Generating West Nile Virus from an Infectious Clone

2016 ◽  
pp. 29-43
Author(s):  
Rianna Vandergaast ◽  
Brenda L. Fredericksen
Keyword(s):  
West Nile Virus ◽  
Infectious Clone ◽  
West Nile

scholarly journals Infection, Dissemination, and Transmission of a West Nile Virus Green Fluorescent Protein Infectious Clone byCulex pipiens quinquefasciatusMosquitoes

2010 ◽  
Vol 10 (3) ◽  
pp. 267-274 ◽  
Author(s):  
Charles E. McGee ◽  
Alexandr V. Shustov ◽  
Konstantin Tsetsarkin ◽  
Ilya V. Frolov ◽  
Peter W. Mason ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
West Nile Virus ◽  
Fluorescent Protein ◽  
Infectious Clone ◽  
West Nile ◽  
Green Fluorescent

scholarly journals In Vitro Resistance Selection and In Vivo Efficacy of Morpholino Oligomers against West Nile Virus

2007 ◽  
Vol 51 (7) ◽  
pp. 2470-2482 ◽  
Author(s):  
Tia S. Deas ◽  
Corey J. Bennett ◽  
Susan A. Jones ◽  
Mark Tilgner ◽  
Ping Ren ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Binding Site ◽  
Viral Genome ◽  
Infectious Clone ◽  
Encephalitis Virus ◽  
West Nile ◽  
In Vivo Efficacy ◽  
Conserved Sequence

ABSTRACT We characterize in vitro resistance to and demonstrate the in vivo efficacy of two antisense phosphorodiamidate morpholino oligomers (PMOs) against West Nile virus (WNV). Both PMOs were conjugated with an Arg-rich peptide. One peptide-conjugated PMO (PPMO) binds to the 5′ terminus of the viral genome (5′-end PPMO); the other targets an essential 3′ RNA element required for genome cyclization (3′ conserved sequence I [3′ CSI] PPMO). The 3′ CSI PPMO displayed a broad spectrum of antiflavivirus activity, suppressing WNV, Japanese encephalitis virus, and St. Louis encephalitis virus, as demonstrated by reductions in viral titers of 3 to 5 logs in cell cultures, likely due to the absolute conservation of the 3′ CSI PPMO-targeted sequences among these viruses. The selection and sequencing of PPMO-resistant WNV showed that the 5′-end-PPMO-resistant viruses contained two to three mismatches within the PPMO-binding site whereas the 3′ CSI PPMO-resistant viruses accumulated mutations outside the PPMO-targeted region. The mutagenesis of a WNV infectious clone demonstrated that the mismatches within the PPMO-binding site were responsible for the 5′-end PPMO resistance. In contrast, a U insertion or a G deletion located within the 3′-terminal stem-loop of the viral genome was the determinant of the 3′ CSI PPMO resistance. In a mouse model, both the 5′-end and 3′ CSI PPMOs (administered at 100 or 200 μg/day) partially protected mice from WNV disease, with minimal to no PPMO-mediated toxicity. A higher treatment dose (300 μg/day) caused toxicity. Unconjugated PMOs (3 mg/day) showed neither efficacy nor toxicity, suggesting the importance of the peptide conjugate for efficacy. The results suggest that a modification of the peptide conjugate composition to reduce its toxicity yet maintain its ability to effectively deliver PMO into cells may improve PMO-mediated therapy.

scholarly journals In vitro and in vivo characterization of a West Nile virus MAD78 infectious clone

2014 ◽  
Vol 159 (11) ◽  
pp. 3113-3118 ◽  
Author(s):  
Katherine L. Hussmann ◽  
Rianna Vandergaast ◽  
Susan Park Ochsner ◽  
Albert C. Huang ◽  
Michael Gale ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Infectious Clone ◽  
West Nile ◽  
In Vivo Characterization

scholarly journals Envelope Protein Glycosylation Status Influences Mouse Neuroinvasion Phenotype of Genetic Lineage 1 West Nile Virus Strains

2005 ◽  
Vol 79 (13) ◽  
pp. 8339-8347 ◽  
Author(s):  
David W. C. Beasley ◽  
Melissa C. Whiteman ◽  
Shuliu Zhang ◽  
Claire Y.-H. Huang ◽  
Bradley S. Schneider ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Amino Acid ◽  
North American ◽  
Envelope Protein ◽  
Infectious Clone ◽  
Protein Glycosylation ◽  
Genetic Lineage ◽  
West Nile ◽  
Old World ◽  
Highly Virulent

