The myriad roles of RNA structure in the flavivirus life cycle.

As positive-sense RNA viruses, the genomes of flaviviruses serve as the template for all stages of the viral life cycle, including translation, replication, and infectious particle production. Yet, they encode just 10 proteins, suggesting that the structure and dynamics of the viral RNA itself helps shepherd the viral genome through these stages. Herein, we highlight advances in our understanding of flavivirus RNA structural elements through the lens of their impact on the viral life cycle. We highlight how RNA structures impact translation, the switch from translation to replication, negative- and positive-strand RNA synthesis, and virion assembly. Consequently, we describe three major themes regarding the roles of RNA structure in flavivirus infections: 1) providing a layer of specificity; 2) increasing the functional capacity; and 3) providing a mechanism to support genome compaction. While the interactions described herein are specific to flaviviruses, these themes appear to extend more broadly across RNA viruses.

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.

E-M349E and NS2A/2B-P1(T) are compensatory mutations of rDTMUV-NS2AB-P1P1'(AA), which regain virus proliferation by enhancing the virus package and restoring NS2A/2B cleavage.

Duck Tembusu virus (DTMUV) belongs to the Flaviviridae family and mainly infects ducks. The genome of DTMUV is translated into a polyprotein, which is further cleaved into several protein by viral NS2B3 protease and host proteases. Crucially, the cleavage of the NS2A/2B precursor during this process is essential for the formation of replication complexes and viral packaging. Previous research has demonstrated that alanine mutations in NS2A/2B (P1P1' (AA)) result in an attenuated strain (rDTMUV-NS2A/2B-P1P1' (AA)) by disrupting NS2A/2B cleavage. In this study, we investigate the effects of the P1P1' (AA) mutation on the viral life cycle and explore compensatory mutations in rDTMUV-NS2A/2B-P1P1' (AA). Infected ducklings exhibit similar body weight gain and viral tissue loads to DTMUV-WT. Compensatory mutations E-M349E and P1(T) emerge, restoring proliferation levels to those of rDTMUV-WT. Specifically, E-M349E enhances viral packaging, while P1(T) reinstates NS2A/2B proteolysis in vitro. Thus, our findings reveal novel compensatory sites capable of restoring the attenuated DTMUV during polyprotein cleavage and packaging.

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.

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.

Skin tropism during Usutu virus and West Nile virus infection: an amplifying and immunological role.

Usutu virus (USUV) and West Nile virus (WNV) are closely related emerging arboviruses belonging to the Flavivirus genus and posing global public health concerns. Although human infection by these viruses is mainly asymptomatic, both have been associated with neurological disorders such as encephalitis and meningoencephalitis. Since USUV and WNV are transmitted through the bite of an infected mosquito, the skin represents the initial site of virus inoculation and provides the first line of host defense. Although some data on the early stages of WNV skin infection are available, very little is known about USUV. Herein, USUV-skin resident cell interactions were characterized. Using primary human keratinocytes and fibroblasts, an early replication of USUV during the first 24 hours was shown in both skin cells. In human skin explants, a high viral tropism for keratinocytes was observed. USUV infection of these models induced type I and III interferon responses associated with upregulated expression of various interferon-stimulated genes as well as pro-inflammatory cytokine and chemokine genes. Among the four USUV lineages studied, the Europe 2 strain replicated more efficiently in skin cells and induced a higher innate immune response. In vivo, USUV and WNV disseminated quickly from the inoculation site to distal cutaneous tissues. In addition, viral replication and persistence in skin cells were associated with an antiviral response. Taken together, these results provide a better understanding of the pathophysiology of the early steps of USUV infection and suggest that the skin constitutes a major amplifying organ for USUV and WNV infection.IMPORTANCEUsutu virus (USUV) and West Nile virus (WNV) are closely related emerging Flaviviruses transmitted through the bite of an infected mosquito. Since they are directly inoculated within the upper skin layers, the interactions between the virus and skin cells are critical in the pathophysiology of USUV and WNV infection. Here, during the early steps of infection, we showed that USUV can efficiently infect two human resident skin cell types at the inoculation site: the epidermal keratinocytes and the dermal fibroblasts, leading to the induction of an antiviral innate immune response. Moreover, following cutaneous inoculation, we demonstrated that both viruses can rapidly spread, replicate, and persist in all distal cutaneous tissues in mice, a phenomenon associated with a generalized skin inflammatory response. These results highlight the key amplifying and immunological role of the skin during USUV and WNV infection.

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.

Toll-like receptor 4 and lipopolysaccharide from commensal microbes regulate Tembusu virus infection.

