A unified route for flavivirus structures uncovers essential pocket factors conserved across pathogenic viruses.

The epidemic emergence of relatively rare and geographically isolated flaviviruses adds to the ongoing disease burden of viruses such as dengue. Structural analysis is key to understand and combat these pathogens. Here, we present a chimeric platform based on an insect-specific flavivirus for the safe and rapid structural analysis of pathogenic viruses. We use this approach to resolve the architecture of two neurotropic viruses and a structure of dengue virus at 2.5 Å, the highest resolution for an enveloped virion. These reconstructions allow improved modelling of the stem region of the envelope protein, revealing two lipid-like ligands within highly conserved pockets. We show that these sites are essential for viral growth and important for viral maturation. These findings define a hallmark of flavivirus virions and a potential target for broad-spectrum antivirals and vaccine design. We anticipate the chimeric platform to be widely applicable for investigating flavivirus biology.

Flavivirus maturation leads to the formation of an occupied lipid pocket in the surface glycoproteins.

Flaviviruses such as Dengue (DENV) or Zika virus (ZIKV) assemble into an immature form within the endoplasmatic reticulum (ER), and are then processed by furin protease in the trans-Golgi. To better grasp maturation, we carry out cryo-EM reconstructions of immature Spondweni virus (SPOV), a human flavivirus of the same serogroup as ZIKV. By employing asymmetric localised reconstruction we push the resolution to 3.8 Å, enabling us to refine an atomic model which includes the crucial furin protease recognition site and a conserved Histidine pH-sensor. For direct comparison, we also solve structures of the mature forms of SPONV and DENV to 2.6 Å and 3.1 Å, respectively. We identify an ordered lipid that is present in only the mature forms of ZIKV, SPOV, and DENV and can bind as a consequence of rearranging amphipathic stem-helices of E during maturation. We propose a structural role for the pocket and suggest it stabilizes mature E.

Replication-Independent Generation and Morphological Analysis of Flavivirus Replication Organelles.

Positive-strand RNA viruses replicate in distinct membranous structures called replication organelles (ROs). Mechanistic studies of RO formation have been difficult because perturbations affecting viral replication have an impact on viral protein amounts, thus affecting RO biogenesis. Here, we present a detailed guide on how to use a replication-independent expression system, designated pIRO (plasmid-induced replication organelle formation), inducing bona fide flavivirus ROs in transfected cells. This will be useful for mechanistic studies of viral and cellular factors driving flavivirus RO biogenesis. For complete details on the use and execution of this protocol, please refer to Cerikan et al. (2020).

The continued threat of emerging flaviviruses.

Flaviviruses are vector-borne RNA viruses that can emerge unexpectedly in human populations and cause a spectrum of potentially severe diseases including hepatitis, vascular shock syndrome, encephalitis, acute flaccid paralysis, congenital abnormalities and fetal death. This epidemiological pattern has occurred numerous times during the last 70 years, including epidemics of dengue virus and West Nile virus, and the most recent explosive epidemic of Zika virus in the Americas. Flaviviruses are now globally distributed and infect up to 400 million people annually. Of significant concern, outbreaks of other less well-characterized flaviviruses have been reported in humans and animals in different regions of the world. The potential for these viruses to sustain epidemic transmission among humans is poorly understood. In this Review, we discuss the basic biology of flaviviruses, their infectious cycles, the diseases they cause and underlying host immune responses to infection. We describe flaviviruses that represent an established ongoing threat to global health and those that have recently emerged in new populations to cause significant disease. We also provide examples of lesser-known flaviviruses that circulate in restricted areas of the world but have the potential to emerge more broadly in human populations. Finally, we discuss how an understanding of the epidemiology, biology, structure and immunity of flaviviruses can inform the rapid development of countermeasures to treat or prevent human infections as they emerge.

Cryo-EM Studies of Virus-Antibody Immune Complexes.

