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-β 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-β 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.

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.

Japanese encephalitis virus (JEV) is a neurotropic flavivirus that invades the central nervous system and causes neuroinflammation and extensive neuronal cell death. Nucleotide-binding oligomerization domain 1 (NOD1) is a type of pattern recognition receptor that plays a regulatory role in both bacterial and nonbacterial infections. However, the role of NOD1 in JEV-induced neuroinflammation remains undisclosed. In this study, we evaluated the effect of NOD1 activation on the progression of JEV-induced neuroinflammation using a human astrocytic cell line and NOD1 knockout mice. The results showed that JEV infection upregulated the mRNA and protein expression of NOD1, ultimately leading to an enhanced neuroinflammatory response in vivo and in vitro. Inhibition of NOD1 in cultured cells or mice significantly abrogated the inflammatory response triggered by JEV infection. Moreover, compared to the wild-type mice, the NOD1 knockout mice showed resistance to JEV infection. Mechanistically, the NOD1-mediated neuroinflammatory response was found to be associated with increased expression or activation/phosphorylation of downstream receptor-interacting protein 2 (RIPK2), mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), Jun N-terminal protein kinase (JNK), and NF-κB signaling molecules. Thus, NOD1 targeting could be a therapeutic approach to treat Japanese encephalitis. IMPORTANCE Neuroinflammation is the main pathological manifestation of Japanese encephalitis (JE) and the most important factor leading to morbidity and death in humans and animals infected by JEV. An in-depth understanding of the basic mechanisms of neuroinflammation will contribute to research on JE treatment. This study proved that JEV infection can activate the NOD1-RIPK2 signal cascade to induce neuroinflammation through the proven downstream MAPK, ERK, JNK, and NF-κB signal pathway. Thus, our study unveiled NOD1 as a potential target for therapeutic intervention for JE.

Japanese encephalitis virus (JEV) is a typical mosquito-borne flavivirus that can cause central nervous system diseases in humans and animals. Host factors attempt to limit virus replication when the viruses invade the host by using various strategies for replication. It is essential to clarify the host factors that affect the life cycle of JEV and explore its underlying mechanism. Here, we found that USP1-associated factor 1 (UAF1; also known as WD repeat-containing protein 48) modulated JEV replication. We found that JEV propagation significantly increased in UAF1-depleted Huh7 cells. Moreover, we found that knockdown of UAF1 activated cell autophagic flux in further functional analysis. Subsequently, we demonstrated that autophagy can be induced by JEV, which promotes viral replication by inhibiting interferon-stimulated gene (ISG) expression in Huh7 cells. The knockdown of UAF1 reduced ISG expression during JEV infection. To explore the possible roles of autophagy in UAF1-mediated inhibition of JEV propagation, we knocked out ATG7 to generate autophagy-deficient cells and found that depletion of UAF1 failed to promote JEV replication in ATG7 knockout cells. Moreover, in ATG7-deficient Huh7 cells, interference with UAF1 expression did not lead to the induction of autophagy. Taken together, these findings indicate that UAF1 is a critical regulator of autophagy and reveal a mechanism by which UAF1 knockdown activates autophagy to promote JEV replication. IMPORTANCE Host factors play an essential role in virus replication and pathogenesis. Although UAF1 is well known to form complexes with ubiquitin-specific proteases, little is known about the function of the UAF1 protein itself. In this study, we confirmed that UAF1 is involved in JEV replication. Notably, we discovered a novel function for UAF1 in regulating autophagy. Furthermore, we demonstrated that UAF1 modulated JEV replication through its autophagy regulation. This study is the first description of the novel function of UAF1 in regulating autophagy, and it clarifies the underlying mechanism of the antiviral effect of UAF1 against JEV. These results provide a new mechanistic insight into the functional annotation of UAF1 and provide a potential target for increasing virus production during vaccine production.

