A Zika virus-specific IgM elicited in pregnancy exhibits ultrapotent neutralization.

Congenital Zika virus (ZIKV) infection results in neurodevelopmental deficits in up to 14% of infants born to ZIKV-infected mothers. Neutralizing antibodies are a critical component of protective immunity. Here, we demonstrate that plasma IgM contributes to ZIKV immunity in pregnancy, mediating neutralization up to 3 months post-symptoms. From a ZIKV-infected pregnant woman, we isolated a pentameric ZIKV-specific IgM (DH1017.IgM) that exhibited ultrapotent ZIKV neutralization dependent on the IgM isotype. DH1017.IgM targets an envelope dimer epitope within domain II. The epitope arrangement on the virion is compatible with concurrent engagement of all ten antigen-binding sites of DH1017.IgM, a solution not available to IgG. DH1017.IgM protected mice against viremia upon lethal ZIKV challenge more efficiently than when expressed as an IgG. Our findings identify a role for antibodies of the IgM isotype in protection against ZIKV and posit DH1017.IgM as a safe and effective candidate immunotherapeutic, particularly during pregnancy.

Structural constraints link differences in neutralization potency of human anti-Eastern equine encephalitis virus monoclonal antibodies.

Selection and development of monoclonal antibody (mAb) therapeutics against pathogenic viruses depends on certain functional characteristics. Neutralization potency, or the half-maximal inhibitory concentration (IC50) values, is an important characteristic of candidate therapeutic antibodies. Structural insights into the bases of neutralization potency differences between antiviral neutralizing mAbs are lacking. In this report, we present cryo-electron microscopy (EM) reconstructions of three anti-Eastern equine encephalitis virus (EEEV) neutralizing human mAbs targeting overlapping epitopes on the E2 protein, with greater than 20-fold differences in their respective IC50 values. From our structural and biophysical analyses, we identify several constraints that contribute to the observed differences in the neutralization potencies. Cryo-EM reconstructions of EEEV in complex with these Fab fragments reveal structural constraints that dictate intravirion or intervirion cross-linking of glycoprotein spikes by their IgG counterparts as a mechanism of neutralization. Additionally, we describe critical features for the recognition of EEEV by these mAbs including the epitope-paratope interaction surface, occupancy, and kinetic differences in on-rate for binding to the E2 protein. Each constraint contributes to the extent of EEEV inhibition for blockade of virus entry, fusion, and/or egress. These findings provide structural and biophysical insights into the differences in mechanism and neutralization potencies of these antibodies, which help inform rational design principles for candidate vaccines and therapeutic antibodies for all icosahedral viruses.

prM-reactive antibodies reveal a role for partially mature virions in dengue virus pathogenesis.

Cleavage of the flavivirus premembrane (prM) structural protein during maturation can be inefficient. The contribution of partially mature flavivirus virions that retain uncleaved prM to pathogenesis during primary infection is unknown. To investigate this question, we characterized the functional properties of newly-generated dengue virus (DENV) prM-reactive monoclonal antibodies (mAbs) in vitro and using a mouse model of DENV disease. Anti-prM mAbs neutralized DENV infection in a virion maturation state-dependent manner. Alanine scanning mutagenesis and cryoelectron microscopy of anti-prM mAbs in complex with immature DENV defined two modes of attachment to a single antigenic site. In vivo, passive transfer of intact anti-prM mAbs resulted in an antibody-dependent enhancement of disease. However, protection against DENV-induced lethality was observed when the transferred mAbs were genetically modified to inhibit their ability to interact with Fcγ receptors. These data establish that in addition to mature forms of the virus, partially mature infectious prM+ virions can also contribute to pathogenesis during primary DENV infections.

Cryo-EM structures of alphavirus conformational intermediates in low pH-triggered prefusion states.

