The four serotypes of dengue virus cause an estimated 100-200 million cases human disease in tropical regions each year. Infection with one serotype produces durable immunity only to that type. Serotype is conferred by the viral structural proteins, capsid (C), pre-membrane (pre-M) and envelope (E). The dengue viral genome, an 11 kilobase strand of positive-sense RNA, contains the three structural protein genes followed by a series of 7 non-structural protein genes. Earlier, a full- length cDNA copy of the entire dengue type 4 virus (D4) genome was constructed. Transfection of mammalian cells with RNA transcripts from this template yielded D4 virus, designated D4 2A. We subsequently replaced the C-preM-E or preM-E genes of the full-length clone with the structural protein genes of two different strains of dengue type 2 virus (D2). In one construct the D4 preM-E genes were replaced with the preM-E genes of a D2 New Guinea C strain virus isolated from a human patient (D2 NGC parental) and in the other the C-preM-E or preM-E genes of the mouse- neurovirulent mutant selected from the parental virus during serial passage in mouse brain, i.e., D2 NGC neurovirulent. Neither D4 2A nor the chimeric virus (D2 ME parental)/D4 was neurovirulent in suckling mice, whereas chimeric viruses containing the C-preM-E or preM-E genes of D2 NGC neurovirulent produced fatal encephalitis. It appears that some or all of the genetic loci responsible for neurovirulence are located in the structural protein genes. Sequence analysis of the cloned C-preM-E genes of parental and neurovirulent D2 NGC identified 7 mutations in the neurovirulent mutant which result in an amino acid change. Based on this information a series of chimeric viruses were constructed in which single or multiple mutations in pre-M and/or E were substituted into the parental sequence, and the resulting mutants were tested for neurovirulence in mice. A mutation in the N-terminal one- third of E protein which changed the negatively charged glutamic acid in the parental E protein to a positively charged lysine appeared to be sufficient to produce the neurovirulence phenotype.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Intramural Research (Z01)
Project #
1Z01AI000681-02
Application #
3746658
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
1994
Total Cost
Indirect Cost
City
State
Country
United States
Zip Code
Butler, Katherine; Ritter, Jana M; Ellis, Shanon et al. (2016) A Depot Medroxyprogesterone Acetate Dose That Models Human Use and Its Effect on Vaginal SHIV Acquisition Risk. J Acquir Immune Defic Syndr 72:363-71
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Kersh, Ellen N; Ritter, Jana; Butler, Katherine et al. (2015) Relationship of Estimated SHIV Acquisition Time Points During the Menstrual Cycle and Thinning of Vaginal Epithelial Layers in Pigtail Macaques. Sex Transm Dis 42:694-701
Dobard, Charles W; Sharma, Sunita; Cong, Mian-Er et al. (2015) Efficacy of topical tenofovir against transmission of a tenofovir-resistant SHIV in macaques. Retrovirology 12:69
Butler, Katherine; Ritter, Jana; Ellis, Shanon et al. (2015) Analysis of putative mucosal SHIV susceptibility factors during repeated DMPA treatments in pigtail macaques. J Med Primatol 44:286-95
Massud, Ivana; Martin, Amy; Dinh, Chuong et al. (2015) Pharmacokinetic profile of raltegravir, elvitegravir and dolutegravir in plasma and mucosal secretions in rhesus macaques. J Antimicrob Chemother 70:1473-81
Tsegaye, Theodros S; Butler, Katherine; Luo, Wei et al. (2015) Repeated Vaginal SHIV Challenges in Macaques Receiving Oral or Topical Preexposure Prophylaxis Induce Virus-Specific T-Cell Responses. J Acquir Immune Defic Syndr 69:385-94
Dietz Ostergaard, Sharon; Butler, Katherine; Ritter, Jana M et al. (2015) A combined oral contraceptive affects mucosal SHIV susceptibility factors in a pigtail macaque (Macaca nemestrina) model. J Med Primatol 44:97-107

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