No abstract

Three consecutive plaque purifications of four chimeric yellow fever virus-dengue virus (ChimeriVax-DEN) vaccine candidates against dengue virus types 1 to 4 were performed. The genome of each candidate was sequenced by the consensus approach after plaque purification and additional passages in cell culture. Our data suggest that the nucleotide sequence error rate for SP6 RNA polymerase used in the in vitro transcription step to initiate virus replication was as high as 1.34 ؋ 10 ؊4 per copied nucleotide and that the error rate of the yellow fever virus RNA polymerase employed by the chimeras for genome replication in infected cells was as low as 1.9 ؋ 10 ؊7 to 2.3 ؋ 10 ؊7 . Clustering of beneficial mutations that accumulated after multiple virus passages suggests that the N-terminal part of the prM protein, a specific site in the middle of the E protein, and the NS4B protein may be essential for nucleocapsid-envelope interaction during flavivirus assembly.A fundamental feature of RNA viruses is the error-prone nature of replication of their genomes, resulting in adaptation and rapid evolution. Various in vitro, in vivo, and ex vivo methods have been used to estimate mutation rates of viral RNA-dependent RNA polymerases. The generally accepted average value of 10 Ϫ4 to 10 Ϫ5 mutations per nucleotide (nt) per round of RNA replication (reviewed in references 6, 7, 23, and 28) results in substitution of 0.1 to 1 nt per each synthesized genomic RNA molecule of ϳ10 kb. Mutation rates vary between 10 Ϫ3 and 10 Ϫ6 in individual studies, depending on the virus and method used, although none of these methods provide an exact measure of the error rate. In vitro methods that utilize purified viral RNA polymerases may overestimate the error rate due to suboptimal in vitro enzymatic conditions and experimental complexity. In ex vivo methods, such as sequencing of phenotypic mutants generated in cell culture and analysis of monoclonal antibody escape mutants or of rates of reversion of phenotypic markers, the number of rounds of RNA replication in infected cells is not precisely known. Methods that involve a reverse transcription-PCR step with cloning of cDNA in bacteria prior to sequencing can be misleading because of the accumulation of additional mutations introduced by reverse transcription-PCR.The Flavivirus genus of the Flaviviridae family consists of about 70 viruses of which 40 have been associated with human illness (reviewed in reference 2). Flaviviruses are small enveloped plus-strand RNA viruses (reviewed in reference 18). The viral particle contains a nucleocapsid composed of viral RNA and capsid protein C, surrounded by a lipid bilayer in which the envelope protein E and membrane protein M are embedded. The genomic RNA is about 11,000 nt long and contains a single long open reading frame (ORF). Translation of the ORF produces a single polyprotein precursor containing viral proteins in the order: C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-2K-NS4B-NS5, where C through E are the structural components of the virion (prM is a pr...

Expansion of a (CTG) n segment within the 3-untranslated region of the myotonic dystrophy protein kinase gene alters mRNA production. The inherent ability of RNA polymerase II to transcribe (CTG) 17-255 tracts corresponding to DNA from normal, unstable, and affected individuals, and the normal (CGG) 54 fragile X repeat tract, was analyzed using a synchronized in vitro transcription system. Core RNA polymerase II transcribed all repeat units irrespective of repeat length or orientation. However, approximately 50% of polymerases transiently halted transcription (with a half-life of approximately 10 ؎ 1 s) within the first and second CTG repeat unit and a more transient barrier to elongation was observed roughly centered within repeats 6 -9. Transcription within the remainder of the CTG tracts and within the CCG, CGG, and CAG tracts appeared uniform with average transcription rates of 170, 250, 300, and 410 nucleotides/min, respectively. These differences correlated with changes in the sequence-specific transient pausing pattern within the CNG repeat tracts; individual incorporation rates were slower after incorporation of pyrimidine residues. Unexpectedly, approximately 4% of the run-off transcripts were, depending on the repeat sequence, either 15 or 18 nucleotides longer than expected. However, these products were not produced by transcriptional slippage within the repeat tract.

ChimeriVax-dengue (DEN) viruses are live attenuated vaccine candidates. They are constructed by replacing the premembrane (prM) and envelope (E) genes of the yellow fever (YF) 17D virus vaccine with the corresponding genes from wild-type DEN viruses (serotypes 1-4) isolated from humans. In this study, the growth kinetics of ChimeriVax-DEN1-4 and parent viruses (wild-type DEN-1-4 and YF 17D) were assessed in human myeloid dendritic cells (DCs) and in three hepatic cell lines (HepG2, Huh7, and THLE-3). In DC, ChimeriVax-DEN-1-4 showed similar growth kinetics to their parent viruses, wild-type DEN virus (propagated in Vero cells), or YF 17D virus (peak titers ~3-4.5 log(10) plaque-forming units (PFU)/mL at 48-72 hours post-infection). Parent wild-type DEN-1-4 viruses derived from C6/36 mosquito cells did not show any growth at a multiplicity of infection of 0.1 in DCs, except for DEN-2 virus, which grew to a modest titer of 2.5 log(10) PFU/mL at 48 hours post-infection. ChimeriVax-DEN1-4 grew to significantly lower titers (2-5 log(10) PFU/mL) than YF 17D virus in hepatic cell lines THLE-3 and HepG2, but not in Huh7 cells. These experiments suggest that ChimeriVax-DEN1-4 viruses replicate similarly to YF-VAX in DCs, but at a lower level than YF 17D virus in hepatic cell lines. The lack of growth of chimeric viruses in human hepatic cells suggests that these viruses may be less hepatotropic than YF 17D virus vaccine in humans.

St. Louis encephalitis (SLE) and West Nile (WN) flaviviruses are genetically closely related and cocirculate in the United States. Virus neutralization tests provide the most specific means for serodiagnosis of infections with these viruses. However, use of wild-type SLE and WN viral strains for laboratory testing is constrained by the biocontainment requirements. We constructed two highly attenuated yellow fever (YF) virus chimeras that contain the premembrane-envelope (prM-E) protein genes from the virulent MSI-7 (isolated in the United States) or the naturally attenuated CorAn9124 (Argentina) SLE strains. The YF/SLE (CorAn version) virus and the previously constructed YF/WN chimera were shown to specifically distinguish between confirmed human SLE and WN cases in a virus neutralization test using patient sera. These chimeras have the potential for use as diagnostic reagents and vaccines against SLE and WN.