Intrinsic disorder in flaviviral capsid proteins and its role in pathogenesis.

A high level of disorder in many viral proteins is a direct consequence of their small genomes, which makes interaction with multiple binding partners a necessity for infection and pathogenicity. A segment of the flaviviral capsid protein (C), also known as the molecular recognition feature (MoRF), undergoes a disorder-toorder transition upon binding to several protein partners. To understand their role in pathogenesis, MoRFs were identified and their occurrence across different flaviviral capsids were studied. Despite lack of sequence similarities, docking studies of Cs with the host proteins indicate conserved interactions involving MoRFs across members of phylogenetic subclades. Additionally, it was observed from the protein-protein networks that some MoRFs preferentially bind proteins that are involved in specialized functions such as ribosome biogenesis. The findings point to the importance of MoRFs in the flaviviral life cycle, with important consequences for disease progression and suppression of the host immune system. Potentially, they might have impacted the way flaviviruses evolved to infect varied hosts using multiple vectors.

Advancements in Research on Duck Tembusu Virus Infections.

Duck Tembusu Virus (DTMUV) is a pathogen of the Flaviviridae family that causes infections in poultry, leading to significant economic losses in the duck farming industry in recent years. Ducks infected with this virus exhibit clinical symptoms such as decreased egg production and neurological disorders, along with serious consequences such as ovarian hemorrhage, organ enlargement, and necrosis. Variations in morbidity and mortality rates exist across different age groups of ducks. It is worth noting that DTMUV is not limited to ducks alone; it can also spread to other poultry such as chickens and geese, and antibodies related to DTMUV have even been found in duck farm workers, suggesting a potential risk of zoonotic transmission. This article provides a detailed overview of DTMUV research, delving into its genomic characteristics, vaccines, and the interplay with host immune responses. These in-depth research findings contribute to a more comprehensive understanding of the virus's transmission mechanism and pathogenic process, offering crucial scientific support for epidemic prevention and control.

Circulation of West Nile Virus and Usutu Virus in Europe: Overview and Challenges.

West Nile Virus (WNV) and Usutu Virus (USUV) are both neurotropic mosquito-borne viruses belonging to the Flaviviridae family. These closely related viruses mainly follow an enzootic cycle involving mosquitoes as vectors and birds as amplifying hosts, but humans and other mammals can also be infected through mosquito bites. WNV was first identified in Uganda in 1937 and has since spread globally, notably in Europe, causing periodic outbreaks associated with severe cases of neuroinvasive diseases such as meningitis and encephalitis. USUV was initially isolated in 1959 in Swaziland and has also spread to Europe, primarily affecting birds and having a limited impact on human health. There has been a recent expansion of these viruses' geographic range in Europe, facilitated by factors such as climate change, leading to increased human exposure. While sharing similar biological traits, ecology, and epidemiology, there are significant distinctions in their pathogenicity and their impact on both human and animal health. While WNV has been more extensively studied and is a significant public health concern in many regions, USUV has recently been gaining attention due to its emergence in Europe and the diversity of its circulating lineages. Understanding the pathophysiology, ecology, and transmission dynamics of these viruses is important to the implementation of effective surveillance and control measures. This perspective provides a brief overview of the current situation of these two viruses in Europe and outlines the significant challenges that need to be addressed in the coming years.

Indirect interaction between an endemic and an invading pathogen: A case study of Plasmodium and Usutu virus dynamics in a shared bird host population.