Unlike most flaviviruses transmitted by arthropods, Tembusu virus (TMUV) is still active during winter and causes outbreaks in some areas, indicating vector-independent spread of the virus. Gastrointestinal transmission might be one of the possible routes of vector-free transmission, which also means that the virus has to interact with more intestinal bacteria. Here, we found evidence that TMUV indeed can transmit through the digestive tract. Interestingly, using an established TMUV disease model by oral gavage combined with an antibiotic treatment, we revealed that a decrease in intestinal bacteria significantly reduced local TMUV proliferation in the intestine, revealing that the bacterial microbiome is important in TMUV infection. We found that lipopolysaccharide (LPS) present in the outer membrane of Gram-negative bacteria enhanced TMUV proliferation by promoting its attachment. Toll-like receptor 4 (TLR4), a cell surface receptor, can transmit signal from LPS. We confirmed colocalization of TLR4 with TMUV envelope (E) protein as well as their interaction in infected cells. Coherently, TMUV infection of susceptible cells was inhibited by an anti-TLR4 antibody, purified soluble TLR4 protein, and knockdown of TLR4 expression. LPS-enhanced TMUV proliferation could also be blocked by a TLR4 inhibitor. Meanwhile, pretreatment of duck primary cells with TMUV significantly impaired LPS-induced interleukin 6 production. Collectively, our study provides first insights into vector-free transmission mechanisms of flaviviruses.

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.

Aquatic Flaviviruses.

Flaviviruses are positive-sense single-stranded RNA viruses, including some well-known human pathogens such as Zika, dengue, and yellow fever viruses, which are primarily associated with mosquito and tick vectors. The vast majority of flavivirus research has focused on terrestrial environments; however, recent findings indicate that a range of flaviviruses are also present in aquatic environments, both marine and freshwater. These flaviviruses are found in various hosts, including fish, crustaceans, molluscs, and echinoderms. Although the effects of aquatic flaviviruses on the hosts they infect are not all known, some have been detected in farmed species and may have detrimental effects on the aquaculture industry. Exploration of the evolutionary history through the discovery of the Wenzhou shark flavivirus in both a shark and crab host is of particular interest since the potential dual-host nature of this virus may indicate that the invertebrate-vertebrate relationship seen in other flaviviruses may have a more profound evolutionary root than previously expected. Potential endogenous viral elements and the range of novel aquatic flaviviruses discovered thus shed light on virus origins and evolutionary history and may indicate that, like terrestrial life, the origins of flaviviruses may lie in aquatic environments.

The interactions of flaviviruses with cellular receptors: Implications for virus entry.

Flaviviruses are a group of enveloped viruses that enter the host cells through receptor-mediated endocytosis. The entry of flaviviruses into the cells is a multi-step process which involves several host factors that trigger the uptake of the virus. The initial step in the virus life cycle is the interactions between viral envelope proteins and the specific receptors on the surface of host cell. To date, several receptors have been identified such as glycosaminoglycans, tight junction proteins, laminin receptor and phosphatidylserine receptors. Moreover, the viruses may utilize integrins and C-type lectin receptors on the surface of host cells as the initial attachment factors. This mini-review will focus on recent progresses in the understanding of virus attachment, internalization, and membrane fusion with specific emphasis on the cellular receptors.

Roles of ESCRT Proteins ALIX and CHMP4A and Their Interplay with Interferon-Stimulated Gene 15 during Tick-Borne Flavivirus Infection.

Flaviviruses are usually transmitted to humans via mosquito or tick bites. During infection, virus replication and assembly, whose cellular sites are relatively close, are controlled by virus proteins and a diverse range of host proteins. By siRNA-mediated gene silencing, we showed that ALIX and CHMP4A, two members of the host endosomal sorting complex required for transport (ESCRT) protein machinery, are required during flavivirus infection. Using cell lines expressing subgenomic replicons and replicon virus-like particles, we demonstrated specific roles for ALIX and CHMP4A in viral replication and assembly, respectively. Employing biochemical and imaging methodology, we showed that the ESCRT proteins are recruited by a putative specific late (L) domain motif LYXLA within the NS3 protein of tick-borne flaviviruses. Furthermore, to counteract the recruitment of ESCRT proteins, the host cells may elicit defense mechanisms. We found that ectopic expression of the interferon-stimulated gene 15 (ISG15) or the E3 ISG15-protein ligase (HERC5) reduced virus replication by suppressing the positive effects of ALIX and CHMP4A. Collectively, these results have provided new insights into flavivirus-host cell interactions that function as checkpoints, including the NS3 and the ESCRT proteins, the ISG15 and the ESCRT proteins, at essential stages of the virus life cycle. IMPORTANCE Flaviviruses are important zoonotic viruses with high fatality rates worldwide. Here, we report that during infection, the virus employs members of ESCRT proteins for virus replication and assembly. Among the ESCRT proteins, ALIX acts during virus replication, while CHMP4A is required during virus assembly. Another important ESCRT protein, TSG101, is not required for virus production. The ESCRT, complex, ALIX-CHMP4A, is recruited to NS3 through their interactions with the putative L domain motif of NS3, while CHMP4A is recruited to E. In addition, we demonstrate the antiviral mechanism of ISG15 and HERC5, which degrades ALIX and CHIMP4A, indirectly targets virus infection. In summary, we reveal host-dependency factors supporting flavivirus infection, but these factors may also be targeted by antiviral host effector mechanisms.

