Global Consensus Sequence Development and Analysis of Dengue NS3 Conserved Domains

Ayub, Ambreen; Ashfaq, Usman Ali; Idrees, Sobia; Haque, Asma

doi:10.1089/biores.2013.0022

Cited by 4 publications

(4 citation statements)

References 19 publications

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“…The results indicate that compounds 1a and 1b have an antiviral effect in the replicon model and DENV4 infection. The specific inhibition observed in 1a and 1b could be due to the genome variability between serotypes and particularly for NS3, the conserved regions of the protein are few [34]. The same behaviorcan be observed for the positive control L-CAR that reduces the viral load specifically for DENV2 in our results but not DENV4.…”

Section: Antiviral Activity On Dengue Virus Serotypesupporting

confidence: 81%

In Vitro and In Silico Studies of Bis-furyl-pyrrolo[3,4-b]pyridin-5-ones on Dengue Virus

Morales-Salazar,

Garduño-Albino,

Montes-Enríquez

et al. 2024

J. Mex. Chem. Soc.

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A series of six bis-furyl-pyrrolo[3,4-b]pyridin-5-ones synthesized via an Ugi-Zhu reaction coupled to a cascade process [aza Diels-Alder cycloaddition/N-acylation/aromatization] were evaluated in vitro against Dengue virus serotype 4 infection, and the Dengue virus replicon system encoding a Renilla luciferase gen reporter. Also, in silico studies on the non-structural protein 3 (NS3), a flavivirus protease comprising an attractive target for development of therapeutic antivirals bound to non-structural protein 2B (NS3-NS2B) were performed. The in vitro results showed that compounds 1a and 1b reduced the expression of Renilla luciferase in 44.2 and 31.6%, respectively. Additionally, the same compounds decreased viral load, thus revealing their potential activity against Dengue virus serotype 4. From in silico simulations, it was developed a NS3-NS2B model, which was used as a target for the studied molecules. Computational results agree with experimental data, showing that 1a is the best ligand. Finally, a pharmacophoric model was computed for NS3-NS2B, which shows that the ligands need two hydrophobic and one hydrophilic fragment. Such results suggest that two out of the six synthesized bis-furyl-pyrrolo[3,4-b]pyridin-5-ones derivatives presents potential antiviral activity against Dengue virus in vitro. Resumen. Una serie de seis bis-furil-pirrolo[3,4-b]piridin-5-onas sintetizadas vía una reacción Ugi-Zhu acoplada a un proceso en cascada [cicloadición aza Diels-Alder/N-acilación/aromatización] fueron evaluadas in vitro contra infección por el serotipo 4 del virus del dengue y el sistema de replicón del virus del Dengue que codifica un gen reportero de la luciferasa de la Renilla. Además, se realizaron estudios in silico sobre la proteína no estructural 3 (NS3), una proteasa de flavivirus que comprende un blanco atractivo para el desarrollo de antivirales terapéuticos unidos a la proteína no estructural 2B (NS3-NS2B). Los estudios in vitro revelaron que los compuestos 1a y 1b reducen la expresión de Renilla luciferasa en un 44.2 y 31.6%, respectivamente. Adicionalmente, estos compuestos redujeron la carga viral, revelando así su actividad potencial contra el virus del Dengue serotipo 4. Derivado de las simulaciones in silico, se obtuvo un modelo homólogo para NS3-NS2B, el cual fue considerado como blanco de las moléculas estudiadas. Los resultados computacionales correlacionan con los experimentales, mostrando que 1a es el mejor ligando. Finalmente, se generó un modelo farmacofórico para NS3-NS2B, el cual muestra que los ligandos necesitan dos fragmentos hidrofóbicos y uno hidrofílico. Estos resultados demuestran que dos de los seis compuestos que se estudiaron presentan actividad antiviral in vitro.

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Section: Antiviral Activity On Dengue Virus Serotypesupporting

confidence: 81%

In Vitro and In Silico Studies of Bis-furyl-pyrrolo[3,4-b]pyridin-5-ones on Dengue Virus

Morales-Salazar,

Garduño-Albino,

Montes-Enríquez

et al. 2024

J. Mex. Chem. Soc.

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“…While Dengue viruses and Zika viruses often harbour a tryptophan, Usutu virus has a valine and Saint Louis encephalitis virus a phenylalanine at this position [86][87][88][89]. In fact, the 583 position does not localize to the conserved RNA helicase domain [90,91]. At the genomic position 8500 within the NS5 gene, the amino acid change N275D was strongly selected in the pre-existing haplotypes 2a and 2b in lines B and C. N275D was described in two JEV isolates (GenBank: MH753129.1, JN381843.1) and other Flaviviruses like West Nile virus, yellow fever virus, Yokose virus and Murray valley encephalitis virus 22 [92][93][94].…”

Section: Discussionmentioning

confidence: 99%

“…While Dengue viruses and Zika viruses often harbour a tryptophan, Usutu virus has a valine and Saint Louis encephalitis virus a phenylalanine at this position [86–89]. In fact, the 583 position does not localize to the conserved RNA helicase domain [90,91].…”