Pigs are the amplifying hosts of Japanese encephalitis virus (JEV). Currently, the safe and effective live attenuated vaccine made of JEV strain SA14-14-2, which does not express NS1', is widely used in humans and domestic animals to prevent JEV infection. In this study, we constructed the NS1' expression recombinant virus (rA66G) through a single nucleotide mutation in NS2A of JEV strain SA14-14-2. Animal experiments showed that NS1' significantly enhanced JEV infection in pig central nervous system (CNS) and tonsil tissues. Pigs shed virus in oronasal secretions in the JEV rA66G virus inoculation group, indicating that NS1' may facilitate the horizontal transmission of JEV. Additionally, dendritic cells (DCs) and macrophages are the main target cells of JEV infection in pig tonsils, which are an important site of persistent JEV infection. The reduction of major histocompatibility complex class II (MHC II) and activation of inducible nitric oxide synthase (iNOS) in pig tonsils caused by viral infection may create a beneficial environment for persistent JEV infection. These results are of significance for JEV infection in pigs and lay the foundation for future studies of JEV persistent infection in pig tonsils. IMPORTANCE Pigs are amplification hosts for Japanese encephalitis virus (JEV). JEV can persist in the tonsils for months despite the presence of neutralizing antibodies. The present study shows that NS1' increases JEV infection in pig tonsils. In addition, DCs and macrophages in the tonsils are the target cells for JEV infection, and JEV NS1' promotes virus infection in DCs and macrophages. This study reveals a novel function of JEV NS1' protein and lays the foundation for future studies of JEV persistent infection in pig tonsils.

The nonstructural protein 4A (NS4A) of flaviviruses has been implicated as a "central organizer" of the membrane-bound replication complex during virus replication. However, its role in the host responses to virus infection is not understood. Using the yeast-two-hybrid library screen, we identified a multitude of host proteins interacting with the Japanese encephalitis virus (JEV) NS4A protein. Several of these interacting proteins are known to localize to the mitochondria. One of these proteins was PTEN-induced kinase 1 (PINK1), a serine/threonine-protein kinase known for its role in mitophagy. Here, we demonstrate the JEV-NS4A localization to the mitochondria and its interaction with PINK1 in Huh7 cells during JEV infection. The JEV-infected cells showed an enhanced mitophagy flux with a concomitant decline in the mitochondrial mass. We present data showing that JEV-NS4A alone was sufficient to induce mitophagy. Interference with mitochondrial fragmentation and mitophagy resulted in reduced virus propagation. Overall, our study provides the first evidence of mitochondrial quality control dysregulation during JEV infection, largely mediated by its NS4A protein. IMPORTANCE The JEV-infected mammalian cells show an enhanced mitophagy flux with a concomitant decline in the mitochondrial mass. We show that the NS4A protein of JEV localized to the mitochondria and interacted with PINK1 in Huh7 cells during infection with the virus and demonstrate that JEV-NS4A alone is sufficient to induce mitophagy. The study provides the first evidence of mitochondrial quality control dysregulation during JEV infection, largely mediated by its NS4A protein.