Alphaviruses can cause severe human arthritis and encephalitis. During virus infection, structural changes of viral glycoproteins in the acidified endosome trigger virus-host membrane fusion for delivery of the capsid core and RNA genome into the cytosol to initiate virus translation and replication. However, mechanisms by which E1 and E2 glycoproteins rearrange in this process remain unknown. Here, we investigate prefusion cryoelectron microscopy (cryo-EM) structures of eastern equine encephalitis virus (EEEV) under acidic conditions. With models fitted into the low-pH cryo-EM maps, we suggest that E2 dissociates from E1, accompanied by a rotation (∼60°) of the E2-B domain (E2-B) to expose E1 fusion loops. Cryo-EM reconstructions of EEEV bound to a protective antibody at acidic and neutral pH suggest that stabilization of E2-B prevents dissociation of E2 from E1. These findings reveal conformational changes of the glycoprotein spikes in the acidified host endosome. Stabilization of E2-B may provide a strategy for antiviral agent development.

Requirement of a functional ion channel for Sindbis virus glycoprotein transport, CPV-II formation, and efficient virus budding.

Many viruses encode ion channel proteins that oligomerize to form hydrophilic pores in membranes of virus-infected cells and the viral membrane in some enveloped viruses. Alphavirus 6K, human immunodeficiency virus type 1 Vpu (HIV-Vpu), influenza A virus M2 (IAV-M2), and hepatitis C virus P7 (HCV-P7) are transmembrane ion channel proteins that play essential roles in virus assembly, budding, and entry. While the oligomeric structures and mechanisms of ion channel activity are well-established for M2 and P7, these remain unknown for 6K. Here we investigated the functional role of the ion channel activity of 6K in alphavirus assembly by utilizing a series of Sindbis virus (SINV) ion channel chimeras expressing the ion channel helix from Vpu or M2 or substituting the entire 6K protein with full-length P7, in cis. We demonstrate that the Vpu helix efficiently complements 6K, whereas M2 and P7 are less efficient. Our results indicate that while SINV is primarily insensitive to the M2 ion channel inhibitor amantadine, the Vpu inhibitor 5-N, N-Hexamethylene amiloride (HMA), significantly reduces SINV release, suggesting that the ion channel activity of 6K similar to Vpu, promotes virus budding. Using live-cell imaging of SINV with a miniSOG-tagged 6K and mCherry-tagged E2, we further demonstrate that 6K and E2 colocalize with the Golgi apparatus in the secretory pathway. To contextualize the localization of 6K in the Golgi, we analyzed cells infected with SINV and SINV-ion channel chimeras using transmission electron microscopy. Our results provide evidence for the first time for the functional role of 6K in type II cytopathic vacuoles (CPV-II) formation. We demonstrate that in the absence of 6K, CPV-II, which originates from the Golgi apparatus, is not detected in infected cells, with a concomitant reduction in the glycoprotein transport to the plasma membrane. Substituting a functional ion channel, M2 or Vpu localizing to Golgi, restores CPV-II production, whereas P7, retained in the ER, is inadequate to induce CPV-II formation. Altogether our results indicate that ion channel activity of 6K is required for the formation of CPV-II from the Golgi apparatus, promoting glycoprotein spike transport to the plasma membrane and efficient virus budding.

Structure of Usutu virus SAAR-1776 displays fusion loop asymmetry.

Usutu virus (USUV) is an emerging arbovirus in Europe that has been increasingly identified in asymptomatic humans and donated blood samples and is a cause of increased incidents of neuroinvasive human disease. Treatment or prevention options for USUV disease are currently nonexistent, the result of a lack of understanding of the fundamental elements of USUV pathogenesis. Here, we report two structures of the mature USUV virus, determined at a resolution of 2.4 Å, using single-particle cryogenic electron microscopy. Mature USUV is an icosahedral shell of 180 copies of envelope (E) and membrane (M) proteins arranged in the classic herringbone pattern. However, unlike previous reports of flavivirus structures, we observe virus subpopulations and differences in the fusion loop disulfide bond. Presence of a second, unique E glycosylation site could elucidate host interactions, contributing to the broad USUV tissue tropism. The structures provide a basis for exploring USUV interactions with glycosaminoglycans and lectins, the role of the RGD motif as a receptor, and the inability of West Nile virus therapeutic antibody E16 to neutralize the mature USUV strain SAAR-1776. Finally, we identify three lipid binding sites and predict key residues that likely participate in virus stability and flexibility during membrane fusion. Our findings provide a framework for the development of USUV therapeutics and expand the current knowledge base of flavivirus biology.

A Prototype-Pathogen Approach for the Development of Flavivirus Countermeasures.

