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.

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.

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.

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.

The role of duck LGP2 in innate immune response of host anti-tembusu virus.

Laboratory of Genetics and Physiology 2 (LGP2), along with Retinoic Acid Induced Gene-I (RIG-I) and Melanoma Differentiation Associated Gene 5, are members of the retinoic acid-inducible gene-I-like receptors (RLRs) in pattern recognition receptors, playing an important role in the host's innate immunity. Due to lacking a caspase activation and recruitment domain, LGP2 is controversially regarded as a positive or negative regulator in the antiviral response. This study aimed to explore how duck LGP2 (duLGP2) participates in duck innate immunity and its role in countering the duck Tembusu virus (DTMUV). In duck embryo fibroblast cells, the overexpression of duLGP2 significantly reduced the cell's antiviral capacity by inhibiting type I interferon (IFN) production and the expression of downstream IFN-stimulated genes. Conversely, duLGP2 knockdown had the opposite effect. For the first time, we introduced the LGP2 gene fragment into duck embryos using a lentiviral vector to ensure persistent expression and generated gene-edited ducks with LGP2 overexpression. We demonstrated that duLGP2 facilitates DTMUV replication in both in vitro and in vivo experiments, leading to robust inflammatory and antiviral responses. Interestingly, the repressive effects of duLGP2 on type I IFN production were only observed in the early stage of DTMUV infection, with type I IFN responses becoming enhanced as the viral load increased. These results indicate that duLGP2 acts as a negative regulator during the resting state and early stages of DTMUV infection. This study provides a theoretical basis for further research on duck RLRs and developing new anti-DTMUV drugs or vaccine adjuvants.

Flaviviruses in AntiTumor Therapy.

Oncolytic viruses offer a promising approach to tumor treatment. These viruses not only have a direct lytic effect on tumor cells but can also modify the tumor microenvironment and activate antitumor immunity. Due to their high pathogenicity, flaviviruses have often been overlooked as potential antitumor agents. However, with recent advancements in genetic engineering techniques, an extensive history with vaccine strains, and the development of new attenuated vaccine strains, there has been a renewed interest in the Flavivirus genus. Flaviviruses can be genetically modified to express transgenes at acceptable levels, and the stability of such constructs has been greatly improving over the years. The key advantages of flaviviruses include their reproduction cycle occurring entirely within the cytoplasm (avoiding genome integration) and their ability to cross the blood-brain barrier, facilitating the systemic delivery of oncolytics against brain tumors. So far, the direct lytic effects and immunomodulatory activities of many flaviviruses have been widely studied in experimental animal models across various types of tumors. In this review, we delve into the findings of these studies and contemplate the promising potential of flaviviruses in oncolytic therapies.

NS5 hijacks TRAF3 to inhibit type I interferon signaling during duck Tembusu virus infection.

The tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) is a key signaling molecule in the retinoic acid-inducible gene I (RIG-I) signaling pathway and plays an important role in host innate immune regulation. The function of TRAF3 has been extensively studied in mammals, however, the role of TRAF3 in ducks remains unclear. In order to reveal the function of duck TRAF3 (duTRAF3) in the innate immune response induced by virus infection, the TRAF3 homologue of mallard (Anas platyrhynchos) has been cloned and the function of duTRAF3 is investigated in this study. We sequenced duTRAF3 and found that the open reading frame (ORF) region of duTRAF3 is 1704 bp long and encodes 567 amino acids (aa), which has a similar functional domain to the mammalian gene. Analysis of tissue distribution of duTRAF3 in 7-day-old ducks showed that the expression of duTRAF3 was highest in harderian gland, followed by heart and lung. Subsequently, duck Tembusu virus (DTMUV) has been shown to enhance duTRAF3 expression, and overexpression of duTRAF3 inhibits DTMUV replication in a dose-dependent manner. In addition, duTRAF3 activates the transcriptional activity of IFN-α and its downstream interferon-stimulating genes (ISGs) induced after DTMUV infection. In this process, DTMUV non-structural (NS) protein 5 resists this innate immune process by interacting with TRAF3 and inhibiting TRAF3 expression. These data support the conclusion that duTRAF3 is an antiviral protein that plays a key role in the defense against DTMUV invasion. These results lay a theoretical foundation for developing new anti-DTMUV strategies.

Structural and functional basis of low-affinity SAM/SAH-binding in the conserved MTase of the multi-segmented Alongshan virus distantly related to canonical unsegmented flaviviruses.