Infectious disease agents can influence each other's dynamics in shared host populations. We consider such influence for two mosquito-borne infections where one pathogen is endemic at the time that a second pathogen invades. We regard a setting where the vector has a bias towards biting host individuals infected with the endemic pathogen and where there is a cost to co-infected hosts. As a motivating case study, we regard Plasmodium spp., that cause avian malaria, as the endemic pathogen, and Usutu virus (USUV) as the invading pathogen. Hosts with malaria attract more mosquitoes compared to susceptible hosts, a phenomenon named vector bias. The possible trade-off between the vector-bias effect and the co-infection mortality is studied using a compartmental epidemic model. We focus first on the basic reproduction number R0 for Usutu virus invading into a malaria-endemic population, and then explore the long-term dynamics of both pathogens once Usutu virus has become established. We find that the vector bias facilitates the introduction of malaria into a susceptible population, as well as the introduction of Usutu in a malaria-endemic population. In the long term, however, both a vector bias and co-infection mortality lead to a decrease in the number of individuals infected with either pathogen, suggesting that avian malaria is unlikely to be a promoter of Usutu invasion. This proposed approach is general and allows for new insights into other negative associations between endemic and invading vector-borne pathogens.

Duck Tembusu virus NS3 protein induces apoptosis by activating the PERK/PKR pathway and mitochondrial pathway.

IMPORTANCE: Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that replicates well in mosquito, bird, and mammalian cells. An in vivo study revealed that BALB/c mice and Kunming mice were susceptible to DTMUV after intracerebral inoculation. Moreover, there are no reports about DTMUV-related human disease, but antibodies against DTMUV and viral RNA were detected in the serum samples of duck industry workers. This information implies that DTMUV has expanded its host range and poses a threat to mammalian health. Thus, understanding the pathogenic mechanism of DTMUV is crucial for identifying potential antiviral targets. In this study, we discovered that NS3 can induce the mitochondria-mediated apoptotic pathway through the PERK/PKR pathway; it can also interact with voltage-dependent anion channel 2 to induce apoptosis. Our findings provide a theoretical basis for understanding the pathogenic mechanism of DTMUV infection and identifying potential antiviral targets and may also serve as a reference for exploring the pathogenesis of other flaviviruses.

Molecular Determinants of Tissue Specificity of Flavivirus Nonstructural Protein 1 Interaction with Endothelial Cells.

Members of the mosquito-borne flavivirus genus such as dengue (DENV), West Nile (WNV), and Zika (ZIKV) viruses cause distinct diseases and affect different tissues. We previously found that the secreted flaviviral nonstructural protein 1 (NS1) interacts with endothelial cells and disrupts endothelial barrier function in a tissue-specific manner consistent with the disease tropism of the respective viruses. However, the underlying molecular mechanism of this tissue-specific NS1-endothelial cell interaction is not well understood. To elucidate the distinct role(s) that the wing and ß-ladder domains of NS1 play in NS1 interactions with endothelial cells, we constructed flavivirus NS1 chimeras that exchanged the wing and ß-ladder domains in a pairwise manner between DENV, WNV, and ZIKV NS1. We found that both the NS1 wing and ß-ladder domains conferred NS1 tissue-specific endothelial dysfunction, with the wing conferring cell binding and the ß-ladder involved in inducing endothelial hyperpermeability as measured by transendothelial electrical resistance. To narrow down the amino acids dictating cell binding specificity, we utilized the DENV-WNV NS1 chimera and identified residues 91 to 93 (GDI) of DENV NS1 as a molecular motif determining binding specificity. Further, using an in vivo mouse model of localized leak, we found that the GDI motif of the wing domain was essential for triggering DENV NS1-induced vascular leak in mouse dermis. Taken together, we identify molecular determinants of flavivirus NS1 that confer NS1 binding and vascular leak and highlight the importance of the NS1 wing domain for flavivirus pathogenesis. IMPORTANCE Flavivirus NS1 is secreted into the bloodstream from infected cells during a viral infection. Dengue virus NS1 contributes to severe dengue pathology such as endothelial dysfunction and vascular leak independently of the virus. We have shown that multiple flavivirus NS1 proteins result in endothelial dysfunction in a tissue-specific manner consistent with their respective viral tropism. Here, we aimed to identify the molecular determinants that make some, but not other, flavivirus NS1 proteins bind to select endothelial cells in vitro and cause vascular leak in a mouse model. We identified the wing domain of NS1 as a primary determinant conferring differential endothelial dysfunction and vascular leak and narrowed the contributing amino acid residues to a three-residue motif within the wing domain. The insights from this study pave the way for future studies on the effects of flavivirus NS1 on viral dissemination and pathogenesis and offer potential new avenues for antiviral therapies.