Antibodies play critical roles in neutralizing viral infections and are increasingly used as therapeutic drugs and diagnostic tools. Structural studies on virus-antibody immune complexes are important for better understanding the molecular mechanisms of antibody-mediated neutralization and also provide valuable information for structure-based vaccine design. Cryo-electron microscopy (cryo-EM) has recently matured as a powerful structural technique for studying bio-macromolecular complexes. When combined with X-ray crystallography, cryo-EM provides a routine approach for structurally characterizing the immune complexes formed between icosahedral viruses and their antibodies. In this review, recent advances in the structural understanding of virus-antibody interactions are outlined for whole virions with icosahedral T = pseudo 3 (picornaviruses) and T = 3 (flaviviruses) architectures, focusing on the dynamic nature of viral shells in different functional states. Glycoprotein complexes from pleomorphic enveloped viruses are also discussed as immune complex antigens. Improving our understanding of viral epitope structures using virus-based platforms would provide a fundamental road map for future vaccine development.

A recombinant platform for flavivirus vaccines and diagnostics using chimeras of a new insect-specific virus.

Flaviviruses such as dengue, yellow fever, Zika, West Nile, and Japanese encephalitis virus present substantial global health burdens. New vaccines are being sought to address safety and manufacturing issues associated with current live attenuated vaccines. Here, we describe a new insect-specific flavivirus, Binjari virus, which was found to be remarkably tolerant for exchange of its structural protein genes (prME) with those of the aforementioned pathogenic vertebrate-infecting flaviviruses (VIFs). Chimeric BinJ/VIF-prME viruses remained replication defective in vertebrate cells but replicated with high efficiency in mosquito cells. Cryo-electron microscopy and monoclonal antibody binding studies illustrated that the chimeric BinJ/VIF-prME virus particles were structurally and immunologically similar to their parental VIFs. Pilot manufacturing in C6/36 cells suggests that high yields can be reached up to 109.5 cell culture infectious dose/ml or ≈7 mg/liter. BinJ/VIF-prME viruses showed utility in diagnostic (microsphere immunoassays and ELISAs using panels of human and equine sera) and vaccine applications (illustrating protection against Zika virus challenge in murine IFNAR-/- mouse models). BinJ/VIF-prME viruses thus represent a versatile, noninfectious (for vertebrate cells), high-yield technology for generating chimeric flavivirus particles with low biocontainment requirements.

Bagaza Virus in Himalayan Monal Pheasants, South Africa, 2016-2017.

Bagaza virus (BAGV) has not been reported in birds in South Africa since 1978. We used phylogenetic analysis and electron microscopy to identify BAGV as the likely etiology in neurologic disease and death in Himalayan monal pheasants in Pretoria, South Africa. Our results suggest circulation of BAGV in South Africa.

Systematic Analysis of Structure Similarity between Zika Virus and Other Flaviviruses.

Zika virus (ZIKV) infection has caused global concern because of its association with fetal microcephaly and serious neurological complications in adults since 2016. Currently, no specific anti-ZIKV therapy is available to control ZIKV infection. During the last couple of years, the intensive investigation of ZIKV structure has provided significant information for structure-based vaccine and drug design. In this review, we summarized the research progress on the structures of ZIKV and its component proteins. We analyzed the structure identity and the differences between ZIKV and other flaviviruses. This information is crucial to guiding structure-based anti-ZIKV inhibitors and vaccine discovery.

The Many Faces of a Dynamic Virion: Implications of Viral Breathing on Flavivirus Biology and Immunogenicity.

Flaviviruses are arthropod-borne RNA viruses that are a significant threat to global health due to their widespread distribution, ability to cause severe disease in humans, and capacity for explosive spread following introduction into new regions. Members of this genus include dengue, tick-borne encephalitis, yellow fever, and Zika viruses. Vaccination has been a highly successful means to control flaviviruses, and neutralizing antibodies are an important component of a protective immune response. High-resolution structures of flavivirus structural proteins and virions, alone and in complex with antibodies, provide a detailed understanding of viral fusion mechanisms and virus-antibody interactions. However, mounting evidence suggests these structures provide only a snapshot of an otherwise structurally dynamic virus particle. The contribution of the structural ensemble arising from viral breathing to the biology, antigenicity, and immunity of flaviviruses is discussed, including implications for the development and evaluation of flavivirus vaccines.