Japanese encephalitis virus (JEV) genotype V (GV) emerged in China in 2009, then South Korea, and has since spread to other regions in Asia and beyond, raising concern about its pathogenicity and the cross-protection offered by JEV vaccines against different genotypes. In this study, we replaced the structural proteins (C-prM-E) of an attenuated genotype I (GI) SD12-F120 strain with those of a virulent GV XZ0934 strain to construct a recombinant chimeric GI-GV JEV (JEV-GI/V) strain to determine the role of the structural proteins in virulence and cross-protection. The recombinant chimeric virus was highly neurovirulent and neuroinvasive in mice. This demonstrated the determinant role of the structural proteins in the virulence of the GV strain. Intracerebral or intraperitoneal inoculation of mice with JEV-GI/V-E5 harboring a combination of substitutions (N47K, L107F, E138K, H123R, and I176R) in E protein, but not mutants containing single substitution of these residues, resulted in decreased or disappeared mortality, suggesting that these residues synergistically, but not individually, played a role in determining the neurovirulence and neuroinvasiveness of the GV strain. Immunization of mice with attenuated strain JEV-GI/V-E5 provided complete protection and induced high neutralizing antibody titers against parental strain JEV-GI/V, but partial cross-protection and low cross-neutralizing antibodies titers against the heterologous GI and GIII strains in mice, suggesting the reduced cross-protection of JEV vaccines among different genotypes. Overall, these findings suggested the essential role of the structural proteins in determination of the virulence of GV strain, and highlighted the need for a novel vaccine against this newly emerged strain. IMPORTANCE The GV JEV showed an increase in epidemic areas, which exhibited higher pathogenicity in mice than the prevalent GI and GIII strains. We replaced a recombinant chimeric GI-GV JEV (JEV-GI/V) strain to determine the role of the structural proteins in virulence and cross-protection. It was found that the essential role of the structural proteins is to determinethe virulence of the GV strain. It is also suggested that there is reduced cross-protection of JEV vaccines among different genotypes, which provides basic data for subsequent JEV prevention, control, and new vaccine development.

The efficient and accurate diagnosis of dengue, a major mosquito-borne disease, is of primary importance for clinical care, surveillance, and outbreak control. The identification of specific dengue virus serotype 1 (DENV-1) to DENV-4 can help in understanding the transmission dynamics and spread of dengue disease. The four rapid low-resource serotype-specific dengue tests use a simple sample preparation reagent followed by reverse transcription-isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology. Results are obtained directly from clinical sample matrices in 35 min, requiring only a heating block and pipettes for liquid handling. In addition, we demonstrate that the rapid sample preparation step inactivates DENV, improving laboratory safety. Human plasma and serum were spiked with DENV, and DENV was detected with analytical sensitivities of 333 to 22,500 median tissue culture infectious doses (TCID50)/mL. The analytical sensitivities in blood were 94,000 to 333,000 TCID50/mL. Analytical specificity testing confirmed that each test could detect multiple serotype-specific strains but did not respond to strains of other serotypes, closely related flaviviruses, or chikungunya virus. Clinical testing on 80 human serum samples demonstrated test specificities of between 94 and 100%, with a DENV-2 test sensitivity of 100%, detecting down to 0.004 PFU/μL, similar to the sensitivity of the PCR test; the other DENV tests detected down to 0.03 to 10.9 PFU/μL. Collectively, our data suggest that some of our rapid dengue serotyping tests provide a potential alternative to conventional labor-intensive RT-quantitative PCR (RT-qPCR) detection, which requires expensive thermal cycling instrumentation, technical expertise, and prolonged testing times. Our tests provide performance and speed without compromising specificity in human plasma and serum and could become promising tools for the detection of high DENV loads in resource-limited settings. IMPORTANCE The efficient and accurate diagnosis of dengue, a major mosquito-borne disease, is of primary importance for clinical care, surveillance, and outbreak control. This study describes the evaluation of four rapid low-resource serotype-specific dengue tests for the detection of specific DENV serotypes in clinical sample matrices. The tests use a simple sample preparation reagent followed by reverse transcription-isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology. These tests have several advantages compared to RT-qPCR detection, such as a simple workflow, rapid sample processing and turnaround times (35 min from sample preparation to detection), minimal equipment needs, and improved laboratory safety through the inactivation of the virus during the sample preparation step. The low-resource formats of these rapid dengue serotyping tests have the potential to support effective dengue disease surveillance and enhance the diagnostic testing capacity in resource-limited countries with both endemic dengue and intense coronavirus disease 2019 (COVID-19) transmission.