Flaviviruses are a genus within the Flaviviridae family of positive-strand RNA viruses and are transmitted principally through mosquito and tick vectors. These viruses are responsible for hundreds of millions of human infections worldwide per year that result in a range of illnesses from self-limiting febrile syndromes to severe neurotropic and viscerotropic diseases and, in some cases, death. A vaccine against the prototype flavivirus, yellow fever virus, has been deployed for 85 years and is highly effective. While vaccines against some medically important flaviviruses are available, others have proven challenging to develop. The emergence and spread of flaviviruses, including dengue virus and Zika virus, demonstrate their pandemic potential. This review highlights the gaps in knowledge that need to be addressed to allow for the rapid development of vaccines against emerging flaviviruses in the future.

Cryo-EM structure of rhinovirus C15a bound to its cadherin-related protein 3 receptor.

Infection by Rhinovirus-C (RV-C), a species of Picornaviridae Enterovirus, is strongly associated with childhood asthma exacerbations. Cellular binding and entry by all RV-C, which trigger these episodes, is mediated by the first extracellular domain (EC1) of cadherin-related protein 3 (CDHR3), a surface cadherin-like protein expressed primarily on the apical surfaces of ciliated airway epithelial cells. Although recombinant EC1 is a potent inhibitor of viral infection, there is no molecular description of this protein or its binding site on RV-C. Here we present cryo-electron microscopy (EM) data resolving the EC1 and EC1+2 domains of human CDHR3 complexed with viral isolate C15a. Structure-suggested residues contributing to required interfaces on both EC1 and C15a were probed and identified by mutagenesis studies with four different RV-C genotypes. In contrast to most other rhinoviruses, which bind intercellular adhesion molecule 1 receptors via a capsid protein VP1-specific fivefold canyon feature, the CDHR3 EC1 contacts C15a, and presumably all RV-Cs, in a unique cohesive footprint near the threefold vertex, encompassing residues primarily from viral protein VP3, but also from VP1 and VP2. The EC1+2 footprint on C15a is similar to that of EC1 alone but shows that steric hindrance imposed by EC2 would likely prevent multiprotein binding by the native receptor at any singular threefold vertex. Definition of the molecular interface between the RV-Cs and their receptors provides new avenues that can be explored for potential antiviral therapies.

ApoA1 Neutralizes Proinflammatory Effects of Dengue Virus NS1 Protein and Modulates Viral Immune Evasion.

Dengue is a mosquito-borne infectious disease that is highly endemic in tropical and subtropical countries. Symptomatic patients can rapidly progress to severe conditions of hemorrhage, plasma extravasation, and hypovolemic shock, which leads to death. The blood tests of patients with severe dengue typically reveal low levels of high-density lipoprotein (HDL), which is responsible for reverse cholesterol transport (RCT) and regulation of the lipid composition in peripheral tissues. It is well known that dengue virus (DENV) depends on membrane cholesterol rafts to infect and to replicate in mammalian cells. Here, we describe the interaction of DENV nonstructural protein 1 (NS1) with apolipoprotein A1 (ApoA1), which is the major protein component of HDL. NS1 is secreted by infected cells and can be found circulating in the serum of patients with the onset of symptoms. NS1 concentrations in plasma are related to dengue severity, which is attributed to immune evasion and an acute inflammatory response. Our data show that the DENV NS1 protein induces an increase of lipid rafts in noninfected cell membranes and enhances further DENV infection. We also show that ApoA1-mediated lipid raft depletion inhibits DENV attachment to the cell surface. In addition, ApoA1 is able to neutralize NS1-induced cell activation and to prevent NS1-mediated enhancement of DENV infection. Furthermore, we demonstrate that the ApoA1 mimetic peptide 4F is also capable of mediating lipid raft depletion to control DENV infection. Taken together, our results suggest the potential of RCT-based therapies for dengue treatment. These results should motivate studies to assess the importance of RCT in DENV infection in vivo. IMPORTANCE DENV is one of the most relevant mosquito-transmitted viruses worldwide, infecting more than 390 million people every year and leading to more than 20 thousand deaths. Although a DENV vaccine has already been approved, its potential side effects have hampered its use in large-scale immunizations. Therefore, new treatment options are urgently needed to prevent disease worsening or to improve current clinical management of severe cases. In this study, we describe a new interaction of the NS1 protein, one of the major viral components, with a key component of HDL, ApoA1. This interaction seems to alter membrane susceptibility to virus infection and modulates the mechanisms triggered by DENV to evade the immune response. We also propose the use of a mimetic peptide named 4F, which was originally developed for atherosclerosis, as a potential therapy for relieving DENV symptoms.