Perturbation of Alphavirus and Flavivirus Infectivity by Components of the Bacterial Cell Wall.

The impact of the host microbiota on arbovirus infections is currently not well understood. Arboviruses are viruses transmitted through the bites of infected arthropods, predominantly mosquitoes or ticks. The first site of arbovirus inoculation is the biting site in the host skin, which is colonized by a complex microbial community that could possibly influence arbovirus infection. We demonstrated that preincubation of arboviruses with certain components of the bacterial cell wall, including lipopolysaccharides (LPS) of some Gram-negative bacteria and lipoteichoic acids or peptidoglycan of certain Gram-positive bacteria, significantly reduced arbovirus infectivity in vitro. This inhibitory effect was observed for arboviruses of different virus families, including chikungunya virus of the Alphavirus genus and Zika virus of the Flavivirus genus, showing that this is a broad phenomenon. A modest inhibitory effect was observed following incubation with a panel of heat-inactivated bacteria, including bacteria residing on the skin. No viral inhibition was observed after preincubation of cells with LPS. Furthermore, a virucidal effect of LPS on viral particles was noticed by electron microscopy. Therefore, the main inhibitory mechanism seems to be due to a direct effect on the virus particles. Together, these results suggest that bacteria are able to decrease the infectivity of alphaviruses and flaviviruses. IMPORTANCE During the past decades, the world has experienced a vast increase in epidemics of alphavirus and flavivirus infections. These viruses can cause severe diseases, such as hemorrhagic fever, encephalitis, and arthritis. Several alpha- and flaviviruses, such as chikungunya virus, Zika virus, and dengue virus, are significant global health threats because of their high disease burden, their widespread (re-)emergence, and the lack of (good) anti-arboviral strategies. Despite the clear health burden, alphavirus and flavivirus infection and disease are not fully understood. A knowledge gap in the interplay between the host and the arbovirus is the potential interaction with host skin bacteria. Therefore, we studied the effect of (skin) bacteria and bacterial cell wall components on alphavirus and flavivirus infectivity in cell culture. Our results show that certain bacterial cell wall components markedly reduced viral infectivity by interacting directly with the virus particle.

West Nile and Usutu Virus Introduction via Migratory Birds: A Retrospective Analysis in Italy.

The actual contribution of migratory birds in spreading West Nile (WNV) and Usutu virus (USUV) across Europe and from Africa to old countries is still controversial. In this study, we reported the results of molecular and serological surveys on migrating birds sampled during peaks of spring and autumn migration at 11 Italian sites located along important flyways, from 2012 to 2014. A total of 1335 specimens made of individual or pooled sera, and organs from 275 dead birds were tested for WNV and USUV RNA by real time PCR (RT-PCR). Furthermore, sera were tested by serum neutralization assay for detecting WNV and USUV neutralizing antibodies. Molecular tests detected WNV lineage 2 RNA in a pool made of three Song Thrush (Turdus philomelos) sera sampled in autumn, and lineage 1 in kidneys of six trans-Saharan birds sampled in spring. Neutralizing antibodies against WNV and USUV were found in 5.80% (n = 72; 17 bird species) and 0.32% (n = 4; 4 bird species) of the tested sera, respectively. Our results do not exclude the role of migratory birds as potential spreaders of WNV and USUV from Africa and Central Europe to Mediterranean areas and highlight the importance of a more extensive active surveillance of zoonotic viruses.

A quasispecies in a BHK-21 cell-derived virulent Tembusu virus strain contains three groups of variants with distinct virulence phenotypes.

