Ixodes ricinus as potential vector for Usutu virus.

Usutu virus (USUV) is an emerging flavivirus that is maintained in an enzootic cycle with mosquitoes as vectors and birds as amplifying hosts. In Europe, the virus has caused mass mortality of wild birds, mainly among Common Blackbird (Turdus merula) populations. While mosquitoes are the primary vectors for USUV, Common Blackbirds and other avian species are exposed to other arthropod ectoparasites, such as ticks. It is unknown, however, if ticks can maintain and transmit USUV. We addressed this question using in vitro and in vivo experiments and field collected data. USUV replicated in IRE/CTVM19 Ixodes ricinus tick cells and in injected ticks. Moreover, I. ricinus nymphs acquired the virus via artificial membrane blood-feeding and maintained the virus for at least 70 days. Transstadial transmission of USUV from nymphs to adults was confirmed in 4.9% of the ticks. USUV disseminated from the midgut to the haemocoel, and was transmitted via the saliva of the tick during artificial membrane blood-feeding. We further explored the role of ticks by monitoring USUV in questing ticks and in ticks feeding on wild birds in the Netherlands between 2016 and 2019. In total, 622 wild birds and the Ixodes ticks they carried were tested for USUV RNA. Of these birds, 48 (7.7%) carried USUV-positive ticks. The presence of negative-sense USUV RNA in ticks, as confirmed via small RNA-sequencing, showed active virus replication. In contrast, we did not detect USUV in 15,381 questing ticks collected in 2017 and 2019. We conclude that I. ricinus can be infected with USUV and can transstadially and horizontally transmit USUV. However, in comparison to mosquito-borne transmission, the role of I. ricinus ticks in the epidemiology of USUV is expected to be minor.

Is Culex modestus a New Usutu virus vector?

Usutu virus is an emerging pathogen transmitted by mosquitoes. Culex modestus mosquitoes are widespread in Europe, but their role in disease transmission is poorly understood. Recent data from a single infectious mosquito suggested that Culex modestus could be an unrecognized vector for Usutu virus. In this study, our aim was to corroborate this finding using a larger sample size. We collected immature Culex modestus from a reedbed pond in Flemish Brabant, Belgium, and reared them in the laboratory until the third generation. Adult females were then experimentally infected with Usutu virus in a blood meal and incubated at 25 °C for 14 days. The presence of Usutu virus in the saliva, head and body of each female was determined by plaque assay and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). The transmission efficiency was 54% (n = 15/28), confirming that Belgian Culex modestus can experimentally transmit Usutu virus.

The structure of immature tick-borne encephalitis virus supports the collapse model of flavivirus maturation.

We present structures of three immature tick-borne encephalitis virus (TBEV) isolates. Our atomic models of the major viral components, the E and prM proteins, indicate that the pr domains of prM have a critical role in holding the heterohexameric prM3E3 spikes in a metastable conformation. Destabilization of the prM furin-sensitive loop at acidic pH facilitates its processing. The prM topology and domain assignment in TBEV is similar to the mosquito-borne Binjari virus, but is in contrast to other immature flavivirus models. These results support that prM cleavage, the collapse of E protein ectodomains onto the virion surface, the large movement of the membrane domains of both E and M, and the release of the pr fragment from the particle render the virus mature and infectious. Our work favors the collapse model of flavivirus maturation warranting further studies of immature flaviviruses to determine the sequence of events and mechanistic details driving flavivirus maturation.

[The viral protein NS1: a major player in the pathogenesis of orthoflaviviruses]. / La protéine virale NS1 : un acteur majeur de la pathogenèse des orthoflavivirus.

Orthoflaviviruses are enveloped positive-sense RNA viruses comprising numerous human pathogens transmitted by hematophagous arthropods. This includes viruses such as dengue virus, Zika virus, and yellow fever virus. The viral nonstructural protein NS1 plays a central role in the pathogenesis and cycle of these viruses by acting in two different forms: associated with the plasma membrane (NS1m) or secreted outside the cell (NS1s). The versatility of NS1 is evident in its ability to modulate various aspects of the infectious process, from immune evasion to pathogenesis. As an intracellular protein, it disrupts many processes, interfering with signaling pathways and facilitating viral replication in concert with other viral proteins. As a secreted protein, NS1 actively participates in immune evasion, interfering with the host immune system, inhibiting the complement system, facilitating viral dissemination, and disrupting the integrity of endothelial barriers. This review primarily aims to address the role of NS1 in viral pathogenesis associated with orthoflaviviruses.

Advances in Alphavirus and Flavivirus Research.

