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Predicted 5-end stem-loop structures of flaviviruses. The optimal MFOLD-predicted RNA secondary-structure models for mosquitoborne and tick-borne flaviviruses and a flavivirus with no known vector are shown. Results are shown for WNV M12294, yellow fever virus (YFV) NC-002031, tick-borne encephalitis virus (TBEV) U27495, and Modoc virus (MODV) AJ242984.
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The 5′ untranslated region (5′UTR) of the dengue virus (DENV) genome contains two defined elements essential for viral replication.
At the 5′ end, a large stem-loop (SLA) structure functions as the promoter for viral polymerase activity. Next to the SLA,
there is a short stem-loop that contains a cyclization sequence known as the 5′ upstream AUG re...
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... suggesting a common role for this element in different mem- bers of the genus. A comparison of the predicted stem-loop structures present at the 5 ends of mosquito-borne flaviviruses (DENV, yellow fever virus, and WNV), tick-borne encephalitis virus, and a flavivirus with no known vector (Modoc virus) shows similar structural elements (Fig. 5). These structures contain two helical regions resembling S1 and S2 of DENV, which are always separated by a U bulge or a U-U mismatch, and an S3 region interrupted by mismatches in different loca- tions. In addition, all genomes bear a TL as well as an SSL with stems of different lengths (Fig. 5). In general, the 5SL is followed by a ...
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... (Modoc virus) shows similar structural elements (Fig. 5). These structures contain two helical regions resembling S1 and S2 of DENV, which are always separated by a U bulge or a U-U mismatch, and an S3 region interrupted by mismatches in different loca- tions. In addition, all genomes bear a TL as well as an SSL with stems of different lengths (Fig. 5). In general, the 5SL is followed by a short track of U residues, except in the tick- borne encephalitis virus genome. To study whether the con- served elements within the SLA were required for viral repli- cation, we designed new mutations altering each of these common ...
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... ELEMENTS FOR FLAVIVIRUS REPLICATION 1003 vious observations (mutant M342 in reference 11), these re- sults indicate that the function of the SLA in viral replication tolerates large variations within the SSL. Interestingly, great variability can be observed in the sequences and structures of the SSLs of different flavivirus 5SLs (Fig. 5). In addition, an important role for the TL of SLA was also confirmed. Previous studies indicated that the sequence of the TL plays a crucial role in SLA promoter activity (11). Here, we found that mutations altering the sequence of the TL gave rise to spontaneous mutations that rescued viral replication by par- tially reconstituting ...
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Citations
... It is also worth mentioning the presence of four pseudoknot elements, PK1-PK4, located throughout the 3′ UTR, which are required for translation, replication and infectivity ( Fig. 1) (Funk et al., 2010;Manzano et al., 2011;Sztuba-Solinska et al., 2013;Xing et al., 2021). The highly conserved sHP and 3′SL elements, located at the 3′-terminal end, contain the 3′ upstream of AUG region (3′UAR), 3′ downstream of AUG region (3′DAR) I and II and 3′ conserved/cyclization (3′CYC) sequence motifs, which interact with their corresponding partners in the 5′ UTR to achieve the cyclization of the viral genome (Alvarez et al., 2005;Friebe and Harris, 2010;Hahn et al., 1987;Khromykh et al., 2001;Lodeiro et al., 2009;Mandl et al., 1993). Cyclization is required for the recruitment of the NS5 polymerase during the initiation of the minus strand RNA synthesis (Davis et al., 2013;Dong et al., 2008;Filomatori et al., 2006;Liu et al., 2016;Meyer et al., 2020;Zhang et al., 2008), while it interferes with efficient translation initiation by affecting the folding of those structural elements required for the proper ribosome scanning, e.g., cHP (Funk et al., 2010;Manzano et al., 2011;Sztuba--Solinska et al., 2013;Xing et al., 2021). ...
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.
... Dengue virus has four serotypes (DENV1 to DENV4) and can produce clinical illnesses ranging from dengue fever, a broad-spectrum flu-like syndrome, to dengue hemorrhagic fever and lethal disease. Dengue is an acute, swiftly spreading, dengue fever disease that has a self-limited incubation period of 5-7 days, with myalgia, rash, headache, lymphadenopathy, and leukopenia (2). Dengue has been continuously escalating (a 30-fold increase over the past 50 years) to new areas and re-emerging in areas where it had recently been controlled. ...
