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Related post: LAB/BRANCH Laboratory of Infectious Diseases SECTION Molecular Viral Biology Section INSTrrUTE AND LOCATION NIAID, NIH, Bethesda, MD 20892 TOTAL MAN- YEARS: 1.0 PROFESSIONAL: 1.0 CHECK APPROPRIATE BOX(ES) n (a) Human subjects D (a1) Minors n (a2) Interviews n (b) Human tissues S (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Dengue virus, a flavivirus, causes human disease throughout subtropical and tropical regions of the world. There are four serotypes; infection with one produces durable immunity only to that type. Serotype is specified by the viral structural proteins. The dengue viral genome, an 11 kilobase strand of positive-sense RNA, contains of three structural protein genes followed by a series of 7 non- structural protein genes. Earlier, we constructed a full-length cDNA copy of the entire dengue 4 (D4) genome, RNA transcripts from which were infectious when transfected into mammalian cells. We subsequently replaced the structural protein genes of the full-length clone with the structural protein genes of Dl or D2 virus, and used these templates to create chimeric Dl/4 and D2/4 viruses. The D4/D1 chimeric virus grows well in ceils and makes the structural proteins of Dl virus. Two D4/D2 chimeric viruses have been made, one using a D2 virus isolated from a human patient, the other a mutant of that virus selected for mouse neurovirulence through serial mouse brain passage. The chimeric D4/neurovirulent D2 virus retained Generic Panadol the neurovirulence property of its parent, but the level of neurovirulence was less than that of the parental type 2 virus. This indicated that at least some of the Cheap Panadol genetic loci responsible for neurovirulence are located in the structural protein genes. Sequencing of these genes in the neurovirulent D2 mutant and in its non- neurovirulent parent revealed 7 mutations resulting in amino acid changes. In order to determine which mutation(s) cause neurovirulence, I plan to make a series of chimeric viruses in which these mutations are isolated against a background of the prototype sequence. Elimination of the neurovirulence property may result in a chimeric D4/D2 virus safe for humans. Since the D4/D2 chimeric viruses appear to replicate more slowly than the parental viruses, they may also have properties of attenuation desirable in a vaccine. A set of suitably engineered chimeric viruses might serve together as a safe and effective tetravalent live dengue virus vaccine. 10-69 PHS 6040 (Rev. 1/84) USGOVEFWUEHTPdNniGCITICE: ini OWi-OI DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBUC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl AI 00598-02 LID PERIOD COVERED October 1. 1991 to September 30, 1992 TITLE OF PROJECT (BO characters or less. Title muslfit on or^e line between tha borders.) Dengue Type 4 Virus Mutants Restricted in Polyprotein Buy Panadol Processing PRINCIPAL INVESTIGATOR (List other professional personnel belcm the Principal InvesHgator.) (Name, title, laboratory, and institute affiliation) PI: Hiroshi Kawano, M.D. Visiting Associate LID, NIAID Others: Ching-Juh Lai, Ph.D. Head, MVB Section LID, NIAID COOPERATING UNITS (It any) LAB/BRANCH Laboratory of Infectious Diseases SECTION Molecular Viral Biology Section INSTITUTE AND LOCATION NIAID, NIH, Bethesda, MD 20892 TOTAL MAN- YEARS: 1.0 PROFESSIONAL: 1.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects n (a1) Minors D (a2) Interviews n (b) Human tissues IS (c) Neither SUMMARY OF WORK (Use Standard unreduced type. Do not exceed the space provided.) The dengue virus genome codes for a polyprotein in the Order Panadol Online order of NH2-C-PreM-E-NSl-NS2A- NS2B-NS3-NS4A-NS4B-NS5-COOH that is proteolytically cleaved to generate the 10 individual proteins. Our previous studies indicated that cleavage between NS2B and NS3 is critical for the functional activity of the viral proteinase. Our goal is to elucidate the mechanism of polyprotein processing and apply the information to engineer stable dengue virus mutants that exhibit restriction of viral replication because a subset of such mutants should be less virulent than wild-type virus but still able to achieve a satisfactory level of immunogenicity during infection. This project was initiated to investigate the sequence requirements for proteolytic processing of dengue virus NS2B- NS3. Deletion analysis showed that two amino acids upstream of the NS2B-NS3 junction are sufficient for cleavage to take place. Subsequently, mutants of NS2B-NS3 containing single amino acid substitutions at the cleavage junction were constructed for analysis of their cleavage phenotype. Several substitutions resulted in an intermediate or a low level of cleavage. The finding that mutants of NS2B-NS3 containing substitutions at the cleavage junction exhibited a reduction of cleavage efficiency has provided a body of information for molecular analysis such as Buy Panadol Online engineering of dengue virus mutants that are restricted in growth in a cell culture and Panadol Online perhaps in infected animals. Finally, full-length dengue type 4 cDNA constructs containing single amino acid substitutions at the NS2B-NS3 cleavage junction were prepared and used as templates for in vitro transcription. Recovered dengue virus mutants were verified for the presence of the mutant sequence in genomic RNA. Work is in progress to analyze the growth phenotype of these mutants. A series of deletion constructs of NS2A or NS2B tiiat has been shown to affect poly protein processing will be employed to construct deletion mutants of dengue virus. Deletion mutants should
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