Newly emerging viruses, primarily zoonotic or vector-borne, pose a persistent threat to public health and have led to outbreaks of global concern [...].

Exploring Iguape Virus-A Lesser-Known Orthoflavivirus.

Brazil has earned the moniker "arbovirus hotspot", providing an ideal breeding ground for a multitude of arboviruses thriving in various zoonotic and urban cycles. As the planet warms and vectors expand their habitat range, a nuanced understanding of lesser-known arboviruses and the factors that could drive their emergence becomes imperative. Among these viruses is the Iguape virus (IGUV), a member of the Orthoflavivirus aroaense species, which was first isolated in 1979 from a sentinel mouse in the municipality of Iguape, within the Vale do Ribeira region of São Paulo State. While evidence suggests that IGUV circulates among birds, wild rodents, marsupials, bats, and domestic birds, there is no information available on its pathogenesis in both humans and animals. The existing literature on IGUV spans decades, is outdated, and is often challenging to access. In this review, we have curated information from the known literature, clarifying its elusive nature and investigating the factors that may influence its emergence. As an orthoflavivirus, IGUV poses a potential threat, which demands our attention and vigilance, considering the serious outbreaks that the Zika virus, another neglected orthoflavivirus, has unleashed in the recent past.

Evolution of STAT2 resistance to flavivirus NS5 occurred multiple times despite genetic constraints.

Zika and dengue virus nonstructural protein 5 antagonism of STAT2, a critical interferon signaling transcription factor, to suppress the host interferon response is required for viremia and pathogenesis in a vertebrate host. This affects viral species tropism, as mouse STAT2 resistance renders only immunocompromised or humanized STAT2 mice infectable. Here, we explore how STAT2 evolution impacts antagonism. By measuring the susceptibility of 38 diverse STAT2 proteins, we demonstrate that resistance arose numerous times in mammalian evolution. In four species, resistance requires distinct sets of multiple amino acid changes that often individually disrupt STAT2 signaling. This reflects an evolutionary ridge where progressive resistance is balanced by the need to maintain STAT2 function. Furthermore, resistance may come with a fitness cost, as resistance that arose early in lemur evolution was subsequently lost in some lemur lineages. These findings underscore that while it is possible to evolve resistance to antagonism, complex evolutionary trajectories are required to avoid detrimental host fitness consequences.

Virulence and transmission vary between Usutu virus lineages in Culex pipiens.

Usutu virus (USUV) is a zoonotic arbovirus infecting mainly wild birds. It is transmitted by ornithophilic mosquitoes, mainly of the genus Culex from birds to birds and to several vertebrate dead-end hosts. Several USUV lineages, differing in their virulence have emerged in the last decades and now co-circulate in Europe, impacting human populations. However, their relative transmission and effects on their mosquito vectors is still not known. We thus compared the vector competence and survival of Culex pipiens mosquitoes experimentally infected with two distinct USUV lineages, EU2 and EU3, that are known to differ in their virulence and replication in vertebrate hosts. Infection rate was variable among blood feeding assays but variations between EU2 and EU3 lineages were consistent suggesting that Culex pipiens was equally susceptible to infection by both lineages. However, EU3 viral load increased with viral titer in the blood meal while EU2 viral load was high at all titers which suggest a greater replication of EU2 than EU3 in mosquito. While their relative transmission efficiencies are similar, at least at low blood meal titer, positive correlation between transmission and blood meal titer was observed for EU3 only. Contrary to published results in vertebrates, EU3 induced a higher mortality to mosquitoes (i.e. virulence) than EU2 whatever the blood meal titer. Therefore, we found evidence of lineage-specific differences in vectorial capacity and virulence to both the vector and vertebrate host which lead to balanced propagation of both viral lineages. These results highlight the need to decipher the interactions between vectors, vertebrate hosts, and the diversity of arbovirus lineages to fully understand transmission dynamics.

The glycosylation deficiency of flavivirus NS1 attenuates virus replication through interfering with the formation of viral replication compartments.