... Dengue virus is a member of the genus Flavivirus in the Flaviviridae family. Dengue virus has a single-stranded positive-sense RNA virus, about 11 kb in length, which encodes a long polyprotein that is synthesized a post-translational and host proteases, yielding three structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (2). Dengue is spread by the of Aedes mosquito, mainly in the area where discharge water lines can occur within a few minutes to several weeks depend upon the environment. ...
In Pakistan, dengue fever has undergone significant demographic change over the past two decades, and as a result of globalization and climate change, the disease now poses a greater threat to humanity. By comparing the hematological and biochemical parameters in dengue-positive patients, this study seeks to better understand the ongoing endemic dengue fever in Peshawar, Pakistan. The cross-sectional observational study was conducted from November 2022 to January 2023 at Lady Reading Hospital in Peshawar, Khyber Pakhtunkhwa, Pakistan. A sample of 100 patients who tested positive for dengue was gathered and evaluated. Our findings show that those between 25 to 40 years are more likely to get dengue fever. The elevated alanine transaminase levels and significant p-value in the study indicated a link between dengue infection and liver cell damage. The study also discovered an increase in white blood cell count with marginally lower normal hemoglobin levels, which raises the possibility of an underlying immune system reaction or imbalance. Hematological and biochemical parameter means, however, fell within the normal range.
... The ZIKV 5′ UTR is 106-nt, and the 3′ UTR is 428-nt in length 25 . The 5′ UTR consists of an m7GpppAmpN1 cap structure and the conserved stem loops A and B (SLA and SLB, Fig. 1A) 26,27 . The cap structure in flavivirus genomes is important for cap-dependent translation and protection from cellular 5′-3′ exonucleases 26,28 . ...
Zika virus (ZIKV) is a mosquito-transmitted flavivirus that has caused devastating congenital Zika syndrome (CZS), including microcephaly, congenital malformation, and fetal demise in human newborns in recent epidemics. ZIKV infection can also cause Guillain-Barré syndrome (GBS) and meningoencephalitis in adults. Despite intensive research in recent years, there are no approved vaccines or antiviral therapeutics against CZS and adult Zika diseases. In this report, we developed a novel live-attenuated ZIKV strain (named Z7) by inserting 50 RNA nucleotides (nt) into the 5' untranslated region (UTR) of a pre-epidemic ZIKV Cambodian strain, FSS13025. We used this particular ZIKV strain as it is attenuated in neurovirulence, immune antagonism, and mosquito infectivity compared with the American epidemic isolates. Our data demonstrate that Z7 replicates efficiently and produces high titers without causing apparent cytopathic effects (CPE) in Vero cells or losing the insert sequence, even after ten passages. Significantly, Z7 induces robust humoral and cellular immune responses that completely prevent viremia after a challenge with a high dose of an American epidemic ZIKV strain PRVABC59 infection in type I interferon (IFN) receptor A deficient (Ifnar1-/-) mice. Moreover, adoptive transfer of plasma collected from Z7 immunized mice protects Ifnar1-/- mice from ZIKV (strain PRVABC59) infection. These results suggest that modifying the ZIKV 5' UTR is a novel strategy to develop live-attenuated vaccine candidates for ZIKV and potentially for other flaviviruses.
... The 5′ CS, which is an 11 nucleotides long (134-UCA AUA UGCUG), mediates long-range RNA-RNA interactions between the ends of the RNA for the genome cyclization. The cyclization of the viral RNA occurs when the 5′UAR and 5′CS hybridize with the counterparts in the 3′ UTR, which is a process required for transferring the viral polymerase from the 5′ SLA to the 3′ ends to initiate genome replication (Fig. 2B) [53,[76][77][78][79][80][81]. ...
