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.

RNF123 Mediates Ubiquitination and Degradation of SOCS1 To Regulate Type I Interferon Production during Duck Tembusu Virus Infection.

Many RING domain E3 ubiquitin ligases play critical roles in fine-tuning the innate immune response, yet little is known about their regulatory role in flavivirus-induced innate immunity. In previous studies, we found that the suppressor of cytokine signaling 1 (SOCS1) protein mainly undergoes lysine 48 (K48)-linked ubiquitination. However, the E3 ubiquitin ligase that promotes the K48-linked ubiquitination of SOCS1 is unknown. In the present study, we found that RING finger protein 123 (RNF123) binds to the SH2 domain of SOCS1 through its RING domain and facilitates the K48-linked ubiquitination of the K114 and K137 residues of SOCS1. Further studies found that RNF123 promoted the proteasomal degradation of SOCS1 and promoted Toll-like receptor 3 (TLR3)- and interferon (IFN) regulatory factor 7 (IRF7)-mediated type I IFN production during duck Tembusu virus (DTMUV) infection through SOCS1, ultimately inhibiting DTMUV replication. Overall, these findings demonstrate a novel mechanism by which RNF123 regulates type I IFN signaling during DTMUV infection by targeting SOCS1 degradation. IMPORTANCE In recent years, posttranslational modification (PTM) has gradually become a research hot spot in the field of innate immunity regulation, and ubiquitination is one of the critical PTMs. DTMUV has seriously endangered the development of the waterfowl industry in Southeast Asian countries since its outbreak in 2009. Previous studies have shown that SOCS1 is modified by K48-linked ubiquitination during DTMUV infection, but E3 ubiquitin ligase catalyzing the ubiquitination of SOCS1 has not been reported. Here, we identify for the first time that RNF123 acts as an E3 ubiquitin ligase that regulates TLR3- and IRF7-induced type I IFN signaling during DTMUV infection by targeting the K48-linked ubiquitination of the K114 and K137 residues of SOCS1 and the proteasomal degradation of SOCS1.

Duck Tembusu Virus Inhibits Type I Interferon Production through the JOSD1-SOCS1-IRF7 Negative-Feedback Regulation Pathway.

Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that mainly causes a decrease in egg production in infected waterfowl. Similar to other members of the Flaviviridae family, it can proliferate in most mammalian cells and may also pose a potential threat to nonavian animals. In previous studies, we found that DTMUV infection can upregulate suppressor of cytokine signaling 1 (SOCS1) to inhibit type I interferon (IFN) production and promote virus replication, but the specific mechanism is unclear. Furthermore, little is known about the regulatory role of ubiquitination during flavivirus infection. In this study, we found that activation of Toll-like receptor 3 (TLR3) signaling rather than type I IFN stimulation led to the upregulation of SOCS1 during DTMUV infection. Further studies revealed that JOSD1 stabilized SOCS1 expression by binding to the SH2 domain of SOCS1 and mediating its deubiquitination. In addition, JOSD1 also inhibited type I IFN production through SOCS1. Finally, SOCS1 acts as an E3 ubiquitin ligase that binds to IFN regulatory factor 7 (IRF7) through its SH2 domain and mediates K48-linked ubiquitination and proteasomal degradation of IRF7, ultimately inhibiting type I IFN production mediated by IRF7 and promoting viral proliferation. These results will enrich and deepen our understanding of the mechanism by which DTMUV antagonizes the host interferon system. IMPORTANCE DTMUV is a newly discovered flavivirus that seriously harms the poultry industry. In recent years, there have been numerous studies on the involvement of ubiquitination in the regulation of innate immunity. However, little is known about the involvement of ubiquitination in the regulation of flavivirus-induced type I IFN signaling. In this study, we found that SOCS1 was induced by TLR3 signaling during DTMUV infection. Furthermore, we found for the first time that duck SOCS1 protein was also modified by K48-linked polyubiquitination, whereas our previous study found that SOCS1 was upregulated during DTMUV infection. Further studies showed that JOSD1 stabilized SOCS1 expression by mediating the deubiquitination of SOCS1. While SOCS1 acts as a negative regulator of cytokines, we found that DTMUV utilized SOCS1 to mediate the ubiquitination and proteasomal degradation of IRF7 and ultimately inhibit type I IFN production, thereby promoting its proliferation.

