a surge in spent LFP battery disposal. Improper handling of waste LFP batteries could result in adverse consequences, including environmental degradation and the mismanagement of valuable secondary resources. This paper presents a comprehensive examination of waste LFP battery treatment methods, encompassing a holistic analysis of their recycling impact across five dimensions: resources, energy, environment, economy, and society. The recycling of waste LFP batteries is not only crucial for reducing the environmental pollution caused by hazardous components but also enables the valuable components to be efficiently recycled, promoting resource utilization. This, in turn, benefits the sustainable development of the energy industry, contributes to economic gains, stimulates social development, and enhances employment rates. Therefore, the recycling of discarded LFP batteries is both essential and inevitable. In addition, the roles and responsibilities of various stakeholders, including governments, corporations, and communities, in the realm of waste LFP battery recycling are also scrutinized, underscoring their pivotal engagement and collaboration. Notably, this paper concentrates on surveying the current research status and technological advancements within the waste LFP battery lifecycle, and juxtaposes their respective merits and drawbacks, thus furnishing a comprehensive evaluation and foresight for future progress.

Crystal structure of the dimerized of porcine circovirus type II replication-related protein Rep'.

Porcine circovirus type 2 (PCV2) can cause porcine circovirus-associated disease (PCVAD), which causes significant economic losses to the global pig industry annually. There are no effective antiviral drugs used to control and treat PCV2, and prevention is mainly obtained through vaccination. PCV2 genome replicates through the rolling circle replication (RCR) mechanism involving Rep and Rep', so analyzing the holistic structure of Rep and Rep' will help us better understand the replication process of PCV2. However, there are no reports on the integral structure of Rep' and Rep, which seriously hinders the research of the viral replication. By using the x-ray diffraction method, the structure of the Rep' dimer was resolved by us in this study. Structural analysis revealed that Rep' is a dimer formed by the interaction of the C-terminal domain. The two Rep' form a positively charged groove, which may play an essential role in the viral binding of dsDNA. Together, this study help to understand the replication process of the virus and may also provide new insights into the development of antiviral drugs.

Loss-of-function mutations of SOX17 lead to YAP/TEAD activation-dependent malignant transformation in endometrial cancer.

Aberrant hyperactivation of the Hippo pathway effector YAP/TEAD complex causes tissue overgrowth and tumorigenesis in various cancers, including endometrial cancer (EC). The transcription factor SOX17 (SRY [sex-determining region Y]-box 17) is frequently mutated in EC; however, SOX17 mutations are rare in other cancer types. The molecular mechanisms underlying SOX17 mutation-induced EC tumorigenesis remain poorly understood. Here, we demonstrate that SOX17 serves as a tumor suppressor to restrict the proliferation, migration, invasion, and anchorage-independent growth of EC cells, partly by suppressing the transcriptional outputs of the Hippo-YAP/TEAD pathway. SOX17 binds to TEAD transcription factors through its HMG domain and attenuates the DNA-binding ability of TEAD. SOX17 loss by inactivating mutations leads to the malignant transformation of EC cells, which can be reversed by small-molecule inhibitors of YAP/TEAD or cabozantinib, an FDA-approved drug targeting the YAP/TEAD transcriptional target AXL. Our findings reveal novel molecular mechanisms underlying Hippo-YAP/TEAD pathway-driven EC tumorigenesis, and suggest potential therapeutic strategies targeting the Hippo-YAP/TEAD pathway in SOX17-mutated EC.

Tortuosity of branch retinal artery is more associated with the genesis and progress of diabetic retinopathy.

