scholarly journals Functional Analysis of Mosquito-Borne Flavivirus Conserved Sequence Elements within 3′ Untranslated Region of West Nile Virus by Use of a Reporting Replicon That Differentiates between Viral Translation and RNA Replication

2003 ◽  
Vol 77 (18) ◽  
pp. 10004-10014 ◽  
Author(s):  
Michael K. Lo ◽  
Mark Tilgner ◽  
Kristen A. Bernard ◽  
Pei-Yong Shi
Keyword(s):  
West Nile Virus ◽  
Untranslated Region ◽  
Rna Replication ◽  
West Nile ◽  
Subgenomic Replicon ◽  
Viral Translation ◽  
Reading Frame ◽  
Conserved Sequence ◽  
Sequence Elements ◽  
In Cells

ABSTRACT We have developed a reporting replicon of West Nile virus (WNV) that could be used to quantitatively distinguish viral translation and RNA replication. A Renilla luciferase (Rluc) gene was fused in-frame with the open reading frame of a subgenomic replicon in the position where the viral structural region was deleted, resulting in RlucRep. Transfection of BHK cells with RlucRep RNA yielded two distinctive Rluc signal peaks, one between 2 and 10 h and the other after 26 h posttransfection. By contrast, only the 2- to 10-h Rluc signal peak was observed in cells transfected with a mutant replicon containing an inactivated viral polymerase NS5 (RlucRep-NS5mt). Immunofluorescence and real-time reverse transcriptase PCR assays showed that the levels of viral protein expression and RNA replication increased in cells transfected with the RlucRep but not in those transfected with the RlucRep-NS5mt. These results suggest that the Rluc signal that occurred at 2 to 10 h posttransfection reflects viral translation of the input replicon, while the Rluc activity after 26 h posttransfection represents RNA replication. Using this system, we showed that mutations of conserved sequence (CS) elements within the 3′ untranslated region of the mosquito-borne flaviviruses did not significantly affect WNV translation but severely diminished or completely abolished RNA replication. Mutations of CS1 that blocked the potential base pairing with a conserved sequence in the 5′ region of the capsid gene (5′CS) abolished RNA replication. Restoration of the 5′CS-CS1 interaction rescued viral replication. Replicons containing individual deletions of CS2, repeated CS2 (RCS2), CS3, or RCS3 were viable, but their RNA replication was dramatically compromised. These results demonstrate that genome cyclization through the 5′CS-CS1 interaction is essential for WNV RNA replication, whereas CS2, RCS2, CS3, and RCS3 facilitate, but are dispensable for, WNV replication.

scholarly journals The non-primate hepacivirus 5′ untranslated region possesses internal ribosomal entry site activity

2013 ◽  
Vol 94 (12) ◽  
pp. 2657-2663 ◽  
Author(s):  
Hazel Stewart ◽  
Cheryl Walter ◽  
Dale Jones ◽  
Sinead Lyons ◽  
Peter Simmonds ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Internal Ribosomal Entry Site ◽  
Rna Replication ◽  
Entry Site ◽  
Subgenomic Replicon ◽  
Stem Loop ◽  
Long Range Interactions ◽  
Ribosomal Entry ◽  
Internal Translation ◽  
And Function

The 5′ untranslated region (5′UTR) of the recently described non-primate hepacivirus (NPHV) contains a region with sequence homology to the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) and GB virus B (GBV-B). Here, we demonstrated internal translation initiation by the NPHV 5′UTR in a bicistronic vector. An RNA stem–loop upstream of the NPHV IRES was structurally distinct from corresponding regions in HCV and GBV-B, and was not required for IRES function. Insertion of the NPHV stem–loop into the corresponding region of the HCV 5′UTR within the HCV subgenomic replicon significantly impaired RNA replication, indicating that long-range interactions between the 5′UTR and cis-acting downstream elements within the NPHV genome are not interchangeable with those of HCV. Despite similarities in IRES structure and function between hepaciviruses, replication elements in the NPHV 5′UTR appear functionally distinct from those of HCV.

scholarly journals Design of in vitro Transcribed mRNA Vectors for Research and Therapy

2019 ◽  
Vol 73 (5) ◽  
pp. 391-394
Author(s):  
Marina Tusup ◽  
Lars E. French ◽  
Mara De Matos ◽  
David Gatfield ◽  
Thomas Kundig ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Messenger Rna ◽  
Untranslated Region ◽  
Open Reading Frame ◽  
Purification Method ◽  
Reading Frame ◽  
Mrna Purification ◽  
Functional Regions ◽  
In Cells