Cell Strain-Derived Induced Pluripotent Stem Cells as an Isogenic Approach To Investigate Age-Related Host Response to Flaviviral Infection.

The expansion of the geographical footprint of dengue viruses (DENVs) and their mosquito vectors have affected more than half of the global population, including older adults who appear to show elevated risk of severe dengue. Despite this epidemiological trend, how aging contributes to increased dengue pathogenesis is poorly understood. A limitation has been the lack of useful in vitro experimental approaches; cell lines commonly used for infection studies are immortal and hence do not age. Cell strains such as WI-38 and MRC-5 with diploid genomes do age with in vitro passaging, but these cell strains were isolated decades ago and are now mostly highly passaged. Here, we show that reprogramming of cell strains with finite life span into induced pluripotent stem cells (iPSCs), followed by conversion back into terminally differentiated cells, can be an approach to derive genetically identical cells at different stages of aging. The iPSC-derived differentiated cells were susceptible to wild-type DENV infection and produced greater levels of type I interferon expression with increased passaging, despite similar levels of infection. In contrast, infection with the attenuated DENV-2 PDK53 and YF17D-204 strains showed reduced and increased levels of infection with increasing passages, respectively; the latter could be clinically pertinent, as YF17D-204 vaccination in older adults is associated with increased risk of severe adverse outcome. The differences in infection susceptibility and host response collectively suggest the potential of iPSC-derived cell strains as a genetically controlled approach to understanding how aging impacts viral pathogenesis. IMPORTANCE Aging has been a risk factor for poor clinical outcome in several infectious diseases, including dengue. However, age-dependent responses to dengue and other flaviviral infection or vaccination have remained incompletely understood due partly to lack of suitable laboratory tools. We thus developed an in vitro approach to examine age-related changes in host response to flaviviral infection. Notably, this approach uses cell strains with diploid rather than aneuploidic genomes, which are unstable. Conversion of these cells into iPSCs ensures sustainability of this resource, and reprogramming back into terminally differentiated cells would, even with a limited number of passages, produce cells at different stages of aging for infection studies. Our findings suggest that this in vitro system has the potential to serve as a genetically controlled approach to define the age-related response to flavivirus infection.

Zinc Finger Protein ZFP36L1 Inhibits Flavivirus Infection by both 5'-3' XRN1 and 3'-5' RNA-Exosome RNA Decay Pathways.

Zinc-finger protein 36, CCCH type-like 1 (ZFP36L1), containing tandem CCCH-type zinc-finger motifs with an RNA-binding property, plays an important role in cellular RNA metabolism mainly by RNA decay pathways. Recently, we demonstrated that human ZFP36L1 has potent antiviral activity against influenza A virus infection. However, its role in the host defense response against flaviviruses has not been addressed. Here, we demonstrate that ZFP36L1 functions as a host innate defender against flaviviruses, including Japanese encephalitis virus (JEV) and dengue virus (DENV). Overexpression of ZFP36L1 reduced JEV and DENV infection, and ZFP36L1 knockdown enhanced viral replication. ZFP36L1 destabilized the JEV genome by targeting and degrading viral RNA mediated by both 5'-3' XRN1 and 3'-5' RNA-exosome RNA decay pathways. Mutation in both zinc-finger motifs of ZFP36L1 disrupted RNA-binding and antiviral activity. Furthermore, the viral RNA sequences specifically recognized by ZFP36L1 were mapped to the 3'-untranslated region of the JEV genome with the AU-rich element (AUUUA) motif. We extend the function of ZFP36L1 to host antiviral defense by directly binding and destabilizing the viral genome via recruiting cellular mRNA decay machineries. IMPORTANCE Cellular RNA-binding proteins are among the first lines of defense against various viruses, particularly RNA viruses. ZFP36L1 belongs to the CCCH-type zinc-finger protein family and has RNA-binding activity; it has been reported to bind directly to the AU-rich elements (AREs) of a subset of cellular mRNAs and then lead to mRNA decay by recruiting mRNA-degrading enzymes. However, the antiviral potential of ZFP36L1 against flaviviruses has not yet been fully demonstrated. Here, we reveal the antiviral potential of human ZFP36L1 against Japanese encephalitis virus (JEV) and dengue virus (DENV). ZFP36L1 specifically targeted the ARE motif within viral RNA and triggered the degradation of viral RNA transcripts via cellular degrading enzymes 5'-3' XRN1 and 3'-5' RNA exosome. These findings provide mechanistic insights into how human ZFP36L1 serves as a host antiviral factor to restrict flavivirus replication.