Alongshan virus (ALSV), a newly discovered member of unclassified Flaviviridae family, is able to infect humans. ALSV has a multi-segmented genome organization and is evolutionarily distant from canonical mono-segmented flaviviruses. The virus-encoded methyltransferase (MTase) plays an important role in viral replication. Here we show that ALSV MTase readily binds S-adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH) but exhibits significantly lower affinities than canonical flaviviral MTases. Structures of ALSV MTase in the free and SAM/SAH-bound forms reveal that the viral enzyme possesses a unique loop-element lining side-wall of the SAM/SAH-binding pocket. While the equivalent loop in flaviviral MTases half-covers SAM/SAH, contributing multiple hydrogen-bond interactions; the pocket-lining loop of ALSV MTase is of short-length and high-flexibility, devoid of any physical contacts with SAM/SAH. Subsequent mutagenesis data further corroborate such structural difference affecting SAM/SAH-binding. Finally, we also report the structure of ALSV MTase bound with sinefungin, an SAM-analogue MTase inhibitor. These data have delineated the basis for the low-affinity interaction between ALSV MTase and SAM/SAH and should inform on antiviral drug design.

New insight into flavivirus maturation from structure/function studies of the yellow fever virus envelope protein complex.

IMPORTANCE: All enveloped viruses enter cells by fusing their envelope with a target cell membrane while avoiding premature fusion with membranes of the producer cell-the latter being particularly important for viruses that bud at internal membranes. Flaviviruses bud in the endoplasmic reticulum, are transported through the TGN to reach the external milieu, and enter other cells via receptor-mediated endocytosis. The trigger for membrane fusion is the acidic environment of early endosomes, which has a similar pH to the TGN of the producer cell. The viral particles therefore become activated to react to mildly acidic pH only after their release into the neutral pH extracellular environment. Our study shows that for yellow fever virus (YFV), the mechanism of activation involves actively knocking out the fusion brake (protein pr) through a localized conformational change of the envelope protein upon exposure to the neutral pH external environment. Our study has important implications for understanding the molecular mechanism of flavivirus fusion activation in general and points to an alternative way of interfering with this process as an antiviral treatment.

Investigation of Flaviviruses Emerging in Brazil as Etiology Factor in Nonsyndromic Orofacial Cleft.

Brazil has one of the largest forest areas on the planet and the potential for the emergence of new diseases. In turn, orofacial clefts, especially cleft lip and or palate (CL/P), are characterized as congenital malformations and may be associated with genetic and environmental factors. The present study aimed to investigate in silico the flavivirus's potential to emerge in Brazil as an etiology of CL/P. A scoring method was created based on literature and nucleotide similarity analysis. An integrative analysis of the literature was performed to answer the questions through the databases PubMed/MEDLINE, SciELO, LILACS, and Google Scholar to have a more significant number of results. The software Basic Local Alignment Search Tool-BLAST 2.12.0, through the Genomic + Transcript Databases (Human Genomic plus Transcript Human G+T), was selected to find similarities with human sequences associated with CL/P. The viral sequences used were obtained from the National Center for Biotechnology Information Virus-NCBI Virus, in which only complete and referential genomes were selected. The flavivirus that emerged in Brazil and presented a high potential to cause CL/P was the Iguape virus strain (species Aroa virus ), followed by the Cacipacore virus and the Rocio virus strain (species Ilheus virus ) with medium potential to cause CL/P. In conclusion, we suggest among the virus evaluated that the Iguape virus presented a high potential of causing CL/P. As prevention, the control of arthropods and the hospital diffusion on viral dynamics, mainly in the CL/P context and other congenital malformations, are indicated.

The anti-immune dengue subgenomic flaviviral RNA is present in vesicles in mosquito saliva and is associated with increased infectivity.

Mosquito transmission of dengue viruses to humans starts with infection of skin resident cells at the biting site. There is great interest in identifying transmission-enhancing factors in mosquito saliva in order to counteract them. Here we report the discovery of high levels of the anti-immune subgenomic flaviviral RNA (sfRNA) in dengue virus 2-infected mosquito saliva. We established that sfRNA is present in saliva using three different methods: northern blot, RT-qPCR and RNA sequencing. We next show that salivary sfRNA is protected in detergent-sensitive compartments, likely extracellular vesicles. In support of this hypothesis, we visualized viral RNAs in vesicles in mosquito saliva and noted a marked enrichment of signal from 3'UTR sequences, which is consistent with the presence of sfRNA. Furthermore, we show that incubation with mosquito saliva containing higher sfRNA levels results in higher virus infectivity in a human hepatoma cell line and human primary dermal fibroblasts. Transfection of 3'UTR RNA prior to DENV2 infection inhibited type I and III interferon induction and signaling, and enhanced viral replication. Therefore, we posit that sfRNA present in salivary extracellular vesicles is delivered to cells at the biting site to inhibit innate immunity and enhance dengue virus transmission.