The use of in vitro transcribed messenger RNA (ivt mRNA) for vaccination, gene therapy and cell reprograming has become increasingly popular in research and medicine. This method can be used in vitro (transfected in cells) or administered naked or formulated (lipoplexes, polyplexes, and lipopolyplexes that deliver the RNA to specific organs, such as immune structures, the lung or liver) and is designed to be an immunostimulatory or immunosilent agent. This vector contains several functional regions (Cap, 5' untranslated region, open reading frame, 3' untranslated region and poly-A tail) that can all be optimised to generate a highly efficacious ivt mRNA. In this study, we review these aspects and report on the effect of the ivt mRNA purification method on the functionality of this synthetic transient genetic vector.

scholarly journals Genetic characterization of Bagaza virus (BAGV) isolated in India and evidence of anti-BAGV antibodies in sera collected from encephalitis patients

2009 ◽  
Vol 90 (11) ◽  
pp. 2644-2649 ◽  
Author(s):  
Vijay P. Bondre ◽  
Gajanan N. Sapkal ◽  
Prasanna N. Yergolkar ◽  
Pradip V. Fulmali ◽  
Vasudha Sankararaman ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Japanese Encephalitis ◽  
Neutralizing Antibodies ◽  
Complement Fixation Test ◽  
West Nile ◽  
Reading Frame ◽  
Frame Sequence ◽  
Mosquito Pool ◽  
Kerala State

During investigations into the outbreak of encephalitis in 1996 in the Kerala state in India, an arbovirus was isolated from a Culex tritaeniorhynchus mosquito pool. It was characterized as a Japanese encephalitis and West Nile virus cross-reactive arbovirus by complement fixation test. A plaque reduction–neutralization test was performed using hyperimmune sera raised against the plaque-purified arbovirus isolate. The sera did not show reactivity with Japanese encephalitis virus and were weakly reactive with West Nile virus. Complete open reading frame sequence analysis characterized the arbovirus as Bagaza virus (BAGV), with 94.80 % nucleotide identity with African BAGV strain DakAr B209. Sera collected from the encephalitic patients during the acute phase of illness showed 15 % (8/53) positivity for anti-BAGV neutralizing antibodies. This is the first report of the isolation of BAGV from India. The presence of anti-BAGV neutralizing antibodies suggests that the human population has been exposed to BAGV.

scholarly journals West Nile virus genome cyclization and RNA replication require two pairs of long-distance RNA interactions

Virology ◽  
2008 ◽  
Vol 373 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Bo Zhang ◽  
Hongping Dong ◽  
David A. Stein ◽  
Patrick L. Iversen ◽  
Pei-Yong Shi
Keyword(s):  
West Nile Virus ◽  
Rna Replication ◽  
Virus Genome ◽  
Long Distance ◽  
West Nile

scholarly journals trans-Packaged West Nile Virus-Like Particles: Infectious Properties In Vitro and in Infected Mosquito Vectors

2004 ◽  
Vol 78 (21) ◽  
pp. 11605-11614 ◽  
Author(s):  
Frank Scholle ◽  
Yvette A. Girard ◽  
Qizu Zhao ◽  
Stephen Higgs ◽  
Peter W. Mason
Keyword(s):  
West Nile Virus ◽  
Cell Lines ◽  
Neutralizing Antibodies ◽  
Fat Body ◽  
Infected Mosquito ◽  
Wild Type Virus ◽  
Coding Region ◽  
West Nile ◽  
Subgenomic Replicon ◽  
Virus Like Particles

ABSTRACT A trans-packaging system for West Nile virus (WNV) subgenomic replicon RNAs (repRNAs), deleted for the structural coding region, was developed. WNV repRNAs were efficiently encapsidated by the WNV C/prM/E structural proteins expressed in trans from replication-competent, noncytopathic Sindbis virus-derived RNAs. Infectious virus-like particles (VLPs) were produced in titers of up to 109 infectious units/ml. WNV VLPs established a single round of infection in a variety of different cell lines without production of progeny virions. The infectious properties of WNV and VLPs were indistinguishable when efficiencies of infection of a number of different cell lines and inhibition of infection by neutralizing antibodies were determined. To investigate the usefulness of VLPs to address biological questions in vivo, Culex pipiens quinquefasciatus mosquitoes were orally and parenterally infected with VLPs, and dissected tissues were analyzed for WNV antigen expression. Antigen-positive cells in midguts of orally infected mosquitoes were detected as early as 2 days postinfection and as late as 8 days. Intrathoracic inoculation of VLPs into mosquitoes demonstrated a dose-dependent pattern of infection of secondary tissues and identified fat body, salivary glands, tracheal cells, and midgut muscle as susceptible WNV VLP infection targets. These results demonstrate that VLPs can serve as a valuable tool for the investigation of tissue tropism during the early stages of infection, where virus spread and the need for biosafety level 3 containment complicate the use of wild-type virus.