Previous studies resulted in the isolation of a low-virulence plaque-purified variant from the third passage (P3) in BHK-21 cells of a Tembusu virus (TMUV) isolate, suggesting the presence of viral quasispecies in the P3 culture. To confirm this notion, the fourth passage virus (P4) was prepared by infecting BHK-21 cells with P3 for isolation of more variants. We isolated 10 plaque-purified viruses. Comparative genome sequence analysis identified six of the 10 viruses as genetically different variants, which harbored a total of eight amino acid differences in the envelope, NS1, NS3, and NS5 proteins. When tested in a 2-day-old Pekin duck model, P4 caused 80 % mortality, belonging to a high-virulence TMUV strain. Out of the six genetically different variants, two presented high-virulence, one exhibited moderate-virulence, and three displayed low-virulence, causing 60 %-70 %, 40 %, and 10 % mortalities, respectively. These results demonstrate that P4 contains at least three groups of variants with distinct virulence phenotypes. Analysis of links between the eight residues and virulence of the six variants identified NS1 protein residue 183 and NS5 protein residues 275 and/or 287 as novel determinants of TMUV virulence. The analysis also provided a new clue for future studies on the molecular basis of TMUV virulence in terms of genetic interaction of different proteins. Overall, our study provides direct evidence to suggest that TMUV exists in in vitro culture of a virulent isolate as a quasispecies, which may enhance our understanding of molecular mechanism of TMUV virulence.

Flavivirus Persistence in Wildlife Populations.

A substantial number of humans are at risk for infection by vector-borne flaviviruses, resulting in considerable morbidity and mortality worldwide. These viruses also infect wildlife at a considerable rate, persistently cycling between ticks/mosquitoes and small mammals and reptiles and non-human primates and humans. Substantially increasing evidence of viral persistence in wildlife continues to be reported. In addition to in humans, viral persistence has been shown to establish in mammalian, reptile, arachnid, and mosquito systems, as well as insect cell lines. Although a considerable amount of research has centered on the potential roles of defective virus particles, autophagy and/or apoptosis-induced evasion of the immune response, and the precise mechanism of these features in flavivirus persistence have yet to be elucidated. In this review, we present findings that aid in understanding how vector-borne flavivirus persistence is established in wildlife. Research studies to be discussed include determining the critical roles universal flavivirus non-structural proteins played in flaviviral persistence, the advancement of animal models of viral persistence, and studying host factors that allow vector-borne flavivirus replication without destructive effects on infected cells. These findings underscore the viral-host relationships in wildlife animals and could be used to elucidate the underlying mechanisms responsible for the establishment of viral persistence in these animals.

Host selection pattern and flavivirus screening of mosquitoes in a disturbed Colombian rainforest.

Studies on the feeding behavior of hematophagous insects, particularly those of medical importance, are relevant for tracking possible pathogen transmission routes and identifying biases in the choice of vertebrates. We evaluated host selection of blood-feeding mosquitoes in a disturbed forest in the Magdalena Medio valley in Colombia from March 2017 to April 2018, after the introduction of Zika virus to the Americas from the 2015-2016 outbreak. We estimated vertebrate diversity and collected blood-engorged female mosquitoes. Genomic DNA/RNA was extracted from the mosquito's abdomen for vertebrate host identification and pathogen detection. We performed conventional PCR and sequencing, using universal primers targeting vertebrate regions of the eukaryotic mitochondrial genome to determine bloodmeal host. Additionally, we tested for the presence of flaviviruses in all mosquito samples with RT-PCR. Based on the identity and quantity of detected bloodmeals, we performed mosquito-vertebrate interaction network analysis and estimated topology metrics. In total, we collected 292 engorged female mosquitoes representing 20 different species. Bloodmeal analyses identified 26 vertebrate species, the majority of which were mammals (N = 16; 61.5%). No flaviviruses of medical importance were detected from the samples. Although feeding patterns varied, network analyses showed a high degree of specialization by mosquitoes and revealed ecological and phylogenetic relationships among the host community. We conclude that host selection or preference by mosquitoes is species specific.

West Nile and Usutu Viruses' Surveillance in Birds of the Province of Ferrara, Italy, from 2015 to 2019.

