An evolutionarily conserved ubiquitin ligase drives infection and transmission of flaviviruses.

Mosquito-borne flaviviruses such as dengue (DENV) and Zika (ZIKV) cause hundreds of millions of infections annually. The single-stranded RNA genome of flaviviruses is translated into a polyprotein, which is cleaved equally into individual functional proteins. While structural proteins are packaged into progeny virions and released, most of the nonstructural proteins remain intracellular and could become cytotoxic if accumulated over time. However, the mechanism by which nonstructural proteins are maintained at the levels optimal for cellular fitness and viral replication remains unknown. Here, we identified that the ubiquitin E3 ligase HRD1 is essential for flaviviruses infections in both mammalian hosts and mosquitoes. HRD1 directly interacts with flavivirus NS4A and ubiquitylates a conserved lysine residue for ER-associated degradation. This mechanism avoids excessive accumulation of NS4A, which otherwise interrupts the expression of processed flavivirus proteins in the ER. Furthermore, a small-molecule inhibitor of HRD1 named LS-102 effectively interrupts DENV2 infection in both mice and Aedes aegypti mosquitoes, and significantly disturbs DENV transmission from the infected hosts to mosquitoes owing to reduced viremia. Taken together, this study demonstrates that flaviviruses have evolved a sophisticated mechanism to exploit the ubiquitination system to balance the homeostasis of viral proteins for their own advantage and provides a potential therapeutic target to interrupt flavivirus infection and transmission.

Single-Dose Immunogenic DNA Vaccines Coding for Live-Attenuated Alpha- and Flaviviruses.

Single-dose, immunogenic DNA (iDNA) vaccines coding for whole live-attenuated viruses are reviewed. This platform, sometimes called immunization DNA, has been used for vaccine development for flavi- and alphaviruses. An iDNA vaccine uses plasmid DNA to launch live-attenuated virus vaccines in vitro or in vivo. When iDNA is injected into mammalian cells in vitro or in vivo, the RNA genome of an attenuated virus is transcribed, which starts replication of a defined, live-attenuated vaccine virus in cell culture or the cells of a vaccine recipient. In the latter case, an immune response to the live virus vaccine is elicited, which protects against the pathogenic virus. Unlike other nucleic acid vaccines, such as mRNA and standard DNA vaccines, iDNA vaccines elicit protection with a single dose, thus providing major improvement to epidemic preparedness. Still, iDNA vaccines retain the advantages of other nucleic acid vaccines. In summary, the iDNA platform combines the advantages of reverse genetics and DNA immunization with the high immunogenicity of live-attenuated vaccines, resulting in enhanced safety and immunogenicity. This vaccine platform has expanded the field of genetic DNA and RNA vaccines with a novel type of immunogenic DNA vaccines that encode entire live-attenuated viruses.

Role of long non-coding RNA DLY6E in regulating TMUV infection.

Long non-coding RNA (lncRNA) is a type of RNA with a length greater than 200 nt and lacking coding ability. In recent years, a considerable number of lncRNAs have been found to have important functions. The lncRNA plays an important role in growth and development, body metabolism, immune function, and regulation of viral replication. A lncRNA, MSTRG8505.2, was screened and named lncRNA DLY6E, which was a new duck-derived lncRNA. The lncRNADLY6E in this study has a complex secondary structure, specifically distributed in the heart, liver and other organs. The expression of lncRNA DLY6E was significantly up-regulated after TMUV infection, which was time-dependent and non-dose-dependent. Overexpression of three structural proteins and seven non-structural proteins of TMUV in DEF cells showed no significant difference in the expression of lncRNADLY6E. Meanwhile, using lipopolysaccharides (LPS) and poly (I:C) to stimulate DEF cells, the results showed that the induced expression of lncRNA DLY6E was associated with the dsRNA-related TLR3/RIG-I/MDA5 pathway rather than the LPS activated signaling pathway. To further explore the function of lncRNA DLY6E, an eukaryotic expression vector was constructed. Overexpression of lncRNA DLY6E in DEF cells can increase the replication of TMUV. After overexpression of lncRNADLY6E, the transcriptional level of its target gene LY6E was detected, and the results showed that lncRNADLY6E did not act through its target gene. Overexpression of lncRNA DLY6E significantly inhibited the mRNA levels of OAS, Mx and PKR, suggesting that lncRNA DLY6E may promote the virus by inhibiting the transcription of antiviral proteins in innate immunity. This phenomenon provides new ideas for the prevention and control of TMUV, which is worth further thinking and exploration.