Ultrastructure of duck Tembusu virus observed by electron microscopy with negative staining.

Duck Tembusu virus (DTMUV) is a newly emerging enveloped flavivirus. This study shows the ultrastructure of DTMUV using viral purification, negative staining and electron microscopy. Electron microscopic examinations revealed mature DTMUV particles with 50 to 75 nm in diameter and typical enveloped flavivirus structure that consists of the internal nucleocapsid, an inner layer of lipid bilayer and an external layer of E glycoprotein ectodomain. Particles appear to be mostly spherical. In particular, RNA core is deep colored and dense, both capsid and lipid bilayer are clearly visible, the capsid forms regular hexagon, and E glycoprotein ectodomain forms a fringe instead of visible spikes. Thus, this report about the clear ultrastructure of the DTMUV particles will be the major driving forces behind structural biology of DTMUV.

Establishment of Baculovirus-Expressed VLPs Induced Syncytial Formation Assay for Flavivirus Antiviral Screening.

The baculovirus-insect cell expression system has been widely used for heterologous protein expression and virus-like particles (VLPs) expression. In this study, we established a new method for antiviral screening targeting to glycoprotein E of flaviviruses based on the baculovirus expression system. ZIKV is a mosquito-borne flavivirus and has posed great threat to the public health. It has been reported that ZIKV infection was associated with microcephaly and serious neurological complications. Our study showed that either ZIKV E or prME protein expressed in insect cells can form VLPs and induce membrane fusion between insect cells. Therefore, the E protein, which is responsible for receptor binding, attachment, and virus fusion during viral entry, achieved proper folding and retained its fusogenic ability in VLPs when expressed in this system. The syncytia in insect cells were significantly reduced by the anti-ZIKV-E specific polyclonal antibody in a dose-dependent manner. AMS, a thiol-conjugating reagent, was also shown to have an inhibitory effect on the E protein induced syncytia and inhibited ZIKV infection by blocking viral entry. Indeed the phenomenon of syncytial formation induced by E protein expressed VLPs in insect cells is common among flaviviruses, including Japanese encephalitis virus (JEV), Dengue virus type 2 (DENV-2), and tick-borne encephalitis virus (TBEV). This inhibition effect on syncytial formation can be developed as a novel, safe, and simple antiviral screening approach for inhibitory antibodies, peptides, or small molecules targeting to E protein of ZIKV and other flaviviruses.

The bright and the dark side of human antibody responses to flaviviruses: lessons for vaccine design.

Zika and dengue viruses belong to the Flavivirus genus, a close group of antigenically related viruses that cause significant arthropod-transmitted diseases throughout the globe. Although infection by a given flavivirus is thought to confer lifelong protection, some of the patient's antibodies cross-react with other flaviviruses without cross-neutralizing. The original antigenic sin phenomenon may amplify such antibodies upon subsequent heterologous flavivirus infection, potentially aggravating disease by antibody-dependent enhancement (ADE). The most striking example is provided by the four different dengue viruses, where infection by one serotype appears to predispose to more severe disease upon infection by a second one. A similar effect was postulated for sequential infections with Zika and dengue viruses. In this review, we analyze the molecular determinants of the dual antibody response to flavivirus infection or vaccination in humans. We highlight the role of conserved partially cryptic epitopes giving rise to cross-reacting and poorly neutralizing, ADE-prone antibodies. We end by proposing a strategy for developing an epitope-focused vaccine approach to avoid eliciting undesirable antibodies while focusing the immune system on producing protective antibodies only.