Innate Immune Antagonism of Mosquito-Borne Flaviviruses in Humans and Mosquitoes.

Mosquito-borne viruses of the Flavivirus genus (Flaviviridae family) pose an ongoing threat to global public health. For example, dengue, Japanese encephalitis, West Nile, yellow fever, and Zika viruses are transmitted by infected mosquitoes and cause severe and fatal diseases in humans. The means by which mosquito-borne flaviviruses establish persistent infection in mosquitoes and cause disease in humans are complex and depend upon a myriad of virus-host interactions, such as those of the innate immune system, which are the main focus of our review. This review also covers the different strategies utilized by mosquito-borne flaviviruses to antagonize the innate immune response in humans and mosquitoes. Given the lack of antiviral therapeutics for mosquito-borne flaviviruses, improving our understanding of these virus-immune interactions could lead to new antiviral therapies and strategies for developing refractory vectors incapable of transmitting these viruses, and can also provide insights into determinants of viral tropism that influence virus emergence into new species.

Regulation of MDA5-dependent anti-Tembusu virus innate immune responses by LGP2 in ducks.

Melanoma differentiation associated factor 5 (MDA5), which belongs to the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) family, has been proved to be a key pattern recognition receptor of innate antiviral signaling in duck, which plays an important role in anti-Tembusu virus (TMUV) infection. However, laboratory of genetics and physiology 2 (LGP2), the third member of RLRs family, the regulatory function on antiviral innate immunity of MDA5 is currently unclear. In this study, we investigated the subcellular localization of duck LGP2 (duLGP2) and confirmed that it is an important regulator of the duMDA5-mediated host innate antiviral immune response. The present experimental data demonstrate that the overexpression of duLGP2 inhibits duMDA5 downstream transcriptional factor (IRF-7, IFN-ß, and NF-κB) promoter activity, and duMDA5-mediated type I IFNs and ISGs expression were significantly suppressed by duLGP2 regardless of viral infection in vitro. The inhibition of duLGP2 on the antiviral activity of duMDA5 ultimately leads to an increase in viral replication. However, the overexpression of duLGP2 promotes expression of mitochondrial antiviral-signaling protein (MAVS) and duMDA5-mediated proinflammatory cytokines. This study provides a new rationale support for the duLGP2 regulates duMDA5-mediated anti-viral immune signaling pathway theory in duck.

Roles of Non-Structural Protein 4A in Flavivirus Infection.

Infections with viruses in the genus Flavivirus are a worldwide public health problem. These enveloped, positive sense single stranded RNA viruses use a small complement of only 10 encoded proteins and the RNA genome itself to remodel host cells to achieve conditions favoring viral replication. A consequence of the limited viral armamentarium is that each protein exerts multiple cellular effects, in addition to any direct role in viral replication. The viruses encode four non-structural (NS) small transmembrane proteins (NS2A, NS2B, NS4A and NS4B) which collectively remain rather poorly characterized. NS4A is a 16kDa membrane associated protein and recent studies have shown that this protein plays multiple roles, including in membrane remodeling, antagonism of the host cell interferon response, and in the induction of autophagy, in addition to playing a role in viral replication. Perhaps most importantly, NS4A has been implicated as playing a critical role in fetal developmental defects seen as a consequence of Zika virus infection during pregnancy. This review provides a comprehensive overview of the multiple roles of this small but pivotal protein in mediating the pathobiology of flaviviral infections.

Single-Cell Analysis of the In Vivo Dynamics of Host Circulating Immune Cells Highlights the Importance of Myeloid Cells in Avian Flaviviral Infection.