Recruitment of the 40S ribosomal subunit by the West Nile virus 3' UTR promotes the cross-talk between the viral genomic ends for translation regulation.

Flaviviral RNA genomes are composed of discrete RNA structural units arranged in an ordered fashion and grouped into complex folded domains that regulate essential viral functions, e.g. replication and translation. This is achieved by adjusting the overall structure of the RNA genome via the establishment of inter- and intramolecular interactions. Translation regulation is likely the main process controlling flaviviral gene expression. Although the genomic 3' UTR is a key player in this regulation, little is known about the molecular mechanisms underlying this role. The present work provides evidence for the specific recruitment of the 40S ribosomal subunit by the 3' UTR of the West Nile virus RNA genome, showing that the joint action of both genomic ends contributes the positioning of the 40S subunit at the 5' end. The combination of structural mapping techniques revealed specific conformational requirements at the 3' UTR for 40S binding, involving the highly conserved SL-III, 5'DB, 3'DB and 3'SL elements, all involved in the translation regulation. These results point to the 40S subunit as a bridge to ensure cross-talk between both genomic ends during viral translation and support a link between 40S recruitment by the 3' UTR and translation control.

The Role of Noncoding RNA in the Transmission and Pathogenicity of Flaviviruses.

Noncoding RNAs (ncRNAs) constitute a class of RNA molecules that lack protein-coding capacity. ncRNAs frequently modulate gene expression through specific interactions with target proteins or messenger RNAs, thereby playing integral roles in a wide array of cellular processes. The Flavivirus genus comprises several significant members, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV), which have caused global outbreaks, resulting in high morbidity and mortality in human populations. The life cycle of arthropod-borne flaviviruses encompasses their transmission between hematophagous insect vectors and mammalian hosts. During this process, a complex three-way interplay occurs among the pathogen, vector, and host, with ncRNAs exerting a critical regulatory influence. ncRNAs not only constitute a crucial regulatory mechanism that has emerged from the coevolution of viruses and their hosts but also hold potential as antiviral targets for controlling flavivirus epidemics. This review introduces the biogenesis of flavivirus-derived ncRNAs and summarizes the regulatory roles of ncRNAs in viral replication, vector-mediated viral transmission, antiviral innate immunity, and viral pathogenicity. A profound comprehension of the interplay between ncRNAs and flaviviruses will help formulate efficacious prophylactic and therapeutic strategies against flavivirus-related diseases.

Temperature affects viral kinetics and vectorial capacity of Aedes aegypti mosquitoes co-infected with Mayaro and Dengue viruses.

BACKGROUND: Increasing global temperatures and unpredictable climatic extremes have contributed to the spread of vector-borne diseases. The mosquito Aedes aegypti is the main vector of multiple arboviruses that negatively impact human health, mostly in low socioeconomic areas of the world. Co-circulation and co-infection of these viruses in humans have been increasingly reported; however, how vectors contribute to this alarming trend remains unclear. METHODS: Here, we examine single and co-infection of Mayaro virus (D strain, Alphavirus) and dengue virus (serotype 2, Flavivirus) in Ae. aegypti adults and cell lines at two constant temperatures, moderate (27 °C) and hot (32 °C), to quantify vector competence and the effect of temperature on infection, dissemination and transmission, including on the degree of interaction between the two viruses. RESULTS: Both viruses were primarily affected by temperature but there was a partial interaction with co-infection. Dengue virus quickly replicates in adult mosquitoes with a tendency for higher titers in co-infected mosquitoes at both temperatures, and mosquito mortality was more severe at higher temperatures in all conditions. For dengue, and to a lesser extent Mayaro, vector competence and vectorial capacity were higher at hotter temperature in co- vs. single infections and was more evident at earlier time points (7 vs. 14 days post infection) for Mayaro. The temperature-dependent phenotype was confirmed in vitro by faster cellular infection and initial replication at higher temperatures for dengue but not for Mayaro virus. CONCLUSIONS: Our study suggests that contrasting kinetics of the two viruses could be related to their intrinsic thermal requirements, where alphaviruses thrive better at lower temperatures compared to flaviviruses. However, more studies are necessary to clarify the role of co-infection at different temperature regimes, including under more natural temperature settings.