Levels of Circulating NS1 Impact West Nile Virus Spread to the Brain.

Dengue virus (DENV) and West Nile virus (WNV) are arthropod-transmitted flaviviruses that cause systemic vascular leakage and encephalitis syndromes, respectively, in humans. However, the viral factors contributing to these specific clinical disorders are not completely understood. Flavivirus nonstructural protein 1 (NS1) is required for replication, expressed on the cell surface, and secreted as a soluble glycoprotein, reaching high levels in the blood of infected individuals. Extracellular DENV NS1 and WNV NS1 interact with host proteins and cells, have immune evasion functions, and promote endothelial dysfunction in a tissue-specific manner. To characterize how differences in DENV NS1 and WNV NS1 might function in pathogenesis, we generated WNV NS1 variants with substitutions corresponding to residues found in DENV NS1. We discovered that the substitution NS1-P101K led to reduced WNV infectivity in the brain and attenuated lethality in infected mice, although the virus replicated efficiently in cell culture and peripheral organs and bound at wild-type levels to brain endothelial cells and complement components. The P101K substitution resulted in reduced NS1 antigenemia in mice, and this was associated with reduced WNV spread to the brain. Because exogenous administration of NS1 protein rescued WNV brain infectivity in mice, we conclude that circulating WNV NS1 facilitates viral dissemination into the central nervous system and impacts disease outcomes. IMPORTANCE Flavivirus NS1 serves as an essential scaffolding molecule during virus replication but also is expressed on the cell surface and is secreted as a soluble glycoprotein that circulates in the blood of infected individuals. Although extracellular forms of NS1 are implicated in immune modulation and in promoting endothelial dysfunction at blood-tissue barriers, it has been challenging to study specific effects of NS1 on pathogenesis without disrupting its key role in virus replication. Here, we assessed WNV NS1 variants that do not affect virus replication and evaluated their effects on pathogenesis in mice. Our characterization of WNV NS1-P101K suggests that the levels of NS1 in the circulation facilitate WNV dissemination to the brain and affect disease outcomes. Our findings facilitate understanding of the role of NS1 during flavivirus infection and support antiviral strategies for targeting circulating forms of NS1.

Structural basis of a potent human monoclonal antibody against Zika virus targeting a quaternary epitope.

Zika virus (ZIKV) is a major human pathogen and member of the Flavivirus genus in the Flaviviridae family. In contrast to most other insect-transmitted flaviviruses, ZIKV also can be transmitted sexually and from mother to fetus in humans. During recent outbreaks, ZIKV infections have been linked to microcephaly, congenital disease, and Guillain-Barré syndrome. Neutralizing antibodies have potential as therapeutic agents. We report here a 4-Å-resolution cryo-electron microscopy structure of the ZIKV virion in complex with Fab fragments of the potently neutralizing human monoclonal antibody ZIKV-195. The footprint of the ZIKV-195 Fab fragment expands across two adjacent envelope (E) protein protomers. ZIKV neutralization by this antibody is presumably accomplished by cross-linking the E proteins, which likely prevents formation of E protein trimers required for fusion of the viral and cellular membranes. A single dose of ZIKV-195 administered 5 days after virus inoculation showed marked protection against lethality in a stringent mouse model of infection.

Flaviviruses have imperfect icosahedral symmetry.

Flaviviruses assemble initially in an immature, noninfectious state and undergo extensive conformational rearrangements to generate mature virus. Previous cryo-electron microscopy (cryo-EM) structural studies of flaviviruses assumed icosahedral symmetry and showed the concentric organization of the external glycoprotein shell, the lipid membrane, and the internal nucleocapsid core. We show here that when icosahedral symmetry constraints were excluded in calculating the cryo-EM reconstruction of an immature flavivirus, the nucleocapsid core was positioned asymmetrically with respect to the glycoprotein shell. The core was positioned closer to the lipid membrane at the proximal pole, and at the distal pole, the outer glycoprotein spikes and inner membrane leaflet were either perturbed or missing. In contrast, in the asymmetric reconstruction of a mature flavivirus, the core was positioned concentric with the glycoprotein shell. The deviations from icosahedral symmetry demonstrated that the core and glycoproteins have varied interactions, which likely promotes viral assembly and budding.