Ducks are an economically important waterfowl but a natural reservoir for some zoonotic pathogens, such as influenza virus and flaviviruses. Our understanding of the duck immune system and its interaction with viruses remains incomplete. In this study, we constructed the transcriptomic landscape of duck circulating immune cells, the first line of defense in the arthropod-borne transmission of arboviruses, using high-throughput single-cell transcriptome sequencing, which defined 14 populations of peripheral blood leukocytes (PBLks) based on distinct molecular signatures and revealed differences in the clustering of PBLks between ducks and humans. Taking advantage of in vivo sex differences in the susceptibility of duck PBLks to avian tembusu virus (TMUV) infection, a mosquito-borne flavivirus newly emerged from ducks with a broad host range from mosquitos to mammals, a comprehensive comparison of the in vivo dynamics of duck PBLks upon TMUV infection between sexes was performed at the single-cell level. Using this in vivo model, we discovered that TMUV infection reprogrammed duck PBLks differently between sexes, driving the expansion of granulocytes and priming granulocytes and monocytes for antiviral immune activation in males but decreasing the antiviral immune activity of granulocytes and monocytes by restricting their dynamic transitions from steady states to antiviral states with a decrease in the abundance of circulating monocytes in females. This study provides insights into the initial immune responses of ducks to arthropod-borne flaviviral infection and provides a framework for studying duck antiviral immunity.

Duck Tembusu Virus Infection Promotes the Expression of Duck Interferon-Induced Protein 35 to Counteract RIG-I Antiviral Signaling in Duck Embryo Fibroblasts.

Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that has caused a substantial drop in egg production and severe neurological disorders in domestic waterfowl. Several studies have revealed that viral proteins encoded by DTMUV antagonize host IFN-mediated antiviral responses to facilitate virus replication. However, the role of host gene expression regulated by DTMUV in innate immune evasion remains largely unknown. Here, we utilized a stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics analysis of DTMUV-infected duck embryo fibroblasts (DEFs) to comprehensively investigate host proteins involved in DTMUV replication and innate immune response. A total of 250 differentially expressed proteins were identified from 2697 quantified cellular proteins, among which duck interferon-induced protein 35 (duIFI35) was dramatically up-regulated due to DTMUV infection in DEFs. Next, we demonstrated that duIFI35 expression promoted DTMUV replication and impaired Sendai virus-induced IFN-ß production. Moreover, duIFI35 was able to impede duck RIG-I (duRIG-I)-induced IFN-ß promoter activity, rather than IFN-ß transcription mediated by MDA5, MAVS, TBK1, IKKϵ, and IRF7. Importantly, we found that because of the specific interaction with duIFI35, the capacity of duRIG-I to recognize double-stranded RNA was significantly impaired, resulting in the decline of duRIG-I-induced IFN-ß production. Taken together, our data revealed that duIFI35 expression stimulated by DTMUV infection disrupted duRIG-I-mediated host antiviral response, elucidating a distinct function of duIFI35 from human IFI35, by which DTMUV escapes host innate immune response, and providing information for the design of antiviral drug.

Previous Usutu Virus Exposure Partially Protects Magpies (Pica pica) against West Nile Virus Disease But Does Not Prevent Horizontal Transmission.

The mosquito-borne flaviviruses USUV and WNV are known to co-circulate in large parts of Europe. Both are a public health concern, and USUV has been the cause of epizootics in both wild and domestic birds, and neurological cases in humans in Europe. Here, we explore the susceptibility of magpies to experimental USUV infection, and how previous exposure to USUV would affect infection with WNV. None of the magpies exposed to USUV showed clinical signs, viremia, or detectable neutralizing antibodies. After challenge with a neurovirulent WNV strain, neither viremia, viral titer of WNV in vascular feathers, nor neutralizing antibody titers of previously USUV-exposed magpies differed significantly with respect to magpies that had not previously been exposed to USUV. However, 75% (6/8) of the USUV-exposed birds survived, while only 22.2% (2/9) of those not previously exposed to USUV survived. WNV antigen labeling by immunohistochemistry in tissues was less evident and more restricted in magpies exposed to USUV prior to challenge with WNV. Our data indicate that previous exposure to USUV partially protects magpies against a lethal challenge with WNV, while it does not prevent viremia and direct transmission, although the mechanism is unclear. These results are relevant for flavivirus ecology and contention.