Low-temperature culture enhances production of flavivirus virus-like particles in mammalian cells.

Flavivirus virus-like particles (VLPs) exhibit a striking structural resemblance to viral particles, making them highly adaptable for various applications, including vaccines and diagnostics. Consequently, increasing VLPs production is important and can be achieved by optimizing expression plasmids and cell culture conditions. While attempting to express genotype III (GIII) Japanese encephalitis virus (JEV) VLPs containing the G104H mutation in the envelope (E) protein, we failed to generate VLPs in COS-1 cells. However, VLPs production was restored by cultivating plasmid-transfected cells at a lower temperature, specifically 28 °C. Furthermore, we observed that the enhancement in JEV VLPs production was independent of amino acid mutations in the E protein. The optimal condition for JEV VLPs production in plasmid-transfected COS-1 cells consisted of an initial culture at 37 °C for 6 h, followed by a shift to 28 °C (37/28 °C) for cultivation. Under 37/28 °C cultivation conditions, flavivirus VLPs production significantly increased in various mammalian cell lines regardless of whether its expression was transiently transfected or clonally selected cells. Remarkably, clonally selected cell lines expressing flavivirus VLPs consistently achieved yields exceeding 1 µg/ml. Binding affinity analyses using monoclonal antibodies revealed similar binding patterns for VLPs of genotype I (GI) JEV, GIII JEV, West Nile virus (WNV), and dengue virus serotype 2 (DENV-2) produced under both 37 °C or 37/28 °C cultivation conditions. In summary, our study demonstrated that the production of flavivirus VLPs can be significantly improved under 37/28 °C cultivation conditions without affecting the conformational structure of the E protein. KEYPOINTS: • Low-temperature culture (37/28 °C) enhances production of flavivirus VLPs. • Flavivirus VLPs consistently achieved yields exceeding 1 µg/ml. • 37/28 °C cultivation did not alter the structure of flavivirus VLPs.

Haemovigilance survey and screening strategy for arthropod-borne viruses in blood donors from Argentina.

Arthropod-borne viruses (arboviruses) count among emerging infections, which represent a major challenge for transfusion safety worldwide. To assess the risk of arboviruses-transmission by transfusion (ATT), we performed a survey to evaluate the potential threat for transfusion safety. Samples were retrospectively and randomly collected from donors who donated during the peak of dengue incidence in Cordoba (years: 2016 and 2019-2022). A cost-efficient strategy for molecular screening was implemented with a nucleic acid test (NAT) configured with Flavivirus and Alphavirus-universal degenerated primers targeting conserved gene regions. Besides, we evaluated the neutralizing antibody (NAb) prevalence by plaque reduction neutralization test (PRNT). A total of 1438 samples were collected. Among the NAT-screened samples, one resulted positive for Flavivirus detection. Subsequent sequencing of the PCR product revealed Saint Louis Encephalitis Virus (SLEV) infection (GeneBank accession number OR236721). NAb prevalence was 2.95% for anti-Dengue, 9.94% anti-SLEV, 1.09% anti-West Nile Virus, and 0% anti-Chikungunya. One of the NAb-positive samples also resulted positive for IgM against SLEV but negative by ARN detection. This is the first haemovigilance study developed in Argentina that evaluates the potential risk of ATT and the first research to determine the prevalence of NAb against Flavivirus through PNRT to avoid possible cross-reactions between Ab against Flavivirus. Herein, the finding of one SLEV-viremic donor and the detection of anti-SLEV IgM in a different donor demonstrated a potential threat for transfusion safety and emphasized the need for increased vigilance and proactive measures to ensure the safety of blood supplies.

A jingmenvirus RNA-dependent RNA polymerase structurally resembles the flavivirus counterpart but with different features at the initiation phase.