Zika virus can directly infect and damage the auditory and vestibular components of the embryonic chicken inner ear.

BACKGROUND: Sensorineural hearing loss is an understudied consequence of congenital Zika syndrome, and balance disorders are essentially unreported to date. Also lacking is information about the susceptibility and the pathogenesis of the developing inner ear following Zika virus (ZIKV) exposure. To address this, ZIKV was delivered directly into the otic cup/otocyst of chicken embryos and infection of inner ear tissues was evaluated using immunohistochemistry. RESULTS: After injections on embryonic days 2 to 5, ZIKV infection was observed in 90% of the samples harvested 2 to 8 days later; however, the degree of infection was highly variable across individuals. ZIKV was detected in all regions of the inner ear, associated ganglia, and in the surrounding periotic mesenchyme. Detection of virus peaked earlier in the ganglion and vestibular compartments, and later in the cochlea. ZIKV infection increased cell death robustly in the auditory ganglion, and modestly in the auditory sensory organ. Macrophage accumulation was found to overlap with dense viral infection in some tissues. Additionally, dysmorphogenesis of the semicircular canals and ganglion was observed for a subset of injection conditions. CONCLUSIONS: This article presents evidence of direct ZIKV infection of developing inner ear epithelium and shows previously unknown inner ear dysmorphogenesis phenotypes.

Dynamic remodeling of lipids coincides with dengue virus replication in the midgut of Aedes aegypti mosquitoes.

We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several flaviviruses transmitted by this vector, our results highlight biochemical choke points that could be targeted to disrupt transmission of multiple pathogens by these mosquitoes.

Zika Virus Structure, Maturation, and Receptors.

The emergence of Zika virus (ZIKV) as a major public health threat has focused research on understanding virus biology and developing a suite of strategies for disease intervention. Recent advances in cryoelectron microscopy have accelerated structure-function studies of flaviviruses and of ZIKV in particular. Structures of the mature and immature ZIKV have demonstrated its similarity with other known flaviviruses such as dengue and West Nile viruses. However, ZIKV's unique pathobiology demands an explanation of how its structure, although similar to its flavivirus relatives, is sufficiently unique to address questions of receptor specificity, transmission, and antigenicity. Progress in defining the immunodominant epitopes and how neutralizing antibodies bind to them will provide great insight as vaccines progress through clinical trials. Identification of host receptors will substantially illuminate the interesting ZIKV tropism and provide insights into pathogenesis. Although the answers to all of these questions are not yet available, rapid progress in combining structural biology with other techniques is revealing the similarities and the differences in virion structure and function between ZIKV and related flaviviruses.

Structure-guided insights on the role of NS1 in flavivirus infection.

We highlight the various domains of the flavivirus virulence factor NS1 and speculate on potential implications of the NS1 3D structure in understanding its role in flavivirus pathogenesis. Flavivirus non-structural protein 1 (NS1) is a virulence factor with dual functions in genome replication and immune evasion. Crystal structures of NS1, combined with reconstructions from electron microscopy (EM), provide insight into the architecture of dimeric NS1 on cell membranes and the assembly of a secreted hexameric NS1-lipid complex found in patient sera. Three structural domains of NS1 likely have distinct roles in membrane association, replication complex assembly, and immune system avoidance. A conserved hydrophobic inner face is sequestered either on the membrane or in the interior of the secreted hexamer and contains regions implicated in viral replication. The exposed variable outer face is presented to cellular and secreted components of the immune system in infected patients and contains candidate regions for immune system modulation. We anticipate that knowledge of the distinct NS1 domains and assembly will lead to advances in elucidating virus-host interactions mediated through NS1 and in dissecting the role of NS1 in viral genome replication.

Neutralizing antibodies can initiate genome release from human enterovirus 71.