Dengue fever is a dengue virus infection, emerging rapidly and posing public health threat worldwide, primarily in tropical and subtropical countries. Nearly half of the world's population is now at risk of contracting the dengue virus, including new countries with no previous history-like Ethiopia. However, little is known about the epidemiology and impact of the disease in different countries. This is especially true in countries, where cases have recently begun to be reported. This review aims to summarize epidemiology, biology, pathogenesis, clinical manifestations, and diagnosis of dengue virus infection and its trend in Ethiopia. It may help countries, where dengue fever is not yet on the public health list-like Ethiopia to alert healthcare workers to consider the disease for diagnosis and treatment. The review retrieved and incorporated 139 published and organizational reports showing approximately 390 million new infections. About 100 million of these infections develop the clinical features of dengue, and thousands of people die annually from severe dengue fever in 129 countries. It is caused by being bitten by a dengue virus-infected female mosquito, primarily Aedes aegypti and, lesser, Ae. albopictus. Dengue virus is a member of the Flavivirus genus of the Flaviviridae family and has four independent but antigen-related single-stranded positive-sense RNA virus serotypes. The infection is usually asymptomatic but causes illnesses ranging from mild febrile illness to fatal dengue hemorrhagic fever or shock syndrome. Diagnosis can be by detecting the virus genome using nucleic acids amplification tests or testing NS1 antigen and/or anti-dengue antibodies from serum, plasma, circulating blood cells, or other tissues. Dengue cases and outbreaks have increased in recent decades, with a significant public health impact. Ethiopia has had nearly annual outbreaks since 2013, devastating an already fragmented health system and economy. Standardization of medication, population-level screening for early diagnosis and prompt treatment, and minimization of mosquito bites reduce overall infection and mortality rates.
... The 5 0 UTR of the dengue virus (DENV) is the most well-studied of all known human pathogenic flaviviruses. The topology of the DENV 5 0 UTR is essential for efficient cyclization and viral replication [22,23,35,36]. The 5 0 UTR of DENV contains a Y shaped stem loop A (SLA) structure, which resembles that of MBFV and TBFV. ...
... The 5 0 UTR of DENV contains a Y shaped stem loop A (SLA) structure, which resembles that of MBFV and TBFV. Elements within the SLA (namely SL1-2 and S0, Fig 1) have been characterized for DENV, West Nile virus and ZIKA [1,10,[35][36][37]. Disruption in the 5' RNA elements (SLA and SLB) influence virus pathogenicity. ...
Tick-borne Encephalitis Virus (TBEV) is an emerging flavivirus that causes neurological disorders including viral encephalitis of varying severity. Whilst secondary RNA structures within the 5' untranslated regions (UTRs) of many flaviviruses determine both virus replication and pathogenic outcomes in humans, these elements have not been systematically investigated for TBEV. In this study, we investigated the role of predicted RNA secondary elements of the first 107 nucleotides (nts) of the viral genome forming the stem-loop A (SLA). Experiments were performed in replicons and infectious TBEV system. This region comprises three distinct structures: 5' stem 0 (S0), stem-loop 1 (SL1) and stem-loop 2 (SL2). S0 was found to be essential for virus infection as mutations in the lower stem of this region significantly reduced virus replication. Point mutations in SL1 that preserved the Y-shape confirmation delayed viral RNA replication but did not abolish virus infectivity. Deletion of SL2 did not abolish infectivity but had a negligible effect on virus propagation. No correlation was observed between in vitro translation efficiency and virus infectivity, suggesting that the 5'UTR functions independently to virus translation. Together, these findings reveal distinct RNA elements within the 5'UTR that are essential for the stability and replication of viral RNA. We further identify changes in RNA folding that lead to altered TBEV infectivity and pathogenesis.
... However, the most severe forms of the disease are Dengue hemorrhagic fever (DHF) and the Dengue shock syndrome (DSS) characterized by thrombocytopenia and vascular leakage leading to hypervolemia, with high mortality without prompt management. Although severe Dengue disease has historically been associated with pediatric populations in areas of hyper-endemia (12,13), recent trends indicate that adults may also be at risk (14,15). ...
