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Virology. 2007 Jun 5;362(2):374-83. Epub 2007 Feb 6.

Construction of an infectious cDNA clone for a Brazilian prototype strain of dengue virus type 1: characterization of a temperature-sensitive mutation in NS1.

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Department of Pathology, University of Texas Medical Branch, 3.206B Mary Moody Northen Pavilion, 301 University Boulevard, Galveston, TX 77555-0436, USA.


To help understand the mechanism of pathogenesis of dengue virus (DV), we set out to create an infectious cDNA of the Brazilian prototype strain of DV serotype 1 (DV1-BR/90). PCR-amplified fragments of DV1-BR/90 cDNA were readily assembled into a subgenomic cDNA that could be used to produce replicating RNAs (replicons), lacking the structural protein-encoding regions of the genome. However, assembly of a cDNA capable of producing infectious virus was only possible using a bacterial artificial chromosome plasmid, indicating that DV1 sequences were especially difficult to propagate in E. coli. While characterizing our cDNA we discovered a fortuitous temperature-sensitive mutation in the NS1 encoding region. Using our infectious cDNA and a renilla luciferase-expressing replicon we were able to demonstrate that this mutation produced a defect in RNA replication at 37 degrees C, demonstrating that the DV1 NS1 protein plays an essential role in RNA replication.

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