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.

Systematic analysis of protein identity between Zika virus and other arthropod-borne viruses.

OBJECTIVE: To analyse the proportions of protein identity between Zika virus and dengue, Japanese encephalitis, yellow fever, West Nile and chikungunya viruses as well as polymorphism between different Zika virus strains. METHODS: We used published protein sequences for the Zika virus and obtained protein sequences for the other viruses from the National Center for Biotechnology Information (NCBI) protein database or the NCBI virus variation resource. We used BLASTP to find regions of identity between viruses. We quantified the identity between the Zika virus and each of the other viruses, as well as within-Zika virus polymorphism for all amino acid k-mers across the proteome, with k ranging from 6 to 100. We assessed accessibility of protein fragments by calculating the solvent accessible surface area for the envelope and nonstructural-1 (NS1) proteins. FINDINGS: In total, we identified 294 Zika virus protein fragments with both low proportion of identity with other viruses and low levels of polymorphisms among Zika virus strains. The list includes protein fragments from all Zika virus proteins, except NS3. NS4A has the highest number (190 k-mers) of protein fragments on the list. CONCLUSION: We provide a candidate list of protein fragments that could be used when developing a sensitive and specific serological test to detect previous Zika virus infections.

Flavivirus structural heterogeneity: implications for cell entry.

The explosive spread of Zika virus is the most recent example of the threat imposed to human health by flaviviruses. High-resolution structures are available for several of these arthropod-borne viruses, revealing alternative icosahedral organizations of immature and mature virions. Incomplete proteolytic maturation, however, results in a cloud of highly heterogeneous mosaic particles. This heterogeneity is further expanded by a dynamic behavior of the viral envelope glycoproteins. The ensemble of heterogeneous and dynamic infectious particles circulating in infected hosts offers a range of alternative possible receptor interaction sites at their surfaces, potentially contributing to the broad flavivirus host-range and variation in tissue tropism. The potential synergy between heterogeneous particles in the circulating cloud thus provides an additional dimension to understand the unanticipated properties of Zika virus in its recent outbreaks.

Correlating Flavivirus virulence and levels of intrinsic disorder in shell proteins: protective roles vs. immune evasion.

Computational analyses revealed correlations between the intrinsic disorder propensity of shell proteins and case fatality rates (CFRs) among Flaviviruses and within at least two Flavivirus species, such as tick-borne encephalitis virus (TBEV) and dengue virus (DENV). The shell proteins analyzed in this study are capsid (C) and membrane (PrM, Pr, and M) proteins. The highest correlations can be found when regression analyses were conducted using Pr (Flavivirus: r(2) = 0.78, p < 0.01) or M (Flavivirus: r(2) = 0.91, p < 0.01) as an independent variable with C and CFR as co-explanatory and dependent variables, respectively. Interestingly, while predicted intrinsic disorder levels (PIDs) of both C and M are positively correlated with the virulence, the PIDs of Pr and CFR are negatively correlated. This is likely due to the fact that the Pr portion of PrM plays various roles in protecting the virion from damage, whereas M and C are assisted by greater potential in binding promiscuity as a result of greater disorder. The C protein of yellow fever virus (YFV), which is the most virulent virus in the sample, has the highest PID levels, whereas the second most virulent TBEV FE subtype has the second highest PID score due to its C protein, and the least virulent West Nile virus (WNV) has the least disordered C protein. This knowledge can be used while working on the development and identification of attenuated strains for vaccine. Curiously, unlike Flaviviruses, a disordered outer shell was described for hepatitis C virus (HCV), human immunodeficiency virus (HIV), and human simplex virus 2 (HSV-2), which currently have no effective vaccine.

Mercadeo Virus: A Novel Mosquito-Specific Flavivirus from Panama.