Dronabinol as an answer to flavivirus infections: an in-silico investigation.

Flavivirus infections are common in several parts of the world. Two major types of flaviviruses are dengue and zika viruses. Both these two viral infections have caused many fatalities around the world. There is an absence of a vaccine and an effective medication against these viruses. In this study, we analyzed the ability of dronabinol to act as a potential cure against these viral infections. We performed the docking of dronabinol with several viral proteins followed by molecular dynamics simulation, MM/PBSA and PCA analysis. We checked the ability of the polyphenol dronabinol to interfere with the binding of viral helicases to their cellular targets. We performed 2 D-QSAR studies, drug likeliness, ADMET and target prediction studies. From our study, we observed that dronabinol had the best docking ability against the helicase proteins of dengue and zika. Molecular dynamics simulation and MM/PBSA investigation confirmed the stability of the binding while PCA investigation showed a lowering of molecular motions in response to dronabinol docking to the helicases. Dronabinol interfered in the binding of the helicases to RNA. 2 D QSAR studies revealed a low IC50 value for dronabinol. Dronabinol showed favorable drug-likeness, ADMET properties and target prediction results. Thus we propose dronabinol be further investigated in-vitro as a cure against dengue and zika virus infections.Communicated by Ramaswamy H. Sarma.

Peptide Microarrays for Flavivirus Diagnosis.

Flavivirus are the most alarming prevalent viruses worldwide due to its vast impact on public health. Most early symptoms of diseases caused by Flavivirus are similar among each other and to other febrile illnesses making the clinical differential diagnosis challenging. In addition, due to cross-reactivity and a relatively limited persistence of viral RNA in infected individuals, the current available diagnosis strategies fail to efficiently provide a differential viral identification. In this context, virus-specific tests are essential to improve patient care, as well as to facilitate disease surveillance and the effective control of transmission. Here, we describe the use of protein microarrays as an effective tool for screening peptides differentially recognized by anti-Yellow Fever virus antibodies induced by vaccination or by natural viral infection.

Structure and function of the NS5 methyltransferase domain from Usutu virus.

Usutu virus (USUV), is a mosquito-borne flavivirus currently spreading outside the African continent producing substantial avian mortality. In contrast, infected humans could exhibit mild neurological symptoms or remain asymptomatic. As in other flaviviruses, the capped USUV genome encodes three structural and seven non-structural (NS) proteins. Among the NS proteins, NS5 plays crucial roles in virus replication, harbouring the capping and methyltransferase (MTase) activities in its N-terminal domain and the RNA-dependent RNA polymerase (RdRP) activity at the C-terminus. In this work, we present the first structural and functional characterization of the USUV MTase domain. The first structure of the USUV MTase has been determined in complex with its natural ligands (S-adenosyl-L-methionine [SAM]) and S-adenosyl-L-homocysteine [SAH]) at 2.2 Å resolution, showing a molecular dimer in the crystal asymmetric unit. One molecule is bound to the methyl donor SAM while the second is bound to the reaction by-product SAH. Both molecules are almost identical and also show a high structural similarity to the MTase domains of other flaviviruses. The structure of the USUV MTase bound to the inhibitor sinefungin at 1.8 Å resolution is also described. Careful comparisons of the interactions in the SAM-binding cavity prompt us to hypothesize about the strength and weakness of the structure-based design of antivirals directed to the SAM/SAH binding site that could be effective to deal with this threat.

Duck TRIM35 Promotes Tembusu Virus Replication by Interfering with RIG-I-Mediated Antiviral Signaling in Duck Embryo Fibroblasts.