Flavivirus infections induce a Golgi stress response in vertebrate and mosquito cells.

The stress of the Golgi apparatus is an autoregulatory mechanism that is induced to compensate for greater demand in the Golgi functions. No examples of Golgi stress responses due to physiological stimuli are known. Furthermore, the impact on this organelle of viral infections that occupy the vesicular transport during replication is unknown. In this work, we evaluated if a Golgi stress response is triggered during dengue and Zika viruses replication, two flaviviruses whose replicative cycle is heavily involved with the Golgi complex, in vertebrate and mosquito cells. Using GM-130 as a Golgi marker, and treatment with monensin as a positive control for the induction of the Golgi stress response, a significant expansion of the Golgi cisternae was observed in BHK-21, Vero E6 and mosquito cells infected with either virus. Activation of the TFE3 pathway was observed in the infected cells as indicated by the translocation from the cytoplasm to the nucleus of TFE3 and increased expression of pathway targeted genes. Of note, no sign of activation of the stress response was observed in CRFK cells infected with Feline Calicivirus (FCV), a virus released by cell lysis, not requiring vesicular transport. Finally, dilatation of the Golgi complex and translocation of TFE3 was observed in vertebrate cells expressing dengue and Zika viruses NS1, but not NS3. These results indicated that infections by dengue and Zika viruses induce a Golgi stress response in vertebrate and mosquito cells due to the increased demand on the Golgi complex imposed by virion and NS1 processing and secretion.

A human-blood-derived microRNA facilitates flavivirus infection in fed mosquitoes.

Hematophagous arthropods, such as mosquitoes, naturally carry and transmit hundreds of arboviruses to humans. Blood meal is a predominant physical interface that shapes cross-species communications among humans, bloodsuckers, and arboviruses. Here, we identify a human-blood-derived microRNA, hsa-miR-150-5p, that interferes with a mosquito antiviral system to facilitate flavivirus infection and transmission. hsa-miR-150-5p is acquired with a blood meal into the mosquito hemocoel and persists for a prolonged time there. The agomir of hsa-miR-150-5p enhances, whereas the antagomir represses flaviviral infection in mosquitoes and transmission from mice to mosquitoes. Mechanistic studies indicate that hsa-miR-150-5p hijacks the mosquito Argonaute-1-mediated RNA interference system to suppress the expression of some chymotrypsins with potent virucidal activity. Mosquito chymotrypsins are essential for resisting systemic flavivirus infection in hemocoel tissues. Chymotrypsin homologs potentially targeted by miR-150-5p are also found in other hematophagous arthropods, demonstrating a conserved miR-150-5p-mediated cross-species RNAi mechanism that might determine flaviviral transmissibility in nature.

Widespread Circulation of Flaviviruses in Horses and Birds in Northeastern Spain (Catalonia) between 2010 and 2019.

The surveillance for West Nile virus (WNV) in Catalonia (northeastern Spain) has consistently detected flaviviruses not identified as WNV. With the aim of characterizing the flaviviruses circulating in Catalonia, serum samples from birds and horses collected between 2010 and 2019 and positive by panflavivirus competition ELISA (cELISA) were analyzed by microneutralization test (MNT) against different flaviviruses. A third of the samples tested were inconclusive by MNT, highlighting the limitations of current diagnostic techniques. Our results evidenced the widespread circulation of flaviviruses, in particular WNV, but also Usutu virus (USUV), and suggest that chicken and horses could serve as sentinels for both viruses. In several regions, WNV and USUV overlapped, but no significant geographical aggregation was observed. Bagaza virus (BAGV) was not detected in birds, while positivity to tick-borne encephalitis virus (TBEV) was sporadically detected in horses although no endemic foci were observed. So far, no human infections by WNV, USUV, or TBEV have been reported in Catalonia. However, these zoonotic flaviviruses need to be kept under surveillance, ideally within a One Health framework.

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.