Background: Dengue is still a public health problem in tropical countries. This disease, which had almost disappeared in some areas of the world, has become re-emergent in certain parts of the world including Africa. The aim of this study is to determine the seroprevalence and evolution of Dengue virus (DENV) infection from 2020 to 2021 at the Hospital Saint Camille de Ouagadougou (HOSCO), Burkina Faso. Methodology: This was a descriptive analytical study of patients seen in general practice with febrile syndrome referred for serological diagnosis of Dengue at the HOSCO laboratory over a period of 2 years (January 1, 2020-December 31, 2021). The "Dengue Duo (AgNS1/IgM/IgG)" kit from SD Bioline was used for the rapid diagnosis through the detection of NS1 antigen and IgM/IgG antibodies in plasma. Data were analysed with SPSS version 20.0 software. Association between demographic data and prevalence of DENV infection was determined by Chi square test and odds ratio (with 95% confidence interval). P value less than 0.05 was considered statistical significance. Results: A total of 2957 patients aged 0-94 years were referred for serological diagnosis of DENV infection at the HOSCO laboratory over the period 2020-2021, comprising 56.3% females and 43.7% males. The overall prevalence of acute DENV infection (NS1Ag positive) was 5.4% (159/2957), with 2.4% (41/1700) in 2020 and 9.4% (118/1257) in 2021 (OR=4.192, 95% CI=2.915-6.028, p<0.0001). The prevalence of acute DENV infection of 7.0% (91/1292) in the males was significantly higher than 4.1% (68/1665) in the females (OR=1.779, 95% CI=1.288-2.458, p=0.0005), and also significantly higher in age groups 20-29 years (7.6%), 10-19 years (6.9%) and 40-49 years (5.8%) than other age groups (X 2 =14.928, p=0.0107). The overall prevalence of DENV IgM and IgG antibodies was 3.2% and 37.3% respectively. The prevalence of DENV IgG antibodies was significantly higher in males (44.0%) than females (32.1%) (OR=1.667, 95%CI=1.434-1.938, p<0.0001) and in age groups 30-39 (43.4%), 40-49 (44.0%) and >50 years (49.3%) than other age groups (X 2 =121.0, p<0.0001), indicating that past exposure to DENV infection is higher among males and older age groups. The peak of DENV infection was between October and November with 84.3% (134/159) of NS1Ag positivity occurring during this period. Conclusion: The present study reports a high prevalence of acute Dengue virus infection in patients from October to November. To eradicate Dengue which has become a tropical silent epidemic, interventions such as vector control, availability of and accessibility to diagnostic tests, and good therapeutic management are of great importance.
... during viral infection [21][22][23]. The ORF encodes a polyprotein consisting of ~3400 amino acids (Fig. 2b), which is cleaved by both viral and host proteases to form three SPs -the capsid (C), membrane/pre-membrane (M/prM), and envelope (E), proteins -and seven NSPs: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. ...
Japanese encephalitis virus (JEV), a single-stranded, enveloped RNA virus, is a health concern across Asian countries, associated with severe neurological disorders, especially in children. Primarily, pigs, bats, and birds are the natural hosts for JEV, but humans are infected incidentally. JEV requires a few host proteins for its entry and replication inside the mammalian host cell. The endoplasmic reticulum (ER) plays a significant role in JEV genome replication and assembly. During this process, the ER undergoes stress due to its remodelling and accumulation of viral particles and unfolded proteins, leading to an unfolded protein response (UPR). Here, we review the overall strategy used by JEV to infect the host cell and various cytopathic effects caused by JEV infection. We also highlight the role of JEV structural proteins (SPs) and non-structural proteins (NSPs) at various stages of the JEV life cycle that are involved in up- and downregulation of different host proteins and are potentially relevant for developing efficient therapeutic drugs.
Graphical abstract
... In particular, the highly conserved stem-loop A (SLA) in the 5 ′ -UTR, close to the 5 ′end of the genome, functions as a "promoter" for the NS5 protein to initiate minus-strand synthesis (Filomatori et al. 2006;Gebhard et al. 2011;Choi 2021). The interaction between SLA and NS5 is critical for RNA synthesis and was confirmed to be direct by biochemical studies; removal of SLA and mutation of certain nucleotides drastically decreases RNA replication (Filomatori et al. 2006;Lodeiro et al. 2009). Consistent with its essential functional role, the sequence and secondary structure of the SLA are highly conserved (Fig. 1). ...
... S1, S2), and to avoid dimer formation through the side stem-loop, as described next. The top terminal loop is an important site for SLA function (Filomatori et al. 2006Lodeiro et al. 2009); thus the top loop of DenvTSL was retained as wild type. ...
... The fact that DENV3 has a single U bulge in the bottom stem and mutational studies of the U-UU bulge implies that Ura63 is critical for SLA promoter function, but the two remaining nucleotides are not. Deletion or mutations of the U-rich bulge largely impair viral RNA replication in vivo, but the effect on the binding affinity to RdRp and its in vitro activity is insignificant (Lodeiro et al. 2009;Filomatori et al. 2011). This suggests that the U-rich bulge interacts with another protein, which is important for viral replication in infected cells. ...
