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Journal of Virology, April 2002, p. 3318-3328, Vol. 76, No. 7
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.7.3318-3328.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Derivation and Characterization of a Dengue Type 1 Host Range-Restricted Mutant Virus That Is Attenuated and Highly Immunogenic in Monkeys

Lewis Markoff,^1* Xiaou Pang,¹ Huo-shu Houng,² Barry Falgout,¹ Raymond Olsen,³ Estella Jones,³ and Stephanie Polo¹

Laboratory of Vector-Borne Virus Diseases, Office of Vaccines Research and Review,¹ Division of Veterinary Services, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, Maryland 20892,³ Department of Virus Diseases, Walter Reed Institute of Research, Silver Spring, Maryland 20910-7500²

Received 20 August 2001/ Accepted 12 December 2001

We recently described the derivation of a dengue serotype 2 virus (DEN2mutF) that exhibited a host range-restricted phenotype; it was severely impaired for replication in cultured mosquito cells (C6/36 cells). DEN2mutF virus had selected mutations in genomic sequences predicted to form a 3' stem-loop structure (3'-SL) that is conserved among all flavivirus species. The 3'-SL constitutes the downstream terminal 95 nucleotides of the 3' noncoding region in flavivirus RNA. Here we report the introduction of these same mutational changes into the analogous region of an infectious DNA derived from the genome of a human-virulent dengue serotype 1 virus (DEN1), strain Western Pacific (DEN1WP). The resulting DEN1 mutant (DEN1mutF) exhibited a host range-restricted phenotype similar to that of DEN2mutF virus. DEN1mutF virus was attenuated in a monkey model for dengue infection in which viremia is taken as a correlate of human virulence. In spite of the markedly reduced levels of viremia that it induced in monkeys compared to DEN1WP, DEN1mutF was highly immunogenic. In addition, DEN1mutF-immunized monkeys retained high levels of neutralizing antibodies in serum and were protected from challenge with high doses of the DEN1WP parent for as long as 17 months after the single immunizing dose. Phenotypic revertants of DEN1mutF and DEN2mutF were each detected after a total of 24 days in C6/36 cell cultures. Complete nucleotide sequence analysis of DEN1mutF RNA and that of a revertant virus, DEN1mutFRev, revealed that (i) the DEN1mutF genome contained no additional mutations upstream from the 3'-SL compared to the DEN1WP parent genome and (ii) the DEN1mutFRev genome contained de novo mutations, consistent with our previous hypothesis that the defect in DEN2mutF replication in C6/36 cells was at the level of RNA replication. A strategy for the development of a tetravalent dengue vaccine is discussed.

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* Corresponding author. Mailing address: Laboratory of Vector-Borne Virus Diseases, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Building 29A, Room 1B17, Bethesda, MD 20892. Phone: (301) 827-1886. Fax: (301) 496-1810. E-mail: markoff{at}cber.fda.gov.

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Journal of Virology, April 2002, p. 3318-3328, Vol. 76, No. 7
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.7.3318-3328.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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