Objective: The purpose of this study is to investigate the potential of using the tortuosity of branch retinal artery as a more promising indicator for early detection and accurate assessment of diabetic retinopathy (DR). Design and method: The diagnoses, consisting of whether DR or not as well as DR severity, were given by ophthalmologists upon the assessment of those fundus images from 495 diabetic patients. Meanwhile, benefiting from those good contrast and high optical resolution fundus images taken by confocal scanning laser ophthalmoscope, the branch arteries, branch veins, main arteries and main veins in retina can be segmented independently, and the tortuosity values of them were further extracted to investigate their potential correlations with DR genesis and progress based on one-way ANOVA test. Results: For both two comparisons, i.e., between non-DR group and DR group as well as among groups with different DR severity levels, larger tortuosity increments were always observed in retinal arteries and the increments in branch retinal vessels were even larger. Furthermore, it was newly found that branch arterial tortuosity was significantly associated with both DR genesis (p=0.030) and DR progress (p<0.001). Conclusion: Based on this cohort study of 495 diabetic patients without DR and with different DR severity, the branch arterial tortuosity has been found to be more closely associated with DR genesis as well as DR progress. Therefore, the branch arterial tortuosity is expected to be a more direct and specific indicator for early detection of DR as well as accurate assessment of DR severity, which can further guide timely and rational management of DR to prevent from visual impairment or even blindness resulting from DR.

Therapeutic exosomes loaded with SERPINA5 attenuated endometrial cancer cell migration via the integrin ß1/FAK signaling pathway.

BACKGROUND: Metastasis is still the major cause of endometrial cancer (EC)-related death. Because of their biological function and regenerative properties, exosomes have been applied to therapeutic regimens. SERPINA5 expression is downregulated in several tumors and linked to tumor cell migration and invasion. However, the expression and biological functions of SERPINA5 in EC remain unclear. METHODS: The levels of SERPINA5 in plasma exosomes were determined with ELISAs. SERPINA5 expression in EC and its relationship with survival outcomes were analyzed using the TCGA database and clinical EC tissue samples. The effect of SERPINA5 overexpression or exosomal SERPINA5 on EC metastasis was examined by cell migration and invasion assays in vitro. Mechanistically, overexpression of SERPINA5 or high exosomal SERPINA5 levels mediated the regulation of the integrin ß1/FAK signaling pathway in EC cell lines. The therapeutic effect of exosomal SERPINA5 was determined with xenograft models. RESULTS: This study revealed that the level of exosomal SERPINA5 was increased in the circulating plasma of EC patients. In addition, the expression of SERPINA5 was decreased in EC patients with distant metastasis, and low expression of SERPINA5 indicated worse survival. In addition, SERPINA5 was elevated in normal tissues adjacent to EC tumors. Moreover, overexpression of SERPINA5 inhibited metastatic potential of EC cell lines in vitro. Furthermore, SERPINA5 loaded on secreted exosomes reduced the metastatic ability of EC cells. Notably, overexpression of SERPINA5 or high exosomal SERPINA5 levels suppressed EC metastatic potential by suppressing integrin ß1/FAK signaling pathway activation. Finally, exosomal SERPINA5 impeded tumor growth and metastasis in xenograft models. CONCLUSIONS: Our findings revealed that a low level of SERPINA5 expression indicated poor survival outcomes in EC and that exogenous SERPINA5 loading of exosomes may be a novel therapeutic strategy for metastatic EC.

To Identify Adenomatous Polyposis Coli Gene Mutation as a Predictive Marker of Endometrial Cancer Immunotherapy.

The adenomatous polyposis coli (APC) gene is the chromatin-remodeling-related gene and a typical tumor suppressor. Patients with a high expression of programmed death-ligand 1 (PD-L1) or a high level of tumor mutational burden (TMB) may benefit from immunotherapy in endometrial cancer (EC). This study aimed to demonstrate the role of APC in the diagnosis and immunotherapy treatment of EC. We performed an integrative analysis of a commercial panel including 520 cancer-related genes on 99 tumors from an endometrial cancer cohort in China and DNA-seq data from The Cancer Genome Atlas (TCGA) to identify new gene mutations as endometrial cancer immunotherapy markers. We found that the significant mutant genes that correlated with the PD-L1 expression and TMB were related to the chromatin state and generated a discovery set having 12 mutated genes, including the APC gene, which was identified as a new marker for immunotherapy. Further analysis revealed that tumors with the APC mutation had high TMB, increased expression of PD-L1, and increased lymphocytic infiltration. Next, we verified that APC has an inactive mutation in EC, which may affect the immune response, including PD-L1 expression, microsatellite instability, and lymphocytic infiltrate. Furthermore, patients with the APC mutation had longer overall survival. Our study demonstrates that APC could play an important role in enhancing the response to endometrial cancer treatment, particularly immunotherapy.