Jingmenviruses are a category of emerging segmented viruses that have garnered global attention in recent years, and are close relatives of the flaviviruses in the Flaviviridae family. One of their genome segments encodes NSP1 homologous to flavivirus NS5. NSP1 comprises both the methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRP) modules playing essential roles in viral genome replication and capping. Here we solved a 1.8-Å resolution crystal structure of the NSP1 RdRP module from Jingmen tick virus (JMTV), the type species of jingmenviruses. The structure highly resembles flavivirus NS5 RdRP despite a sequence identity less than 30%. NSP1 RdRP enzymatic properties were dissected in a comparative setting with several representative Flaviviridae RdRPs included. Our data indicate that JMTV NSP1 produces characteristic 3-mer abortive products similar to the hepatitis C virus RdRP, and exhibits the highest preference of terminal initiation and shorter-primer usage. Unlike flavivirus NS5, JMTV RdRP may require the MTase for optimal transition from initiation to elongation, as an MTase-less NSP1 construct produced more 4-5-mer intermediate products than the full-length protein. Taken together, this work consolidates the evolutionary relationship between the jingmenvirus group and the Flaviviridae family, providing a basis to the further understanding of their viral replication/transcription process.

Detection of anti-premembrane antibody as a specific marker of four flavivirus serocomplexes and its application to serosurveillance in endemic regions.

In the past few decades, several emerging/re-emerging mosquito-borne flaviviruses have resulted in disease outbreaks of public health concern in the tropics and subtropics. Due to cross-reactivities of antibodies recognizing the envelope protein of different flaviviruses, serosurveillance remains a challenge. Previously we reported that anti-premembrane (prM) antibody can discriminate between three flavivirus infections by Western blot analysis. In this study, we aimed to develop a serological assay that can discriminate infection or exposure with flaviviruses from four serocomplexes, including dengue (DENV), Zika (ZIKV), West Nile (WNV) and yellow fever (YFV) viruses, and explore its application for serosurveillance in flavivirus-endemic countries. We employed Western blot analysis including antigens of six flaviviruses (DENV1, 2 and 4, WNV, ZIKV and YFV) from four serocomplexes. We tested serum samples from YF-17D vaccinees, and from DENV, ZIKV and WNV panels that had been confirmed by RT-PCR or by neutralization assays. The overall sensitivity/specificity of anti-prM antibodies for DENV, ZIKV, WNV, and YFV infections/exposure were 91.7%/96.4%, 91.7%/99.2%, 88.9%/98.3%, and 91.3%/92.5%, respectively. When testing 48 samples from Brazil, we identified multiple flavivirus infections/exposure including DENV and ZIKV, DENV and YFV, and DENV, ZIKV and YFV. When testing 50 samples from the Philippines, we detected DENV, ZIKV, and DENV and ZIKV infections with a ZIKV seroprevalence rate of 10%, which was consistent with reports of low-level circulation of ZIKV in Asia. Together, these findings suggest that anti-prM antibody is a flavivirus serocomplex-specific marker and can be employed to delineate four flavivirus infections/exposure in regions where multiple flaviviruses co-circulate.

Cellular nuclear-localized U2AF2 protein is hijacked by the flavivirus 3'UTR for viral replication complex formation and RNA synthesis.

Japanese encephalitis virus (JEV) is a zoonotic pathogen belonging to the Flavivirus genus, causing viral encephalitis in humans and reproductive failure in swine. The 3' untranslated region (3'UTR) of JEV contains highly conservative secondary structures required for viral translation, RNA synthesis, and pathogenicity. Identification of host factors interacting with JEV 3'UTR is crucial for elucidating the underlying mechanism of flavivirus replication and pathogenesis. In this study, U2 snRNP auxiliary factor 2 (U2AF2) was identified as a novel cellular protein that interacts with the JEV genomic 3'UTR (the SL-I, SL-II, SL-III, and DB region) via its 1 to 148 amino acids. JEV infection or JEV 3' UTR on its own triggered the nuclear-localized U2AF2 redistributed to the cytoplasm and colocalized with viral replication complex. U2AF2 also interacts with JEV NS3 and NS5 protein, the downregulation of U2AF2 nearly abolished the formation of flavivirus replication vesicles. The production of JEV protein, RNA, and viral titers were all increased by U2AF2 overexpression and decreased by knockdown. U2AF2 also functioned as a pro-viral factor for Zika virus (ZIKV) and West Nile virus (WNV), but not for vesicular stomatitis virus (VSV). Mechanically, U2AF2 facilitated the synthesis of both positive- and negative-strand flavivirus RNA without affecting viral attachment, internalization or release process. Collectively, our work paves the way for developing U2AF2 as a potential flavivirus therapeutic target.