Section: Discussionmentioning

confidence: 99%

Fitness adaptations of Japanese encephalitis virus in pigs following vector-free serial passaging

Marti,

Nater,

Magalhaes

et al. 2024

Preprint

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Japanese encephalitis virus (JEV) is a zoonotic mosquito-transmitted Flavivirus circulating in birds and pigs. In humans, JEV can cause severe viral encephalitis with high mortality. Considering that vector-free direct virus transmission was observed in pigs, JEV introduction into an immunologically naïve pig population could result in a series of direct transmissions disrupting the alternating host cycling between vertebrates and mosquitoes. To assess the potential consequences of such a realistic scenario, we passaged JEV ten times in pigs. This resulted in higherin vivoviral replication, increased shedding, and stronger innate immune responses in pigs. Nevertheless, the viral tissue tropism remained similar and frequency of direct transmission was not enhanced. Next generation sequencing showed single nucleotide deviations in 10% of the genome during passaging. In total, 25 point mutations were selected to reach a frequency of at least 35% in one of the passages. From these, six mutations resulted in amino acid changes located in the precursor of membrane, the envelope, the non-structural 3 and the non-structural 5 proteins. In a competition experiment with two lines of passaging, the mutation M374L in the envelope protein and N275D in the non-structural protein 5 showed a fitness advantage in pigs. Altogether, the interruption of the alternating host cycle of JEV caused a prominent selection of viral quasispecies as well as selection of de novo mutations associated with fitness gains in pigs, albeit without enhancing direct transmission frequency.Author summaryJapanese encephalitis virus (JEV) represents a major health threat in parts of Asia and Oceania. Primary vertebrate hosts are birds and pigs, but human infection also occurs and can cause severe encephalitis with high mortality. Like other Flaviviruses transmitted by insect bites, JEV requires replication in alternating cycles between mosquitoes on one side and birds or pigs on the other side. However, we previously reported that direct transmissions between pigs in absence of mosquitos can occur. Considering the increased risks for such events after the spread of JEV to a new region with immunologically naïve pigs, the present study was performed to understand if and how a series of direct transmissions would promote JEV adaptations to pigs and change virus-host interactions. Pigs infected with JEV passaged ten times showed enhanced clinical symptoms and stronger antiviral immune response, but luckily no increase in direct transmission was observed. Nevertheless, genomic analysis demonstrated a complete change in dominant virus variants, as well as selection of six viral amino acid changes. This indicates that interruptions of the alternating lifestyle of JEV causes a strong evolutionary pressure, which through fitness adaptations can change the viral characteristics.

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“…The N-terminal (1-180 aa) region of NS3 has been identified as the protease domain, together with a peptidic cofactor NS2B to form the NS2B-NS3Pro complex, which has been considered as a primary target for antiviral drugs. [33][34][35] In our current work, the NS3-crRNA targets nucleotides encoding a 10-aa residue (NS3 55-64aa , FHTMWHVTRG) of a serine protease domain, which is highly conserved among the four DENV serotypes, 36 efficiently inhibiting the replication of DENV. As Cas13a protein undergoes further conformational changes since the target RNA is recognized and bound with the crRNA, the specific cleavage site of Cas13a on target RNA may be dependent on its configuration.…”

Section: Discussionmentioning

confidence: 99%

CRISPR-Cas13a Cleavage of Dengue Virus NS3 Gene Efficiently Inhibits Viral Replication

Wang²,

Dong

et al. 2020

Molecular Therapy - Nucleic Acids

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The CRISPR-Cas9 system has been applied to DNA editing with precision in eukaryotic and prokaryotic systems, but it is unable to edit RNA directly. A recently developed CRISPR-Cas13a system has been shown to be capable of effectively knocking down RNA expression in mammalian and plant cells. In this study, we employ the CRISPR-Cas13a system to achieve reprogrammable inactivation of dengue virus in mammalian cells. Quantitative reverse transcription PCR (qRT-PCR), fluorescence-activated cell sorting (FACS), and plaque assays showed that CRISPR RNA (crRNA) targeting the NS3 region led to the greatest viral inhibition among 10 crRNAs targeting different regions along the dengue viral genomic RNA. Deletions and insertions had also been found adjacent to the NS3 region after NS3-crRNA/ Cas13a complex transfection. Our results demonstrate that the CRISPR-Cas13a system is a novel and effective technology to inhibit dengue viral replication, suggesting that such a programmable method may be further developed into a novel therapeutic strategy for dengue and other RNA viruses.

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Global Consensus Sequence Development and Analysis of Dengue NS3 Conserved Domains

Cited by 4 publications

References 19 publications

In Vitro and In Silico Studies of Bis-furyl-pyrrolo[3,4-b]pyridin-5-ones on Dengue Virus

In Vitro and In Silico Studies of Bis-furyl-pyrrolo[3,4-b]pyridin-5-ones on Dengue Virus

Fitness adaptations of Japanese encephalitis virus in pigs following vector-free serial passaging

CRISPR-Cas13a Cleavage of Dengue Virus NS3 Gene Efficiently Inhibits Viral Replication

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