Identification of a small compound that specifically inhibits Zika virus in vitro and in vivo by targeting the NS2B-NS3 protease.

Zika virus (ZIKV) has rapid become a global threat, but no ZIKV-specific vaccines or drugs are currently available. In this study, inhibitors of ZIKV NS2B-NS3 protease were screened from a library containing 4,452 compound fragments. One of the compounds, 6-bromo-1,2-naphthalenedione, exhibited high specific inhibition against ZIKV NS2B-NS3 protease, but had no inhibitory effects against other viral proteases. A microscale thermophoresis (MST) assay confirmed that the compound bound to ZIKV NS2B-NS3 protein with a binding constant (Kd) of 12.26 µM. Indirect immunofluorescence assays, Western blots, and plaque assays indicated that the compound inhibited virus replication in cells. Virus titer was reduced by more than 75% when the compound was present at 1 µM. A time-of-addition assay showed that inhibition occurred at the virus replication stage, but not at the adsorption or invasion stages. The half cytotoxicity concentration (CC50) of the compound on HeLa, Vero, and BHK-21 cells were 445.44 µM, 123.87 µM, and 123.64 µM, respectively. In vivo tests using infected AG129 mice demonstrated that treatment with the compound reduced mortality by up to 60%. Mice treated with the compound showed a reduction in histopathological lesions in brain, testis, and ovary. Viral RNA, IL-1ß, and IL-6 mRNA levels decreased significantly in these tissues. In summary, this study has identified a small compound with high and specific inhibitory effects on ZIKV. The compound can be used as a therapeutic agent and is also an ideal starting point for drug optimization.

Japanese Encephalitis Virus NS1' Protein Interacts with Host CDK1 Protein to Regulate Antiviral Response.

Type I interferon (IFN-I) is a key component of the host innate immune system. To establish efficient replication, viruses have developed several strategies to escape from the host IFN response. Japanese encephalitis virus (JEV) NS1', a larger NS1-related protein, is known to inhibit the mitochondrial antiviral signaling (MAVS)-mediated IFN-ß induction by increasing the binding of transcription factors (CREB and c-Rel) to the microRNA 22 (miRNA-22) promoter. However, the mechanism by which NS1' induces the recruitment of CREB and c-Rel onto the miRNA-22 promoter is unknown. Here, we found that JEV NS1' protein interacts with the host cyclin-dependent kinase 1 (CDK1) protein. Mechanistically, NS1' interrupts the CDC25C phosphatase-mediated dephosphorylation of CDK1, which prolongs the phosphorylation status of CDK1 and leads to the inhibition of MAVS-mediated IFN-ß induction. Furthermore, the CREB phosphorylation and c-Rel activation through the IκBα phosphorylation were observed to be enhanced upon the augmentation of CDK1 phosphorylation by NS1'. The abrogation of CDK1 activity by a small-molecule inhibitor significantly suppressed the JEV replication in vitro and in vivo. Moreover, the administration of CDK1 inhibitor protected the wild-type mice from JEV-induced lethality but showed no effect on the MAVS-/- mice challenged with JEV. In conclusion, our study provides new insight into the mechanism of JEV immune evasion, which may lead to the development of novel therapeutic options to treat JEV infection. IMPORTANCE Japanese encephalitis virus (JEV) is the main cause of acute human encephalitis in Asia. The unavailability of specific treatment for Japanese encephalitis demands a better understanding of the basic cellular mechanisms that contribute to the onset of disease. The present study identifies a novel interaction between the JEV NS1' protein and the cellular CDK1 protein, which facilitates the JEV replication by dampening the cellular antiviral response. This study sheds light on a novel mechanism of JEV replication, and thus our findings could be employed for developing new therapies against JEV infection.