ABSTRACT The introduction of West Nile virus (WNV) into North America has been associated with relatively high rates of neurological disease and death in humans, birds, horses, and some other animals. Previous studies identified strains in both genetic lineage 1 and genetic lineage 2, including North American isolates of lineage 1, that were highly virulent in a mouse neuroinvasion model, while other strains were avirulent or significantly attenuated (D. W. C. Beasley, L. Li, M. T. Suderman, and A. D. T. Barrett, Virology 296:17-23, 2002). To begin to elucidate the basis for these differences, we compared a highly virulent New York 1999 (NY99) isolate with a related Old World lineage 1 strain, An4766 (ETH76a), which is attenuated for mouse neuroinvasion. Genomic sequencing of ETH76a revealed a relatively small number of nucleotide (5.1%) and amino acid (0.6%) differences compared with NY99. These differences were located throughout the genome and included five amino acid differences in the envelope protein gene. Substitution of premembrane and envelope genes of ETH76a into a NY99 infectious clone backbone yielded a virus with altered in vitro growth characteristics and a mouse virulence phenotype comparable to ETH76a. Further site-specific mutagenesis studies revealed that the altered phenotype was primarily mediated via loss of envelope protein glycosylation and that this was associated with altered stability of the virion at mildly acidic pH. Therefore, the enhanced virulence of North American WNV strains compared with other Old World lineage 1 strains is at least partly mediated by envelope protein glycosylation.

scholarly journals A Putative Lipid-Associating Motif in the West Nile Virus NS4A Protein Is Required for Efficient Virus Replication

2021 ◽  
Vol 9 ◽  
Author(s):  
Andrea Mikulasova ◽  
Leah K. Gillespie ◽  
Rebecca L. Ambrose ◽  
Turgut E. Aktepe ◽  
Alice M. Trenerry ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Virus Replication ◽  
Infectious Clone ◽  
Site Directed Mutagenesis ◽  
Wild Type Virus ◽  
The West ◽  
West Nile ◽  
Replication Complex ◽  
West Nile Virus Strain ◽  
Cellular Markers

Flavivirus replication is intimately associated with re-organized cellular membranes. These virus-induced changes in membrane architecture form three distinct membranous “organelles” that have specific functions during the flavivirus life cycle. One of these structures is the replication complex in which the flaviviral RNA is replicated to produce progeny genomes. We have previously observed that this process is strictly dependent on cellular cholesterol. In this study we have identified a putative cholesterol recognition/interaction amino acid consensus (CRAC) motif within the West Nile virus strain Kunjin virus (WNVKUN) NS4A protein. Site-directed mutagenesis of this motif within a WNVKUN infectious clone severely attenuated virus replication and the capacity of the mutant viruses to form the replication complex. Replication of the mutant viruses also displayed reduced co-localization with cellular markers recruited to replication sites during wild-type virus replication. In addition, we observed that the mutant viruses were significantly impaired in their ability to remodel cytoplasmic membranes. However, after extensive analysis we are unable to conclusively reveal a role for the CRAC motif in direct cholesterol binding to NS4A, suggesting additional complex lipid-protein and protein-protein interactions. We believe this study highlights the crucial role for this region within NS4A protein in recruitment of cellular and viral proteins to specialized subdomains on membrane platforms to promote efficient virus replication.

scholarly journals West Nile virus NS2A protein facilitates virus-induced apoptosis independently of interferon response

2013 ◽  
Vol 94 (2) ◽  
pp. 308-313 ◽  
Author(s):  
Ezequiel Balmori Melian ◽  
Judith H. Edmonds ◽  
Tomoko Kim Nagasaki ◽  
Edward Hinzman ◽  
Nadia Floden ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Amino Acid ◽  
Amino Acid Change ◽  
Infectious Clone ◽  
Single Amino Acid ◽  
Interferon Response ◽  
Lower Percentage ◽  
Chromosomal Dna ◽  
West Nile ◽  
Single Amino Acid Change

The flavivirus NS2A protein is a small, multifunctional protein, involved in replication, virion formation and regulation of the innate immune response. Using the Kunjin strain of West Nile virus (WNVKUN) we previously demonstrated that a single amino acid change from alanine to proline at position 30 of the NS2A protein (A30P) reduced viral cytopathicity in cells and virulence in mice. To further investigate functions of the NS2A protein we have substituted alanine at position 30 with different amino acids (A30 mutants) in a WNVKUN infectious clone. The virulence of mutant viruses in wild-type (WT) and IRF3/IRF7 double-knockout mice was influenced by the amino acid change and ranged from high to low in the order of WT>A30L>A30E>A30P/A30G. Moreover, infection of beta interferon (IFN-β)-deficient Vero cells with A30P virus showed less pronounced chromosomal DNA degradation and lower percentage of cells with positive TUNEL labelling than in WT virus infection, indicating a role for the WT NS2A protein in IFN-independent apoptotic cell death.

West Nile Virus

ASHA Leader ◽  
2004 ◽  
Vol 9 (9) ◽  
pp. 10-13
Author(s):  
Susan Brady ◽  
Rhonda Miserendino ◽  
Noel Rao
Keyword(s):  
West Nile Virus ◽  
West Nile

West Nile Virus Season Off to Slow Start

2005 ◽  
Vol 39 (8) ◽  
pp. 10
Author(s):  
PATRICE WENDLING
Keyword(s):  
West Nile Virus ◽  
West Nile ◽  
Slow Start

Sudden Paralysis Is Red Flag for West Nile Virus

2005 ◽  
Vol 38 (8) ◽  
pp. 55
Author(s):  
MICHELE G. SULLIVAN
Keyword(s):  
West Nile Virus ◽  
West Nile ◽  
Red Flag
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