A Chimeric Classical Insect-Specific Flavivirus Provides Complete Protection Against West Nile Virus Lethal Challenge in Mice.

West Nile virus (WNV), an arthropod-borne flavivirus, can cause severe symptoms, including encephalitis, and death, posing a threat to public health and the economy. However, there is still no approved treatment or vaccine available for humans. Here, we developed a novel vaccine platform based on a classical insect-specific flavivirus (cISF) YN15-283-02, which was derived from Culicoides. The cISF-WNV chimera was constructed by replacing prME structural genes of the infectious YN15-283-02 cDNA clone with those of WNV and successfully rescued in Aedes albopictus cells. cISF-WNV was nonreplicable in vertebrate cells and nonpathogenic in type I interferon receptor (IFNAR)-deficient mice. A single-dose immunization of cISF-WNV elicited considerable Th1-biased antibody responses in C57BL/6 mice, which was sufficient to offer complete protection against lethal WNV challenge with no symptoms. Our studies demonstrated the potential of the insect-specific cISF-WNV as a prophylactic vaccine candidate to prevent infection with WNV.

Infectious clone of a contemporary Tembusu virus and replicons expressing reporter genes or heterologous antigens from poultry viruses.

The novel mosquito-borne Tembusu virus (TMUV, family Flaviviridae) was discovered as the cause of a severe outbreak of egg-drop syndrome affecting ducks in Southeast Asia in 2010. TMUV infection can also lead to high mortality in various additional avian species such as geese, pigeons, and chickens. This study describes the construction of an infectious cDNA clone of a contemporary duck-isolate (TMUV WU2016). The virus recovered after transfection of BHK-21 cells shows enhanced virus replication compared to the mosquito-derived MM1775 strain. Next, the WU2016 cDNA clone was modified to create a SP6 promoter-driven, self-amplifying mRNA (replicon) capable of expressing a range of different reporter genes (Renilla luciferase, mScarlet, mCherry, and GFP) and viral (glyco)proteins of avian influenza virus (AIV; family Orthomyxoviridae), infectious bursal disease virus (IDBV; family Bunyaviridae) and infectious bronchitis virus (IBV; family Coronaviridae). The current study demonstrates the flexibility of the TMUV replicon system, to produce different heterologous proteins over an extended period of time and its potential use as a platform technology for novel poultry vaccines.

Mutations in the 3' non-coding region of a no-known vector flavivirus Yokose virus increased its replication ability in mosquito C6/36 cells.

Yokose virus (YOKV) is a bat-associated no-known vector flavivirus group member. We investigated the replication ability of YOKV in mosquito-derived C6/36 cells. YOKV grew in C6/36 cells, but its kinetics of YOKV was markedly slower than those of other mosquito-borne flaviviruses. Transmission electron microscopy indicated an extremely small number of viral particles in YOKV-infected C6/36 cells. Mosquito-borne Japanese encephalitis virus prM-E-bearing chimeric YOKV failed to propagate efficiently in C6/36 cells. We isolated C6/36-adapted YOKV and identified nucleotide mutations in the adapted YOKV. Mutations detected in the 3' non-coding region of the adapted YOKV were critical for the enhanced proliferation ability of the virus. Moreover, the growth of the original and adapted YOKV in C6/36 cells was remarkably increased by shifting the culture temperature from 28 to 36 °C. Thus, our results demonstrate the potential of YOKV to propagate in mosquito cells and support its classification as a mosquito-borne flavivirus.

Duck Tembusu virus infection activates the MKK3/6-p38 MAPK signaling pathway to promote virus replication.