West Nile (WNV) and Usutu (USUV) viruses are mosquito-borne flaviviruses. Thanks to their importance as zoonotic diseases, a regional plan for surveillance of Arboviruses was implemented in Emilia-Romagna in 2009. The province of Ferrara belongs to the Emilia-Romagna region, and it is an endemic territory for these viruses, with favorable ecological conditions for abundance of mosquitoes and wild birds. From 2015 to 2019, we collected 1842 dead-found birds at a wildlife rehabilitation center, which were analysed by three different PCRs for the detection of WNV and USUV genomes. August was characterized by the highest infection rate for both viruses. Columbiformes scored the highest USUV prevalence (8%), while Galliformes and Strigiformes reported the highest prevalence for WNV (13%). Among Passeriformes (the most populated Order), Turdus merula was the most abundant species and scored the highest prevalence for both viruses. To optimize passive surveillance plans, monitoring should be focused on the summer and towards the avian species more prone to infection by both viruses.

A 1958 Isolate of Kedougou Virus (KEDV) from Ndumu, South Africa, Expands the Geographic and Temporal Range of KEDV in Africa.

The mosquito-borne flavivirus, Kedougou virus (KEDV), first isolated in Senegal in 1972, is genetically related to dengue, Zika (ZIKV) and Spondweni viruses (SPOV). Serological surveillance studies in Senegal and isolation of KEDV in the Central African Republic indicate occurrence of KEDV infections in humans, but to date, no disease has been reported. Here, we assembled the coding-complete genome of a 1958 isolate of KEDV from a pool of Aedes circumluteolus mosquitoes collected in Ndumu, KwaZulu-Natal, South Africa. The AR1071 Ndumu KEDV isolate bears 80.51% pairwise nucleotide identity and 93.34% amino acid identity with the prototype DakAar-D1470 strain and was co-isolated with SPOV through intracerebral inoculation of suckling mice and passage on VeroE6 cells. This historical isolate expands the known geographic and temporal range of this relatively unknown flavivirus, aiding future temporal phylogenetic calibration and diagnostic assay refinement.

Viral and Prion Infections Associated with Central Nervous System Syndromes in Brazil.

Virus-induced infections of the central nervous system (CNS) are among the most serious problems in public health and can be associated with high rates of morbidity and mortality, mainly in low- and middle-income countries, where these manifestations have been neglected. Typically, herpes simplex virus 1 and 2, varicella-zoster, and enterovirus are responsible for a high number of cases in immunocompetent hosts, whereas other herpesviruses (for example, cytomegalovirus) are the most common in immunocompromised individuals. Arboviruses have also been associated with outbreaks with a high burden of neurological disorders, such as the Zika virus epidemic in Brazil. There is a current lack of understanding in Brazil about the most common viruses involved in CNS infections. In this review, we briefly summarize the most recent studies and findings associated with the CNS, in addition to epidemiological data that provide extensive information on the circulation and diversity of the most common neuro-invasive viruses in Brazil. We also highlight important aspects of the prion-associated diseases. This review provides readers with better knowledge of virus-associated CNS infections. A deeper understanding of these infections will support the improvement of the current surveillance strategies to allow the timely monitoring of the emergence/re-emergence of neurotropic viruses.

Replication/Assembly Defective Avian Flavivirus With Internal Deletions in the Capsid Can Be Used as an Approach for Living Attenuated Vaccine.