Antibodies were prepared by immunizing mice with empty, immature particles of human enterovirus 71 (EV71), a picornavirus that causes severe neurological disease in young children. The capsid structure of these empty particles is different from that of the mature virus and is similar to "A" particles encountered when picornaviruses recognize a potential host cell before genome release. The monoclonal antibody E18, generated by this immunization, induced a conformational change when incubated at temperatures between 4 °C and 37 °C with mature virus, transforming infectious virions into A particles. The resultant loss of genome that was observed by cryo-EM and a fluorescent SYBR Green dye assay inactivated the virus, establishing the mechanism by which the virus is inactivated and demonstrating that the E18 antibody has potential as an anti-EV71 therapy. The antibody-mediated virus neutralization by the induction of genome release has not been previously demonstrated. Furthermore, the present results indicate that antibodies with genome-release activity could also be produced for other picornaviruses by immunization with immature particles.

Structure of acidic pH dengue virus showing the fusogenic glycoprotein trimers.

UNLABELLED: Flaviviruses undergo large conformational changes during their life cycle. Under acidic pH conditions, the mature virus forms transient fusogenic trimers of E glycoproteins that engage the lipid membrane in host cells to initiate viral fusion and nucleocapsid penetration into the cytoplasm. However, the dynamic nature of the fusogenic trimer has made the determination of its structure a challenge. Here we have used Fab fragments of the neutralizing antibody DV2-E104 to stop the conformational change of dengue virus at an intermediate stage of the fusion process. Using cryo-electron microscopy, we show that in this intermediate stage, the E glycoproteins form 60 trimers that are similar to the predicted "open" fusogenic trimer. IMPORTANCE: The structure of a dengue virus has been captured during the formation of fusogenic trimers. This was accomplished by binding Fab fragments of the neutralizing antibody DV2-E104 to the virus at neutral pH and then decreasing the pH to 5.5. These trimers had an "open" conformation, which is distinct from the "closed" conformation of postfusion trimers. Only two of the three E proteins within each spike are bound by a Fab molecule at domain III. Steric hindrance around the icosahedral 3-fold axes prevents binding of a Fab to the third domain III of each E protein spike. Binding of the DV2-E104 Fab fragments prevents domain III from rotating by about 130° to the postfusion orientation and thus precludes the stem region from "zipping" together the three E proteins along the domain II boundaries into the "closed" postfusion conformation, thus inhibiting fusion.

A molecularly cloned, live-attenuated japanese encephalitis vaccine SA14-14-2 virus: a conserved single amino acid in the ij Hairpin of the Viral E glycoprotein determines neurovirulence in mice.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes fatal neurological disease in humans, is one of the most important emerging pathogens of public health significance. JEV represents the JE serogroup, which also includes West Nile, Murray Valley encephalitis, and St. Louis encephalitis viruses. Within this serogroup, JEV is a vaccine-preventable pathogen, but the molecular basis of its neurovirulence remains unknown. Here, we constructed an infectious cDNA of the most widely used live-attenuated JE vaccine, SA14-14-2, and rescued from the cDNA a molecularly cloned virus, SA14-14-2MCV, which displayed in vitro growth properties and in vivo attenuation phenotypes identical to those of its parent, SA14-14-2. To elucidate the molecular mechanism of neurovirulence, we selected three independent, highly neurovirulent variants (LD50, <1.5 PFU) from SA14-14-2MCV (LD50, >1.5×105 PFU) by serial intracerebral passage in mice. Complete genome sequence comparison revealed a total of eight point mutations, with a common single G1708→A substitution replacing a Gly with Glu at position 244 of the viral E glycoprotein. Using our infectious SA14-14-2 cDNA technology, we showed that this single Gly-to-Glu change at E-244 is sufficient to confer lethal neurovirulence in mice, including rapid development of viral spread and tissue inflammation in the central nervous system. Comprehensive site-directed mutagenesis of E-244, coupled with homology-based structure modeling, demonstrated a novel essential regulatory role in JEV neurovirulence for E-244, within the ij hairpin of the E dimerization domain. In both mouse and human neuronal cells, we further showed that the E-244 mutation altered JEV infectivity in vitro, in direct correlation with the level of neurovirulence in vivo, but had no significant impact on viral RNA replication. Our results provide a crucial step toward developing novel therapeutic and preventive strategies against JEV and possibly other encephalitic flaviviruses.