Using optimized combinatorial mutagenesis techniques and Digital Imaging Spectroscopy (DIS), we have isolated mutants of the cloned Aequorea victoria green fluorescent protein (GFP) that show red-shifted excitation spectra similar to that of Renilla reniformis GFP. Selective excitation of wild-type versus Red-Shifted GFP (RSGFP) enables spectral separation of these proteins. Six contiguous codons spanning the tyrosine chromophore region were randomized and sequence analysis of the mutants revealed a tyrosineglycine consensus. These mutants will enable the simultaneous analysis of two promoters or proteins per cell or organism. In consideration of the multitude of applications which are developing for GFP alone, we envisage that spectrally shifted fluorescent proteins will be of value to a diversity of research programs, including developmental and cell biology, drug-screening, and diagnostic assays.

Yellow fever (YF) 17D vaccine virus, having a 60-year history of safe and effective use, is an ideal vector to deliver heterologous genes from other medically important flaviviruses. A chimeric YF/Japanese encephalitis (JE) virus (ChimeriVax-JE virus) was constructed by insertion of the premembrane and envelope (prME) genes of an attenuated human vaccine strain (SA14-14-2) of Japanese encephalitis (JE) virus between core and nonstructural (NS) genes of a YF 17D infectious clone. The virus grew to high titers in cell cultures and was not neurovirulent for 3- to 4-week-old mice at doses /=10(3) pfu of ChimeriVax-JE virus were solidly protected against intraperitoneal challenge with a virulent JE virus. Genetic stability of the chimera was assessed by sequential passages in cell cultures or in mouse brain. All attenuating residues and the avirulent phenotype were preserved after 18 passages in cell cultures or 6 passages in mouse brains.

Global genome analysis reveals a vast and dynamic anellovirus landscape within the human virome Graphical abstract Highlights d Anellovirus genomes assembled from longitudinal bloodtransfusion cohorts d Co-infections are common, with a median of six anellovirus lineages per subject d Transmitted anellovirus lineages were observed up to 260 days post-transfusion d Recombination is a key driver in anellovirus genomic diversification

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