In China, the duck industry has been severely impacted by the newly emerging duck Tembusu virus (DTMUV). For DTMUV to successfully infect host cells, it employs several strategies that subvert the host's innate immune response. It has been found that several viral proteins encoded by DTMUV have strategically targeted the crucial molecules of the RIG-I-like Receptor (RLR) signaling pathway to antagonize host antiviral responses. However, it is not well known how the host proteins manipulated by DTMUV contribute to innate immune evasion. The present study reports that duck TRIM35 (duTRIM35) antagonizes DTMUV-induced innate immune responses by targeting duck RIG-I (duRIG-I) in duck embryo fibroblasts. A significant increase in duTRIM35 expression occurred during DTMUV infection. DuTRIM35 overexpression suppressed DTMUV-triggered expression of interferon beta (IFN-ß) and interferon-stimulated genes (ISGs), promoting viral replication, whereas knockdown of duTRIM35 augments the innate immune response, reducing viral replication. Furthermore, duTRIM35 significantly impaired the IFN-ß expression mediated by duRIG-I but not by other RLR signaling molecules. Mechanistically, duTRIM35 interfered with duRIG-I-duTRIM25 interaction and impeded duTRIM25-mediated duRIG-I ubiquitination by interacting with both duRIG-I and duTRIM25. Our findings indicate that duTRIM35 expression induced by DTMUV infection interfered with the duRIG-I-mediated antiviral response, illustrating a novel strategy in which DTMUV can evade the host's innate immunity. IMPORTANCE Duck Tembusu virus (DTMUV), an emerging flavivirus pathogen causing a substantial drop in egg production and severe neurological disorders in duck populations, has led to massive economic losses in the global duck industry. DTMUV has employed various strategies to subvert the host's innate immune response to establish a productive infection in host cells. In this study, we report that duck TRIM35 (duTRIM35) expression was upregulated upon DTMUV infection in vitro and in vivo, and its expression antagonized DTMUV-induced innate immune responses by targeting duck RIG-I (duRIG-I) in duck embryo fibroblasts. Further studies suggest that duTRIM35 interfered with duRIG-I-duTRIM25 interaction and impeded duTRIM25-mediated duRIG-I ubiquitination by interacting with both duRIG-I and duTRIM25. Together, these results revealed that duTRIM35 expression induced by DTMUV infection downregulated duRIG-I-mediated host antiviral response, which elucidated a novel strategy of DTMUV for innate immune evasion.

Eeyarestatin I, an inhibitor of the valosin-containing protein, exhibits potent virucidal activity against the flaviviruses.

Cellular responses to stress generally lead to the activation of the endoplasmic reticulum-associated protein degradation (ERAD) pathway. Several lines of study support that ERAD may be playing a proviral role during flaviviral infection. A key host factor in ERAD is the valosin-containing protein (VCP), an ATPase which ushers ubiquitin-tagged proteins to degradation by the proteasome. VCP exhibits different proviral activities, such as engaging in the biogenesis of viral replication organelles and facilitating flavivirus genome uncoating after the viral particle entry. To investigate the possible antiviral value of drugs targeting VCP, we tested two inhibitors: eeyarestatin I (EEY) and xanthohumol (XAN). Both compounds were highly effective in suppressing Zika virus (ZIKV) and Usutu virus (USUV) replication during infection in cell culture. Further analysis revealed an unexpected virucidal activity for EEY, but not for XAN. Preincubation of ZIKV or USUV with EEY before inoculation to cells resulted in significant decreases in infectivity in a dose- and time-dependent manner. Viral genomes in samples previously treated with EEY were more sensitive to propidium monoazide, an intercalating agent, with 10- to 100-fold decreases observed in viral RNA levels, supporting that EEY affects viral particle integrity. Altogether, these results support that EEY is a strong virucide against two unrelated flaviviruses, encouraging further studies to investigate its potential use as a broad-acting drug or the development of improved derivatives in the treatment of flaviviral infection.

Molecular cloning, functional characterization of duck TRADD and its effect on infection with duck Tembusu virus.

Tumor necrosis factor receptor 1 (TNFR1) associated death domain protein (TRADD) is a pivotal adaptor in TNF signaling pathway and plays an important role in apoptosis and immune regulation. The function of TRADD has been investigated extensively in mammals, however, the role of TRADD in ducks remains obscure. To reveal the function of duck TRADD (duTRADD) in the apoptosis and innate immune response, the TRADD homologue of mallard (Anas platyrhynchos) has been cloned and the function of duTRADD is investigated in this study. We conducted sequence analysis of the duTRADD, the open reading frame (ORF) region of duTRADD gene was 1065 bp, encoding 354 amino acids (aa), which shares similar functional domain to its mammalian counterpart. Tissue distribution profile of duTRADD in 7-day-old ducklings showed that the expression level of the gene was the highest in heart, followed by liver and brain. Accordingly, duck Tembusu virus (DTMUV) has been shown to decrease duTRADD expression, while overexpression of duTRADD inhibited DTMUV replication in a dose-dependent manner. Furthermore, duTRADD activated the transcriptional activity of caspase-3/8/9, the flow cytometry showed that duTRADD significantly induced apoptosis. However, duTRADD showed hardly any effect on the transcriptional activity of IFN-α/ß and its downstream interferon-stimulated genes (ISGs). The current data support the conclusion that duTRADD is a novel pro-apoptotic protein with a critical role in defense against DTMUV invasion. These results lay the theoretical foundation for the development of new anti-DTMUV strategies.