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.

Seroprevalence of Flavivirus Neutralizing Antibodies in Thailand by High-Throughput Neutralization Assay: Endemic Circulation of Zika Virus before 2012.

Thailand is a hyperendemic country for flavivirus infections in Southeast Asia. Although the reporting system for flavivirus surveillance in Thailand is well established, syndromic surveillance tends to underestimate the true epidemiological status of flaviviruses due to the majority of infections being asymptomatic. To accurately understand the prevalence of flaviviruses in endemic regions, we performed neutralization tests against multiple flaviviruses using 147 serum samples from healthy donors collected from four distinct regions in Thailand. Single-round infectious particles (SRIP) for six flaviviruses, dengue virus types 1 to 4 (DENV-1 to -4), Japanese encephalitis virus (JEV), and Zika virus (ZIKV), were used as antigens for developing a safe, high-throughput neutralization assay. Titers of neutralizing antibodies (NAbs) against the six flaviviruses revealed that DENV-1 and DENV-2, followed by ZIKV were the predominant circulating flaviviruses in a total of four regions, whereas the prevalence of NAbs against JEV varied among regions. Although the seroprevalence of ZIKV was low relative to that of DENV-1 and DENV-2, the findings strongly suggested that ZIKV has been circulating at a sustained level in Thailand since before 2012. These findings not only demonstrated the application of an SRIP-neutralization test in a serological study, but also elucidated the circulation and distribution trends of different flaviviruses in Thailand. IMPORTANCE Neutralization tests are the most reliable assay for flavivirus antibody detection; however, these assays are not suitable for high-throughput processing due to their time-consuming and labor-intensive nature. In this study, we developed single-round infectious particles (SRIPs) with a luciferase gene for dengue virus types 1 to 4, Japanese encephalitis virus, and Zika virus for use in a safe, high-throughput neutralization assay. We performed neutralization tests against multiple flaviviruses using 147 serum samples that were collected from healthy donors residing in four distinct regions of Thailand in 2011 to 2012. The assay was useful for surveys of flavivirus seroprevalence. The data revealed that dengue virus type 1 (DENV-1) and DENV-2 were the predominant circulating flaviviruses in Thailand and that Zika virus has been circulating at a sustained level in Thailand since before 2012.

Amelioration of Beta Interferon Inhibition by NS4B Contributes to Attenuating Tembusu Virus Virulence in Ducks.

Our previous studies reported that duck Tembusu virus nonstructural protein 2A (NS2A) is a major inhibitor of the IFNß signaling pathway through competitively binding to STING with TBK1, leading to a reduction in TBK1 phosphorylation. Duck TMUV NS2B3 could cleave and bind STING to subvert the IFNß signaling pathway. Here, we found that overexpression of duck TMUV NS4B could compete with TBK1 in binding to STING, reducing TBK1 phosphorylation and inhibiting the IFNß signaling pathway by using the Dual-Glo® Luciferase Assay System and the NanoBiT protein-protein interaction (PPI) assay. We further identified the E2, M3, G4, W5, K10 and D34 residues in NS4B that were important for its interaction with STING and its inhibition of IFNß induction, which were subsequently introduced into a duck TMUV replicon and an infectious cDNA clone. We found that the NS4B M3A mutant enhanced RNA replication and exhibited significantly higher titer levels than WT at 48-72 hpi but significantly decreased mortality (80%) in duck embryos compared to WT (100%); the NS4B G4A and R36A mutants slightly reduced RNA replication but exhibited the same titer levels as WT. However, the NS4B R36A mutant did not attenuate the virulence in duck embryos, whereas the G4A mutant significantly decreased the mortality (70%) of duck embryos. In addition, the NS4B W5A mutant did not affect viral replication, whereas the D34A mutant slightly reduced RNA replication, and both mutants exhibited significantly lower titer levels than the WT and significantly decreased mortality (90% and 70%, respectively) in duck embryos. Hence, our findings provide new insight into the development of attenuated flaviviruses by targeting the disabling viral strategies used to evade the innate defense mechanisms.