Avian Tembusu virus (TMUV) is a novel flavivirus causing severe egg drop and fatal encephalitis in avian in Asia. In the present study, we screened the structural and functional requirements of TMUV capsid protein (CP) for viral morphogenesis using reverse genetics methods in combination with replicon packaging assays. TMUV-CP showed dramatic functional and structural flexibility, and even though 44 residues were removed from the N-terminus, it was still capable of packaging replicon RNA; in addition, 33 residues were deleted from the C-terminus (containing nearly the entire α4-helix), and infectious particles were still produced, although α4-α4' is supposedly vital for CP dimerization and nucleocapsid formation. We further analyzed two mutants (ΔC20-43 and ΔC64-96 viruses) with relatively large deletions that still replicated well in BHK-21 cells. Our data indicate that internal deletions within CP impaired viral replication or assembly, resulting in attenuated virus proliferation in cells and attenuated virulence in duck embryos, and these deletion mutations are quite stable in cell culture. An in vivo assay indicated that both ΔC20-43 virus and ΔC64-96 virus were highly attenuated in ducklings but still immunogenic. Single-dose immunization with ΔC20-43 virus or ΔC64-96 virus could protect ducklings from a lethal challenge with good antigen clearance. Together, our data shed light on replication/assembly defective TMUV with internal deletions in CP and provide an effective approach to attenuate viral virulence in live vaccines without changing the antigen composition.

Pathology and Pathogenesis of Eurasian Blackbirds (Turdus merula) Naturally Infected with Usutu Virus.

The Usutu virus (USUV) is a mosquito-borne zoonotic flavivirus. Despite its continuous circulation in Europe, knowledge on the pathology, cellular and tissue tropism and pathogenetic potential of different circulating viral lineages is still fragmentary. Here, macroscopic and microscopic evaluations are performed in association with the study of cell and tissue tropism and comparison of lesion severity of two circulating virus lineages (Europe 3; Africa 3) in 160 Eurasian blackbirds (Turdus merula) in the Netherlands. Results confirm hepatosplenomegaly, coagulative necrosis and lymphoplasmacytic inflammation as major patterns of lesions and, for the first time, vasculitis as a novel virus-associated lesion. A USUV and Plasmodium spp. co-infection was commonly identified. The virus was associated with lesions by immunohistochemistry and was reported most commonly in endothelial cells and blood circulating and tissue mononucleated cells, suggesting them as a major route of entry and spread. A tropism for mononuclear phagocytes cells was further supported by viral labeling in multinucleated giant cells. The involvement of ganglionic neurons and epithelial cells of the gastrointestinal tract suggests a possible role of oral transmission, while the involvement of feather follicle shafts and bulbs suggests their use as a diagnostic sample for live bird testing. Finally, results suggest similar pathogenicity for the two circulating lineages.

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.

Isolation of a novel insect-specific flavivirus with immunomodulatory effects in vertebrate systems.

We describe the isolation and characterization of a novel insect-specific flavivirus (ISFV), tentatively named Aripo virus (ARPV), that was isolated from Psorophora albipes mosquitoes collected in Trinidad. The ARPV genome was determined and phylogenetic analyses showed that it is a dual host associated ISFV, and clusters with the main mosquito-borne flaviviruses. ARPV antigen was significantly cross-reactive with Japanese encephalitis virus serogroup antisera, with significant cross-reactivity to Ilheus and West Nile virus (WNV). Results suggest that ARPV replication is limited to mosquitoes, as it did not replicate in the sandfly, culicoides or vertebrate cell lines tested. We also demonstrated that ARPV is endocytosed into vertebrate cells and is highly immunomodulatory, producing a robust innate immune response despite its inability to replicate in vertebrate systems. We show that prior infection or coinfection with ARPV limits WNV-induced disease in mouse models, likely the result of a robust ARPV-induced type I interferon response.

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.

The importance of viral and cellular factors on flavivirus entry.

The flavivirus are emerging and re-emerging arthropod-borne pathogens responsible for significant mortality and morbidity worldwide. The genus comprises more than 70 viruses, and despite genomic and structural similarities, infections by different flaviviruses result in different clinical presentations. In the absence of a safe and effective vaccine against these infections, the search for new strategies to inhibit viral infection is necessary. The life cycle of arboviruses begins with the entry process composed of multiple steps: attachment, internalization, endosomal escape and capsid uncoating. This mini-review describes factors and mechanisms involved in the viral entry as events required to take over the cellular machinery and host factors and cellular pathways commonly used by flaviviruses as possible approaches for developing broad-spectrum antiviral drugs.