Viruses in the genus Flavivirus (family Flaviviridae) include many arthropod-borne viruses of public health and veterinary importance. However, during the past two decades an explosion of novel insect-specific flaviviruses (ISFs), some closely related to vertebrate pathogens, have been discovered. Although many flavivirus pathogens of vertebrates have been isolated from naturally infected mosquitoes in Panama, ISFs have not previously been reported from the country. This report describes the isolation and characterization of a novel ISF, tentatively named Mercadeo virus (MECDV), obtained from Culex spp. mosquitoes collected in Panama. Two MECDV isolates were sequenced and cluster phylogenetically with cell-fusing agent virus (CFAV) and Nakiwogo virus (NAKV) to form a distinct lineage within the insect-specific group of flaviviruses.

Wide detection of Aedes flavivirus in north-eastern Italy--a European hotspot of emerging mosquito-borne diseases.

The pattern of flavivirus infection in mosquitoes belonging to the genera Aedes and Culex collected in two regions of north-eastern Italy (Trentino and Veneto) was assessed. Mosquitoes were collected during 2012 and screened for flaviviruses using a generic reverse transcription-nested-PCR targeted on a region of the non-structural NS5 gene. The phylogenetic analysis was performed on a fragment of ~1000 bp. Virus isolation was attempted in C6/36 insect cell lines and the infected cell cultures were studied by electron microscopy. We detected a wide distribution of Aedes flavivirus (AeFV) in Aedes albopictus, with higher infection prevalence in Trentino than in Veneto. In Culex pipiens collected in Veneto, we detected a new sequence of an insect-specific flavivirus and one of Usutu virus. Interestingly, we detected AeFV in C. pipiens, for the first time to our knowledge, in both regions. Viral isolation in cell culture was successful for AeFV. AeFV sequences found in Veneto showed a high percentage of similarity to those detected in Trentino and to those previously reported in other areas of northern Italy. Co-infections with different flaviviruses were not detected.

Microscopy techniques in flavivirus research.

The Flavivirus genus is composed of many medically important viruses that cause high morbidity and mortality, which include Dengue and West Nile viruses. Various molecular and biochemical techniques have been developed in the endeavour to study flaviviruses. However, microscopy techniques still have irreplaceable roles in the identification of novel virus pathogens and characterization of morphological changes in virus-infected cells. Fluorescence microscopy contributes greatly in understanding the fundamental viral protein localizations and virus-host protein interactions during infection. Electron microscopy remains the gold standard for visualizing ultra-structural features of virus particles and infected cells. New imaging techniques and combinatory applications are continuously being developed to push the limit of resolution and extract more quantitative data. Currently, correlative live cell imaging and high resolution three-dimensional imaging have already been achieved through the tandem use of optical and electron microscopy in analyzing biological specimens. Microscopy techniques are also used to measure protein binding affinities and determine the mobility pattern of proteins in cells. This chapter will consolidate on the applications of various well-established microscopy techniques in flavivirus research, and discuss how recently developed microscopy techniques can potentially help advance our understanding in these membrane viruses.

First isolation of Aedes flavivirus in the Western Hemisphere and evidence of vertical transmission in the mosquito Aedes (Stegomyia) albopictus (Diptera: Culicidae).

We report here the first evidence of vertical transmission of Aedes flavivirus (AEFV) and its first isolation in the Western Hemisphere. AEFV strain SPFLD-MO-2011-MP6 was isolated in C6/36 cells from a pool of male Aedes albopictus mosquitoes that were reared to adults from larvae collected in southwest Missouri, USA, in 2011. Electron micrographs of the virus showed virions of approximately 45nm in diameter with morphological characteristics associated with flaviviruses. The genomic sequence demonstrated that AEFV-SPFLD-MO-2011-MP6 shares a high degree of nucleotide and amino acid sequence identity with the AEFV Narita-21 strain, isolated in Japan in 2003. Intracerebral inoculation of newborn mice with the virus failed to produce observable illness or death and the virus did not replicate in vertebrate cells, consistent with a lack of vertebrate host range.