BACKGROUND: Flavivirus is a challenge all over the world. The replication of flavivirus takes place within membranous replication compartments (RCs) derived from endoplasmic reticulum (ER). Flavivirus NS1 proteins have been proven essential for the formation of viral RCs by remodeling the ER. The glycosylation of flavivirus NS1 proteins is important for viral replication, yet the underlying mechanism remains unclear. METHODS: HeLa cells were used to visualize the ER remodeling effects induced by NS1 expression. ZIKV replicon luciferase assay was performed with BHK-21 cells. rZIKV was generated from BHK-21 cells and the plaque assay was done with Vero Cells. Liposome co-floating assay was performed with purified NS1 proteins from 293T cells. RESULTS: We found that the glycosylation of flavivirus NS1 contributes to its ER remodeling activity. Glycosylation deficiency of NS1, either through N-glycosylation sites mutations or tunicamycin treatment, compromises its ER remodeling activity and interferes with viral RCs formation. Disruption of NS1 glycosylation results in abnormal aggregation of NS1, rather than reducing its membrane-binding activity. Consequently, deficiency in NS1 glycosylation impairs virus replication. CONCLUSIONS: In summary, our results highlight the significance of NS1 glycosylation in flavivirus replication and elucidate the underlying mechanism. This provides a new strategy for combating flavivirus infections.

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.

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.

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.

Dissemination of the Flavivirus Subgenomic Replicon Genome and Viral Proteins by Extracellular Vesicles.

Extracellular vesicles (EVs) such as exosomes have been shown to play physiological roles in cell-to-cell communication by delivering various proteins and nucleic acids. In addition, several studies revealed that the EVs derived from the cells that are infected with certain viruses could transfer the full-length viral genomes, resulting in EVs-mediated virus propagation. However, the possibility cannot be excluded that the prepared EVs were contaminated with infectious viral particles. In this study, the cells that harbor subgenomic replicon derived from the Japanese encephalitis virus and dengue virus without producing any replication-competent viruses were employed as the EV donor. It was demonstrated that the EVs in the culture supernatants of those cells were able to transfer the replicon genome to other cells of various types. It was also shown that the EVs were incorporated by the recipient cells primarily through macropinocytosis after interaction with CD33 and Tim-1/Tim-4 on HeLa and K562 cells, respectively. Since the methods used in this study are free from contamination with infectious viral particles, it is unequivocally indicated that the flavivirus genome can be transferred by EVs from cell to cell, suggesting that this pathway, in addition to the classical receptor-mediated infection, may play some roles in the viral propagation and pathogenesis.

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.

The topological model of NS4B and its TMD3 in duck TMUV proliferation.

Duck Tembusu virus (DTMUV) belongs to the Flaviviridae family and mainly infects ducks. Duck Tembusu virus genome encodes one polyprotein that undergoes cleavage to produce 10 proteins. Among these, NS4B, the largest transmembrane protein, plays a crucial role in the viral life cycle. In this study, we investigated the localization of NS4B and found that it is located in the endoplasmic reticulum, where it co-localizes with DTMUV dsRNA. Subsequently, we confirmed 5 different transmembrane domains of NS4B and discovered that only its transmembrane domain 3 (TMD3) can traverse ER membrane. Then mutations were introduced in the conserved amino acids of NS4B TMD3 of DTMUV replicon and infectious clone. The results showed that V111G, V117G, and I118G mutations enhanced viral RNA replication, while Q104A, T106A, A113L, M116A, H120A, Y121A, and A122G mutations reduced viral replication. Recombinant viruses with these mutations were rescued and studied in BHK21 cells. The findings demonstrated that A113L and H120A mutations led to higher viral titers than the wild-type strain, while Q104A, T106A, V111G, V117G, and Y121A mutations attenuated viral proliferation. Additionally, H120A, M116A, and A122G mutations enhanced viral proliferation. Furthermore, Q104A, T106A, V111G, M116A, V117G, Y121A, and A122G mutants showed reduced viral virulence to 10-d duck embryos. Animal experiments further indicated that all mutation viruses resulted in lower genome copy numbers in the spleen compared to the WT group 5 days postinfection. Our data provide insights into the topological model of DTMUV NS4B, highlighting the essential role of NS4B TMD3 in viral replication and proliferation.

CDK5-mediated rearrangement of vimentin during Duck Tembusu virus infection inhibits viral replication.

Duck Tembusu virus (DTMUV) is a newly emerging pathogen that causes massive economic losses to the poultry industry in China and neighbouring countries. Vimentin, an intermediate filament protein, has been demonstrated to be involved in viral replication during infection. However, the specific role of vimentin in DTMUV replication has not been determined. In this study, we found that overexpression of vimentin in BHK-21 cells can inhibit DTMUV replication. Moreover, DTMUV replication was enhanced after vimentin expression was reduced in BHK-21 cells via small interfering RNA (siRNA). Further research indicated that DTMUV infection had no effect on the transcription or expression of vimentin. However, we found that DTMUV infection induced vimentin rearrangement, and the rearrangement of vimentin was subsequently confirmed to negatively modulate viral replication through the use of a vimentin network disrupting agent. Vimentin rearrangement is closely associated with its phosphorylation. Our experiments revealed that the phosphorylation of vimentin at Ser56 was promoted in the early stage of DTMUV infection. In addition, by inhibiting the phosphorylation of vimentin at Ser56 with a CDK5 inhibitor, vimentin rearrangement was suppressed, and DTMUV replication was significantly enhanced. These results indicated that DTMUV infection induced vimentin phosphorylation and rearrangement through CDK5, resulting in the inhibition of DTMUV replication. In summary, our study reveals a role for vimentin as a negative factor in the process of DTMUV replication, which helps to elucidate the function of cellular proteins in regulating DTMUV replication.