A Sentinel Serological Study in Selected Zoo Animals to Assess Early Detection of West Nile and Usutu Virus Circulation in Slovenia.

Monitoring infectious diseases is a crucial part of preventive veterinary medicine in zoological collections. This zoo environment contains a great variety of animal species that are in contact with wildlife species as a potential source of infectious diseases. Wild birds may be a source of West Nile virus (WNV) and Usutu (USUV) virus, which are both emerging pathogens of rising concern. The aim of this study was to use zoo animals as sentinels for the early detection of WNV and USUV in Slovenia. In total, 501 sera from 261 animals of 84 animal species (including birds, rodents, lagomorphs, carnivores, ungulates, reptiles, equids, and primates) collected for 17 years (2002-2018) were tested for antibodies to WNV and USUV. Antibodies to WNV were detected by indirect immunofluorescence tests in 16 (6.1%) of 261 animals representing 10 species, which were sampled prior to the first active cases of WNV described in 2018 in Slovenia in humans, a horse, and a hooded crow (Corvus cornix). Antibodies to USUV were detected in 14 out of 261 animals tested (5.4%) that were positive prior to the first positive cases of USUV infection in common blackbirds (Turdus merula) in Slovenia. The study illustrates the value of zoological collections as a predictor of future emerging diseases.

Pathogenesis and shedding of Usutu virus in juvenile chickens.

Usutu virus (USUV; family: Flaviviridae, genus: Flavivirus), is an emerging zoonotic arbovirus that causes severe neuroinvasive disease in humans and has been implicated in the loss of breeding bird populations in Europe. USUV is maintained in an enzootic cycle between ornithophilic mosquitos and wild birds. As a member of the Japanese encephalitis serocomplex, USUV is closely related to West Nile virus (WNV) and St. Louis encephalitis virus (SLEV), both neuroinvasive arboviruses endemic in wild bird populations in the United States. An avian model for USUV is essential to understanding zoonotic transmission. Here we describe the first avian models of USUV infection with the development of viremia. Juvenile commercial ISA Brown chickens were susceptible to infection by multiple USUV strains with evidence of cardiac lesions. Juvenile chickens from two chicken lines selected for high (HAS) or low (LAS) antibody production against sheep red blood cells showed markedly different responses to USUV infection. Morbidity and mortality were observed in the LAS chickens, but not HAS chickens. LAS chickens had significantly higher viral titers in blood and other tissues, as well as oral secretions, and significantly lower development of neutralizing antibody responses compared to HAS chickens. Mathematical modelling of virus-host interactions showed that the viral clearance rate is a stronger mitigating factor for USUV viremia than neutralizing antibody response in this avian model. These chicken models provide a tool for further understanding USUV pathogenesis in birds and evaluating transmission dynamics between avian hosts and mosquito vectors.

Effect of TMUV on immune organs of TMUV infected ducklings.

Tembusu Virus (TMUV), a pathogenic member of Flavivirus family, acts as the causative agent of egg-laying and has severely threatened the duck industry over the past few years. Thus far, the pathogenicity of such virus has been extensively studied, whereas TMUV on immune system has been less comprehensively assessed, especially on ducklings that exhibit more susceptible to TMUV attack. Accordingly, in the present study, 5-day-old ducklings were infected with TMUV-TC2B (104 TCID50) via intravenous injection, and mock ones were inoculated with phosphate-buffered saline (PBS) in identical manner as control. At 1 day-post inoculation (dpi), the innate immunity was strongly activated, and reacted rapidly to TMUV invasion, which was reflected as the significantly up-regulated IFN-stimulated genes (ISGs), especially in immune organs (e.g., thymus, bursa of Fabricius (BF) and spleen). Subsequently, under the continuous monitoring, the levels of IgA, IgM and IgG acting as the representative immunoglobulins (Igs) were constantly higher than those of mock ducklings, demonstrating that humoral immunity also played a major role in anti-virus infection. Despite the immune system activated positively, TMUV still caused systemic infection, and in particular, the immune organs were subject to severe damage in the early infection. With our constant observation, the injury of spleen and BF turned out to be getting more serious, and at 6 dpi, TMUV antigen was widely detected in both of two immune organs by immunohistochemistry (IHC) and main histopathological lesion presented as lymphocytopenia. Moreover, the elevated apoptosis rate of splenic lymphocytes and the alteration of immune organ index also revealed the damage of lymphoid organs and similarly, it is worth noting that severe damages were detected in thymus of TMUV-infected ducklings as well. In brief, the present study systematically described the dynamic damage of immune system after being attacked by TMUV and presented insights into the research of pathogenicity.