scholarly journals Identification of Novel Small-Molecule Inhibitors of West Nile Virus Infection

2007 ◽  
Vol 81 (21) ◽  
pp. 11992-12004 ◽  
Author(s):  
Amine O. Noueiry ◽  
Paul D. Olivo ◽  
Urszula Slomczynska ◽  
Yi Zhou ◽  
Ben Buscher ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Yellow Fever ◽  
High Throughput Screening ◽  
Yellow Fever Virus ◽  
The United States ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chemical Library ◽  
Entry Site ◽  
West Nile ◽  
Subgenomic Replicon

ABSTRACT West Nile virus (WNV) has spread throughout the United States and Canada and now annually causes a clinical spectrum of human disease ranging from a self-limiting acute febrile illness to acute flaccid paralysis and lethal encephalitis. No therapy or vaccine is currently approved for use in humans. Using high-throughput screening assays that included a luciferase expressing WNV subgenomic replicon and an NS1 capture enzyme-linked immunosorbent assay, we evaluated a chemical library of over 80,000 compounds for their capacity to inhibit WNV replication. We identified 10 compounds with strong inhibitory activity against genetically diverse WNV and Kunjin virus isolates. Many of the inhibitory compounds belonged to a chemical family of secondary sulfonamides and have not been described previously to inhibit WNV or other related or unrelated viruses. Several of these compounds inhibited WNV infection in the submicromolar range, had selectivity indices of greater than 10, and inhibited replication of other flaviviruses, including dengue and yellow fever viruses. One of the most promising compounds, AP30451, specifically blocked translation of a yellow fever virus replicon but not a Sindbis virus replicon or an internal ribosome entry site containing mRNA. Overall, these compounds comprise a novel class of promising inhibitors for therapy against WNV and other flavivirus infections in humans.

Effective siRNA targeting of the 3′ untranslated region of the West Nile virus genome

2009 ◽  
Vol 82 (3) ◽  
pp. 166-168 ◽  
Author(s):  
Karen G. Anthony ◽  
Fengwei Bai ◽  
Manoj N. Krishnan ◽  
Erol Fikrig ◽  
Raymond A. Koski
Keyword(s):  
West Nile Virus ◽  
Untranslated Region ◽  
Virus Genome ◽  
The West ◽  
West Nile

scholarly journals Potential High-Throughput Assay for Screening Inhibitors of West Nile Virus Replication

2003 ◽  
Vol 77 (23) ◽  
pp. 12901-12906 ◽  
Author(s):  
Michael K. Lo ◽  
Mark Tilgner ◽  
Pei-Yong Shi
Keyword(s):  
West Nile Virus ◽  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Health Priorities ◽  
Stable Cell Line ◽  
West Nile ◽  
Subgenomic Replicon ◽  
Genes Encoding ◽  
High Throughput Assay

ABSTRACT Prevention and treatment of infection by West Nile virus (WNV) and other flaviviruses are public health priorities. We describe a reporting cell line that can be used for high-throughput screening of inhibitors against all targets involved in WNV replication. Dual reporter genes, encoding Renilla luciferase (Rluc) and neomycin phosphotransferase (Neo), were engineered into a WNV subgenomic replicon, resulting in Rluc/NeoRep. Geneticin selection of BHK-21 cells transfected with Rluc/NeoRep yielded a stable cell line that contains persistently replicating replicons. Incubation of the reporting cells with known WNV inhibitors decreased Rluc activity, as well as the replicon RNA level. The efficacies of the inhibitors, as measured by the depression of Rluc activity in the reporting cells, are comparable to those derived from authentic viral infection assays. Therefore, the WNV reporting cell line can be used as a high-throughput assay for anti-WNV drug discovery. A similar approach should be applicable to development of genetics-based antiviral assays for other flaviviruses.

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