Duck Tembusu virus (DTMUV) infection poses a serious threat to ducks, chickens, and geese, causing a range of detrimental effects, including reduced egg production, growth retardation, and even death. These consequences lead to substantial economic losses for the Chinese poultry industry. Although it is established that various viral infections can trigger activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway, the precise role and mechanisms underlying p38 MAPK activation in DTMUV infection remain poorly understood. To address this knowledge gap, we conducted a study to investigate whether the replication of DTMUV necessitates the activation of p38 MAPK. We found that DTMUV infection stimulates activation of the MKK3/6-p38 MAPK pathway, and the activation of p38 MAPK increases with viral titer. Subsequently, the use of the small molecule inhibitor SB203580 significantly reduced DTMUV replication by inhibiting p38 MAPK activity. Furthermore, downregulation of p38 MAPK protein expression by siRNA also inhibited DTMUV replication, whereas transient transfection of p38 MAPK protein promoted DTMUV replication. Interestingly, we found that the DTMUV capsid protein activates p38 MAPK, and there is interaction between DTMUV capsid and p38 MAPK. Finally, we found that DTMUV infection induces elevated mRNA expression of IFN-α, IFN-ß, IFN-γ, IL-1ß, IL-6, and IL-12, which is associated with p38 MAPK activity. These results indicated that virus hijacking of p38 activation is a crucial event for DTMUV replication, and that pharmacological blockade of p38 activation represents a potential anti-DTMUV strategy.

Cytoarchitecture of ex vivo midgut cultures of unfed Ixodes scapularis infected with a tick-borne flavivirus.

A bite from an infected tick is the primary means of transmission for tick-borne flaviviruses (TBFV). Ticks ingest the virus while feeding on infected blood. The traditional view is that the virus first replicates in and transits the tick midgut prior to dissemination to other organs, including salivary glands. Thus, understanding TBFV infection in the tick midgut is a key first step in identifying potential countermeasures against infection. Ex vivo midgut cultures prepared from unfed adult female Ixodes scapularis ticks were viable and remained morphologically intact for more than 8 days. The midgut consisted of two clearly defined cell layers separated by a basement membrane: an exterior network of smooth muscle cells and an internal epithelium composed of digestive generative cells. The smooth muscle cells were arranged in a stellate circumferential pattern spaced at regular intervals along the long axis of midgut diverticula. When the cultures were infected with the TBFV Langat virus (LGTV), virus production increased by two logs with a peak at 96 hours post-infection. Infected cells were readily identified by immunofluorescence staining for the viral envelope protein, nonstructural protein 3 (NS3) and dsRNA. Microscopy of the stained cultures suggested that generative cells were the primary target for virus infection in the midgut. Infected cells exhibited an expansion of membranes derived from the endoplasmic reticulum; a finding consistent with TBFV infected cell cultures. Electron microscopy of infected cultures revealed virus particles in the basolateral region between epithelial cells. These results demonstrated LGTV replication in midgut generative cells of artificially infected, ex vivo cultures of unfed adult female I. scapularis ticks.

A novel tamanavirus (Flaviviridae) of the European common frog (Rana temporaria) from the UK.

Flavivirids are small, enveloped, positive-sense RNA viruses from the family Flaviviridae with genomes of ~9-13 kb. Metatranscriptomic analyses of metazoan organisms have revealed a diversity of flavivirus-like or flavivirid viral sequences in fish and marine invertebrate groups. However, no flavivirus-like virus has been identified in amphibians. To remedy this, we investigated the virome of the European common frog (Rana temporaria) in the UK, utilizing high-throughput sequencing at six catch locations. De novo assembly revealed a coding-complete virus contig of a novel flavivirid ~11.2 kb in length. The virus encodes a single ORF of 3456 aa and 5' and 3' untranslated regions (UTRs) of 227 and 666 nt, respectively. We named this virus Rana tamanavirus (RaTV), as BLASTp analysis of the polyprotein showed the closest relationships to Tamana bat virus (TABV) and Cyclopterus lumpus virus from Pteronotus parnellii and Cyclopterus lumpus, respectively. Phylogenetic analysis of the RaTV polyprotein compared to Flavivirus and Flavivirus-like members indicated that RaTV was sufficiently divergent and basal to the vertebrate Tamanavirus clade. In addition to the Mitcham strain, partial but divergent RaTV, sharing 95.64-97.39 % pairwise nucleotide identity, were also obtained from the Poole and Deal samples, indicating that RaTV is widespread in UK frog samples. Bioinformatic analyses of predicted secondary structures in the 3'UTR of RaTV showed the presence of an exoribonuclease-resistant RNA (xrRNA) structure standard in flaviviruses and TABV. To examine this biochemically, we conducted an in vitro Xrn1 digestion assay showing that RaTV probably forms a functional Xrn1-resistant xrRNA.