Dengue virus, a single-stranded positive sense RNA virus, is the most prevalent mosquito-borne pathogen in the world. Like all RNA viruses, it uses conserved structural elements within its genome to control essential replicative steps. A 70 nucleotides stem-loop RNA structure (called SLA) found at the 5’-end of the genome of all flaviviruses, functions as the promoter for viral replication. This highly conserved structure interacts with the viral polymerase NS5 to initiate RNA synthesis. Here we report the NMR structure of a monomeric SLA from Dengue virus serotype 1, assembled to high-resolution from independently folded structural elements. The DENV1 SLA has an L-shape structure, where the top and side helices are coaxially-stacked and the bottom helix is roughly perpendicular to them. Because the sequence is highly conserved among different flavivirus genomes, it is likely that the three-dimensional fold and local structure of SLA are also conserved among flaviviruses and required for efficient replication. This work provides structural insight into the Dengue promoter and provides the foundation for the discovery of new antiviral drugs that target this essential replicative step.
... removal of SLA and mutation of certain nucleotides drastically decreases RNA replication (Filomatori et al., 2006;Lodeiro et al., 2009). Consistent with its essential functional role, the sequence and secondary structure of the SLA are highly conserved ( Figure 1). ...
... The fact that DENV3 has a single U bulge in the bottom stem and mutational studies of the U-UU bulge imply that Ura63 is critical for SLA promoter function, but the two remaining nucleotides are not. Deletion or mutations of the U-rich bulge largely impair viral RNA replication in vivo, but the effect on the binding affinity to RdRp and its in vitro activity is insignificant (Filomatori et al., 2011;Lodeiro et al., 2009). This suggests that U-rich bulge interacts with another protein which is important for viral replication in infected cells. ...
... ; https://doi.org/10. 1101 The apical loop is important for SLA function and highly conserved; the CAG(X)U sequence is found in all four Dengue serotypes (Figure 1), and mutations impair both viral replication in vivo and RdRp activity in vitro (Filomatori et al., 2011;Filomatori et al., 2006;Lodeiro et al., 2009). Interestingly, mutations in the terminal loop do not significantly affect RdRp binding, suggesting the terminal loop might play an important role in post-binding steps to promote polymerase activity (Filomatori et al., 2011). ...
Dengue virus, a single-stranded positive sense RNA virus, is the most prevalent mosquito-borne pathogen in the world. Like all RNA viruses, it uses conserved structural elements within its genome to control essential replicative steps. A 70 nucleotides stem-loop RNA structure (called SLA) found at the 5'-end of the genome of all flaviviruses, functions as the promoter for viral replication. This highly conserved structure interacts with the viral polymerase NS5 to initiate RNA synthesis. Here we report the NMR structure of a monomeric SLA from Dengue virus serotype 1, assembled to high-resolution from independently folded structural elements. The DENV1 SLA has an L-shape structure, where the top and side helices are coaxially-stacked and the bottom helix is roughly perpendicular to them. Because the sequence is highly conserved among different flavivirus genomes, it is likely that the three-dimensional fold and local structure of SLA are also conserved among flaviviruses and required for efficient replication. This work provides structural insight into the Dengue promoter and provides the foundation for the discovery of new antiviral drugs that target this essential replicative step.
... Fig. S10). However, SLA and SLB of DONV were predicted to 90 nt and 20 nt in length with no intervening poly(U) sequence, which is believed to be important for the virus replication (34). Replacing the SLA or SLB of DONV with that of ZIKV (designated as DONV 5 0 1 to 81 or DONV 5 0 82 to 107) also enhanced RNA replication in 293T cells by real-time PCR and dsRNA staining, yet showed no expression of E protein, implying that the complete 5 0 UTR was important for the viral protein translation (Fig. 4 B-D). ...
... Replacement of 5 0 or 3 0 UTR separately (named as DONV 5 0 and DONV 3 0 ) showed the similar effects in 293T cells, irrespective of the introduction of two noncomplimentary nucleotides between the 5 0 CS and 3 0 CS (Fig. 4A and SI Appendix, Fig. S7A)(33). The 5 0 UTR of ZIKV consists of SLA and SLB, which are 70 nt and 30 nt in length separated by a poly(U) sequence(34,35) (Fig. 4Aand SI Appendix, ...
Significance
Most viruses have a relatively narrow host range. In contrast, vector-borne flaviviruses, such as dengue virus and Zika virus, maintain their transmission cycle between arthropods and vertebrates, belonging to different phyla. How do these viruses adapt to the distinct cellular environments of two phyla? By comparing the single-host insect--specific flavivirus and dual-host Zika virus, we identified three key molecular factors that determine MBF host tropism. This study will greatly increase the understanding of entry, replication, and cross-species evolution of mosquito-borne flaviviruses.