Conserved motifs in the flavivirus NS3 RNA helicase enzyme.

Flaviviruses are a major health concern because over half of the world population is at risk of infection and there are very few antiviral therapeutics to treat diseases resulting from infection. Replication is an essential part of the flavivirus survival. One of the viral proteins, NS3 helicase, is critical for unwinding the double stranded RNA intermediate during flaviviral replication. The helicase performs the unwinding of the viral RNA intermediate structure in an ATP-dependent manner. NS3 helicase is a member of the Viral/DEAH-like subfamily of the superfamily 2 helicase containing eight highly conserved structural motifs (I, Ia, II, III, IV, IVa, V, and VI) localized between the ATP-binding and RNA-binding pockets. Of these structural motifs only three are well characterized for function in flaviviruses (I, II, and VI). The roles of the other structural motifs are not well understood for NS3 helicase function, but comparison of NS3 with other superfamily 2 helicases within the viral/DEAH-like, DEAH/RHA, and DEAD-box subfamilies can be used to elucidate the roles of these structural motifs in the flavivirus NS3 helicase. This review aims to summarize the role of each conserved structural motif within flavivirus NS3 in RNA helicase function. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA in Disease and Development > RNA in Disease.

Mapping of a unique epitope on domain III of the envelope protein of Tembusu virus.

We recently developed a Tembusu virus (TMUV)-specific monoclonal antibody (MAb) 12F11, which was found to recognize a long amino acid sequence between residues 8 and 77 of domain III of the envelope protein (EDIII). Here, the epitope recognized by MAb 12F11 was mapped using alanine substitutions combined with dissociation constant analysis. The findings, and prediction of tertiary structure of TMUV EDIII, showed that the MAb 12F11 epitope contained one critical residue and 13 peripheral residues. Moreover, the antigenic site was shown to span four loops (N-terminal region, AB, BC, and CD) and three ß-strands (A, B, and D). The present work contributes to the understanding of antigenic structure of TMUV envelope protein.

The host range restriction of bat-associated no-known-vector flaviviruses occurs post-entry.

Most flaviviruses are transmitted horizontally between vertebrate hosts by haematophagous arthropods. Others exhibit host ranges restricted to vertebrates or arthropods. Vertebrate-specific flaviviruses are commonly referred to as no-known-vector (NKV) flaviviruses and can be separated into bat- and rodent-associated NKV flaviviruses. Rio Bravo virus (RBV) is one of eight recognized bat-associated NKV (B-NKV) flaviviruses. Studies designed to identify the genetic determinants that condition the host range restriction of B-NKV flaviviruses have never been performed. To investigate whether the host range restriction occurs at the level of attachment or entry, chimeric flaviviruses were created by inserting the pre-membrane and envelope protein genes of RBV into the genetic backbones of yellow fever virus (YFV) and Zika virus (ZIKV), two mosquito-borne flaviviruses associated with human disease. The chimeric viruses infected both vertebrate and mosquito cells. In vertebrate cells, all viruses produced similar mean peak titres, but the chimeric viruses grew more slowly than their parental viruses during early infection. In mosquito cells, the chimeric virus of YFV and RBV grew more slowly than YFV at early post-inoculation time points, but reached a similar mean peak titre. In contrast, the chimeric virus of ZIKV and RBV produced a mean peak titre that was approximately 10-fold lower than ZIKV. The chimeric virus of YFV and RBV produced an intermediate plaque phenotype, while the chimeric virus of ZIKV and RBV produced smaller plaques than both parental viruses. To conclude, we provide evidence that the structural glycoproteins of RBV permit entry into both mosquito and vertebrate cells, indicating that the host range restriction of B-NKV flaviviruses is mediated by a post-attachment/entry event.