A naturally isolated symbiotic bacterium suppresses flavivirus transmission by Aedes mosquitoes.

The commensal microbiota of the mosquito gut plays a complex role in determining the vector competence for arboviruses. In this study, we identified a bacterium from the gut of field Aedes albopictus mosquitoes named Rosenbergiella sp. YN46 (Rosenbergiella_YN46) that rendered mosquitoes refractory to infection with dengue and Zika viruses. Inoculation of 1.6 × 103 colony forming units (CFUs) of Rosenbergiella_YN46 into A. albopictus mosquitoes effectively prevents viral infection. Mechanistically, this bacterium secretes glucose dehydrogenase (RyGDH), which acidifies the gut lumen of fed mosquitoes, causing irreversible conformational changes in the flavivirus envelope protein that prevent viral entry into cells. In semifield conditions, Rosenbergiella_YN46 exhibits effective transstadial transmission in field mosquitoes, which blocks transmission of dengue virus by newly emerged adult mosquitoes. The prevalence of Rosenbergiella_YN46 is greater in mosquitoes from low-dengue areas (52.9 to ~91.7%) than in those from dengue-endemic regions (0 to ~6.7%). Rosenbergiella_YN46 may offer an effective and safe lead for flavivirus biocontrol.

The infection kinetics and transmission potential of two Guaico Culex viruses in Culex quinquefasciatus mosquitoes.

Guaico Culex virus (GCXV) is a newly identified segmented Jingmenvirus from Culex spp. mosquitoes in Central and South America. The genome of GCXV is composed of four or five single-stranded positive RNA segments. However, the infection kinetics and transmission capability of GCXV in mosquitoes remain unknown. In this study, we used reverse genetics to rescue two GCXVs (4S and 5S) that contained four and five RNA segments, respectively, in C6/36 â€‹cells. Further in vitro characterization revealed that the two GCXVs exhibited comparable replication kinetics, protein expression and viral titers. Importantly, GCXV RNAs were detected in the bodies, salivary glands, midguts and ovaries of Culex quinquefasciatus at 4-10 days after oral infection. In addition, two GCXVs can colonize Cx. quinquefasciatus eggs, resulting in positive rates of 15%-35% for the second gonotrophic cycle. In conclusion, our results demonstrated that GCXVs with four or five RNA segments can be detected in Cx. quinquefasciatus eggs during the first and second gonotrophic cycles after oral infection.

Pathogenicity and Interspecies Transmission of Cluster 3 Tembusu Virus Strain TMUV HQ-22 Isolated from Geese.

Since 2010, the Tembusu virus (TMUV) has been highly prevalent in China, causing significant economic losses to the poultry industry. In 2022, a suspected outbreak of TMUV occurred at a goose farm located in Anhui Province. A strain of TMUV, TMUV HQ-22, was isolated from the infected geese. Phylogenetic analysis using the E gene of the HQ-22 strain demonstrated its affiliation with cluster 3, a less commonly reported cluster in comparison to the main circulating cluster, cluster 2. Through a comparison of the envelope (E) protein of HQ-22 with other typical TMUV strains, a mutation at the 157th amino acid position was identified, wherein valine (V) in cluster 3 changed to alanine (A), a characteristic that is unique to cluster 2. These findings highlight the diversity and complexity of the TMUV strains circulating in China. In our experimental analysis, an injection of TMUV HQ-22 into the muscles of 3-day-old goslings resulted in severe neurological symptoms and a mortality rate of 60%. Similarly, the intracranial or intranasal infection of 3-week-old ICR mice with TMUV HQ-22 led to severe neurological symptoms and respective mortality rates of 100% or 10%. In summary, our study isolated a TMUV strain, TMUV HQ-22, from geese that belongs to cluster 3 and exhibits significant pathogenicity in both goslings and ICR mice. These results emphasize the genetic diversity of the TMUV circulating in China and expand the host range beyond mosquitoes to include ducks, chickens, geese, and even mice. It is crucial to not underestimate the risk of TMUV infection in mammals, warranting our utmost attention.