Zika virus (ZIKV) is a mosquito-borne flavivirus that emerged in the Pacific islands in 2007 and spread to the Americas in 2015. The infection remains asymptomatic in most cases but can be associated with severe neurological disorders. Despite massive efforts, no specific drug or vaccine against ZIKV infection is available to date. Claudins are tight-junction proteins that favor the entry of several flaviviruses, including ZIKV. In this study, we identified two peptides derived from the N-terminal sequences of claudin-7 and claudin-1, named CL7.1 and CL1.1, respectively, that inhibited ZIKV infection in a panel of human cell lines. Using cell-to-cell fusion assays, we demonstrated that these peptides blocked the ZIKV E-mediated membrane fusion. A comparison of the antiviral efficacy of CL1.1 and CL7.1 pointed to the importance of the peptide amphipathicity. Electron microscopic analysis revealed that CL1.1 altered the ultrastructure of the viral particles likely by binding the virus lipid envelope. However, amphipathicity could not fully explain the antiviral activity of CL1.1. In silico docking simulations suggested that CL1.1 may also interact with the E protein, near its stem region. Overall, our data suggested that claudin-derived peptides inhibition may be linked to simultaneous interaction with the E protein and the viral lipid envelope. Finally, we found that CL1.1 also blocked infection by yellow fever and Japanese encephalitis viruses but not by HIV-1 or SARS-CoV-2. Our results provide a basis for the future development of therapeutics against a wide range of endemic and emerging flaviviruses. IMPORTANCE Zika virus (ZIKV) is a flavivirus transmitted by mosquito bites that have spread to the Pacific Islands and the Americas over the past decade. The infection remains asymptomatic in most cases but can cause severe neurological disorders. ZIKV is a major public health threat in areas of endemicity, and there is currently no specific antiviral drug or vaccine available. We identified two antiviral peptides deriving from the N-terminal sequences of claudin-7 and claudin-1 with the latter being the most effective. These peptides block the envelope-mediated membrane fusion. Our data suggested that the inhibition was likely achieved by simultaneously interacting with the viral lipid envelope and the E protein. The peptides also inhibited other flaviviruses. These results could provide the basis for the development of therapies that might target a wide array of flaviviruses from current epidemics and possibly future emergences.

The large (L) polymerase proteins of most nonsegmented, negative-stranded (NNS) RNA viruses have conserved methyltransferase motifs, (G)-G-G-D and K-D-K-E, which are important for the stabilization and translation of mRNA. However, the function of the (G)-G-G-D and K-D-K-E motifs in the NNS RNA virus Newcastle disease virus (NDV) remains unclear. We observed G-G-D and K-D-K-E motifs in all NDV genotypes. By using the infection cloning system of NDV rSG10 strain, recombinant NDVs with a single amino acid mutated to alanine in one motif (G-G-D or K-D-K-E) were rescued. The intracerebral pathogenicity index and mean death time assay results revealed that the G-G-D motif and K-D-K-E motif attenuate the virulence of NDV to various degrees. The replication, transcription, and translation levels of the K-D-K-E motif-mutant strains were significantly higher than those of wild-type virus owing to their altered regulation of the affinity between nucleocapsid protein and eukaryotic translation initiation factor 4E. When the infection dose was changed from a multiplicity of infection (MOI) of 10 to an MOI of 0.01, the cell-to-cell spread abilities of G-G-D- and K-D-K-E-mutant strains were reduced, according to plaque assay and dynamic indirect immunofluorescence assay results. Finally, we found that NDV strains with G-G-D or K-D-K-E motif mutations had less pathogenicity in 3-week-old specific-pathogen-free chickens than wild-type NDV. Therefore, these methyltransferase motifs can affect virulence by regulating the translation and cell-to-cell spread abilities of NDV. This work provides a feasible approach for generating vaccine candidates for viruses with methyltransferase motifs. IMPORTANCE Newcastle disease virus (NDV) is an important pathogen that is widespread globally. Research on its pathogenic mechanism is an important means of improving prevention and control efforts. Our study found that a deficiency in its methyltransferase motifs (G-G-D and K-D-K-E motifs) can attenuate NDV and revealed the molecular mechanism by which these motifs affect pathogenicity, which provides a new direction for the development of NDV vaccines. In addition to the (G)-G-G-D and K-D-K-E motifs of many nonsegmented, negative-stranded RNA viruses, similar motifs have been found in dengue virus, Zika virus, Japanese encephalitis virus (JEV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This suggests that such motifs may be present in more viruses. Our finding also provides a molecular basis for the discovery and functional study of (G)-G-G-D and K-D-K-E motifs of other viruses.