Identification of a linear epitope within domain I of Duck Tembusu virus envelope protein using a novel neutralizing monoclonal antibody.

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that caused severe egg drop syndrome in laying ducks in China since 2010, leading to massive economic losses to the duck industry. Although the DTMUV E protein is considered to be critical in inducing the protective immune response, the functional epitopes within this protein remain largely unknown. In the present study, we isolated a DTMUV neutralizing monoclonal antibody (mAb) 3B8 from DTMUV E-immunized mice. Epitope mapping showed that mAb 3B8 recognized a novel linear epitope FSCLGMQNR located on the extreme N-terminal of the domain I (EDI) of E protein. Sequence alignment and Western blot analyses showed that the epitope is greatly conserved with high DTMUV-specificity. Moreover, upon cloning the heavy and light chain variable region sequences of mAb 3B8, we prepared the single-chain variable antibody fragment (scFv) 3B8 by connecting the two chains via a flexible peptide linker. The recombinant scFv 3B8 exhibited antiviral activity against DTMUV infection in vitro and in vivo. Our results provide valuable implications for the development of DTMUV vaccines and therapeutics.

TMEM41B Is a Pan-flavivirus Host Factor.

Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.

Role of the complement system in antibody-dependent enhancement of flavivirus infections.

Flavivirus infections have increased dramatically in the last decades in tropical and subtropical regions of the world. Antibody-dependent enhancement of dengue virus infections has been one of the main hypotheses to explain severity of disease and one of the major challenges to safe and effective vaccine development. In the presence of cross-reactive sub-neutralizing concentrations of anti-dengue antibodies, immune complexes can amplify viral infection in mononuclear phagocytic cells, triggering a cytokine cascade and activating the complement system that leads to severe disease. The complement system comprises a family of plasma and cellular surface proteins that recognize pathogen associated molecular patterns, modified ligands and immune complexes, interacting in a regulated manner and forming an enzymatic cascade. Pathogenic as well as protective effects of complement have been reported in flavivirus infections. This review provides updated knowledge on complement activation during flavivirus infection, including antiviral effects of complement and its regulation, as well as mechanisms of complement evasion and dysregulation of complement activity during viral infection leading to pathogenesis. Particularly, insights into classical pathway activation and its protective role on antibody-dependent enhancement of flavivirus infections are highlighted.

Structural Basis of Zika Virus Specific Neutralization in Subsequent Flavivirus Infections.

Zika virus (ZIKV), a mosquito-borne human flavivirus that causes microcephaly and other neurological disorders, has been a recent focus for the development of flavivirus vaccines and therapeutics. We report here a 4.0 Å resolution structure of the mature ZIKV in complex with ADI-30056, a ZIKV-specific human monoclonal antibody (hMAb) isolated from a ZIKV infected donor with a prior dengue virus infection. The structure shows that the hMAb interactions span across the E protein dimers on the virus surface, inhibiting conformational changes required for the formation of infectious fusogenic trimers similar to the hMAb, ZIKV-117. Structure-based functional analysis, and structure and sequence comparisons, identified ZIKV residues essential for neutralization and crucial for the evolution of highly potent E protein crosslinking Abs in ZIKV. Thus, this epitope, ZIKV's "Achilles heel", defined by the contacts between ZIKV and ADI-30056, could be a suitable target for the design of therapeutic antibodies.