Viral RNA in Mosquitoes (Diptera: Culicidae) Collected between 2019 and 2021 in Germany.

Due to globalisation and climate change, mosquito-borne pathogens are emerging in new areas on all continents, including Europe, which has recently faced outbreaks of dengue, chikungunya and West Nile fever. The present study complements previous investigations to evaluate the circulation of mosquito-borne viruses in Germany, with the aim of identifying potential vector species and risk areas. Mosquitoes collected from 2019 to 2021 and identified to species or species group level were screened for viruses of the families Flaviviridae, Peribunyaviridae and the genus Alphavirus of the family Togaviridae. In total, 22,528 mosquitoes were examined, thus providing the most comprehensive study on West Nile virus (WNV) circulation so far in the German mosquito population. Usutu virus (USUV) RNA was detected in six samples, Sindbis virus (SINV) RNA in 21 samples and WNV RNA in 11 samples. Samples containing RNA of USUV and WNV consisted of mosquitoes collected in the East German federal states of Brandenburg, Saxony and Saxony-Anhalt, while samples with RNA of SINV originated from more widespread locations. Although minimum infection rates have remained relatively low, the intensity of virus circulation appears to be increasing compared to previous studies. Continuous mosquito screening contributes to the early detection of the introduction and spread of mosquito-borne pathogens.

Subgenomic Flaviviral RNAs of Dengue Viruses.

Subgenomic flaviviral RNAs (sfRNAs) are produced during flavivirus infections in both arthropod and vertebrate cells. They are undegraded products originating from the viral 3' untranslated region (3' UTR), a result of the action of the host 5'-3' exoribonuclease, Xrn1, when it encounters specific RNA structures known as Xrn1-resistant RNAs (xrRNAs) within the viral 3' UTR. Dengue viruses generate three to four distinct species of sfRNAs through the presence of two xrRNAs and two dumbbell structures (DBs). The tertiary structures of xrRNAs have been characterized to form a ringlike structure around the 5' end of the viral RNA, effectively inhibiting the activity of Xrn1. The most important role of DENV sfRNAs is to inhibit host antiviral responses by interacting with viral and host proteins, thereby influencing viral pathogenicity, replicative fitness, epidemiological fitness, and transmission. In this review, we aimed to summarize the biogenesis, structures, and functions of DENV sfRNAs, exploring their implications for viral interference.

Antiviral effects of duck type I and type III interferons against Duck Tembusu virus in vitro and in vivo.

Duck Tembusu Virus (DTMUV) is a newly emerging avian flavivirus that causes substantial economic losses to the duck industry in Asia by causing severe egg drop syndrome and fatal encephalitis in domestic ducks. During viral replication, host cells recognize the RNA structures produced by DTMUV, which triggers the production of interferons (IFNs) to inhibit viral replication. However, the function of duck type I and type III IFNs in inhibiting DTMUV infection remains largely unknown. In this study, we expressed and purified recombinant duck IFN-ß (duIFN-ß) and IFN-λ (duIFN-λ) in Escherichia coli and evaluated their antiviral activity against vesicular stomatitis virus (VSV). Furthermore, we found that both duIFN-ß and duIFN-λ activated the ISRE promoter and induced the expression of ZAP, OAS, and RNaseL in duck embryo fibroblasts (DEFs). Notably, duIFN-ß showed faster and more potent induction of ISGs in vitro and in vivo compared to duIFN-λ. Moreover, both duIFN-ß and duIFN-λ showed high potential to inhibit DTMUV infection in DEFs, with duIFN-ß demonstrating better antiviral efficacy than duIFN-λ against DTMUV in ducks. In conclusion, our results revealed that both duIFN-ß and duIFN-λ can induce ISGs production and exhibit significant antiviral activity against DTMUV in vitro and in vivo, providing new insights for the development of antiviral therapeutic strategies in ducks.