Quick and accurate detection of neutralizing antibodies (nAbs) against yellow fever is essential in serodiagnosis during outbreaks for surveillance and to evaluate vaccine efficacy in population-wide studies. All of this requires serological assays that can process a large number of samples in a highly standardized format. Albeit being laborious, time-consuming, and limited in throughput, the classical plaque reduction neutralization test (PRNT) is still considered the gold standard for the detection and quantification of nAbs due to its sensitivity and specificity. Here, we report the development of an alternative fluorescence-based serological assay (SNTFLUO) with an equally high sensitivity and specificity that is fit for high-throughput testing with the potential for automation. Finally, our novel SNTFLUO was cross-validated in several reference laboratories and against international WHO standards, showing its potential to be implemented in clinical use. SNTFLUO assays with similar performance are available for the Japanese encephalitis, Zika, and dengue viruses amenable to differential diagnostics. IMPORTANCE Fast and accurate detection of neutralizing antibodies (nAbs) against yellow fever virus (YFV) is key in yellow fever serodiagnosis, outbreak surveillance, and monitoring of vaccine efficacy. Although classical PRNT remains the gold standard for measuring YFV nAbs, this methodology suffers from inherent limitations such as low throughput and overall high labor intensity. We present a novel fluorescence-based serum neutralization test (SNTFLUO) with equally high sensitivity and specificity that is fit for processing a large number of samples in a highly standardized manner and has the potential to be implemented for clinical use. In addition, we present SNTFLUO assays with similar performance for Japanese encephalitis, Zika, and dengue viruses, opening new avenues for differential diagnostics.

Flaviviruses are important human pathogens worldwide. Diagnostic testing for these viruses is difficult because many of the pathogens require specialized biocontainment. To address this issue, we generated 39 virus-like particle (VLP)- and nonstructural protein 1 (NS1)-secreting stable cell lines in HEK-293 cells of 13 different flaviviruses, including dengue, yellow fever, Japanese encephalitis, West Nile, St. Louis encephalitis, Zika, Rocio, Ilheus, Usutu, and Powassan viruses. Antigen secretion was stable for at least 10 cell passages, as measured by enzyme-linked immunosorbent assays and immunofluorescence assays. Thirty-five cell lines (90%) had stable antigen expression over 10 passages, with three of these cell lines (7%) increasing in antigen expression and one cell line (3%) decreasing in antigen expression. Antigen secretion in the HEK-293 cell lines was higher than in previously developed COS-1 cell line counterparts. These antigens can replace current antigens derived from live or inactivated virus for safer use in diagnostic testing. IMPORTANCE Serological diagnostic testing for flaviviral infections is hindered by the need for specialized biocontainment for preparation of reagents and assay implementation. The use of previously developed COS-1 cell lines secreting noninfectious recombinant viral antigen is limited due to diminished antigen secretion over time. Here, we describe the generation of 39 flaviviral virus-like particle (VLP)- and nonstructural protein 1 (NS1)-secreting stable cell lines in HEK-293 cells representing 13 medically important flaviviruses. Antigen production was more stable and statistically higher in these newly developed cell lines than in their COS-1 cell line counterparts. The use of these cell lines for production of flaviviral antigens will expand serological diagnostic testing of flaviviruses worldwide.