RESUMEN
Hepatitis G virus (HGV)/GB virus C (GBV-C) causes persistent, non-pathogenic infection in a large proportion of the human population. Epidemiological and genetic evidence indicates a long-term association between HGV/GBV-C and related viruses and a range of primate species, and the co-speciation of these viruses with their hosts during primate evolution. Using a combination of covariance scanning and analysis of variability at synonymous sites, we previously demonstrated that the coding regions of HGV/GBV-C may contain extensive secondary structure of undefined function (Simmonds & Smith, Journal of Virology 73, 5787-5794, 1999 ). In this study we have carried out a detailed comparison of the structure of the 3'untranslated region (3'UTR) of HGV/GBV-C with that of the upstream NS5B coding sequence. By investigation of free energies on folding, secondary structure predictive algorithms and analysis of covariance between HGV/GBV-C genotypes 1-4 and the more distantly related HGV/GBV-C chimpanzee variant, we obtained evidence for extensive RNA secondary structure formation in both regions. In particular, the NS5B region contained long stem-loop structures of up to 38 internally paired nucleotides which were evolutionarily conserved between human and chimpanzee HGV/GBV-C variants. The prediction of similar structures in the same region of hepatitis C virus may allow the functions of these structures to be determined with a more tractable experimental model.
Asunto(s)
Regiones no Traducidas 3'/química , Flaviviridae/genética , ARN Viral/química , Secuencia de Bases , Evolución Biológica , Secuencia Conservada , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Secundaria de Proteína , Termodinámica , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genéticaRESUMEN
Comparison of 33 epidemiologically distinct GBV-C/hepatitis G virus complete genome sequences suggests the existence of four major phylogenetic groupings that are equally divergent from the chimpanzee isolate GBV-C(tro) and have distinct geographical distributions. These four groupings are not consistently reproduced by analysis of the virus 5'-noncoding region (5'-NCR), or of individual genes or subgenomic fragments with the exception of the E2 gene as a whole or of 200-600 nucleotide fragments from its 3' half. This region is upstream of a proposed anti-sense reading frame and contains conserved potential RNA secondary structures that may be capable of directing the internal initiation of translation. Phylogenetic analysis of this region from certain South African isolates is consistent with previous analysis of the 5'-NCR suggesting that these belong to a fifth group. The geographical distribution of virus variants is consistent with a long evolutionary history that may parallel that of pre-historic human migrations, implying that the long-term evolution of this RNA virus is extremely slow.
Asunto(s)
Flaviviridae/clasificación , Flaviviridae/genética , Filogenia , Regiones no Traducidas 5' , Animales , Secuencia de Bases , Cartilla de ADN/genética , Femenino , Flaviviridae/aislamiento & purificación , Genoma Viral , Hepatitis Viral Humana/epidemiología , Hepatitis Viral Humana/virología , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Pan troglodytes/virología , Embarazo , ARN Viral/química , ARN Viral/genética , Sudáfrica/epidemiologíaRESUMEN
We have investigated the ability of different subgenomic fragments to reproduce the phylogenetic relationships observed between six complete genome sequences of GBV-C/hepatitis G virus (HGV). While similar relationships were observed following analysis of part of the 5' non-coding region (5'NCR), for the coding region they were not accurately reproduced for some large fragments or for the majority of fragments of 300 or 600 nucleotides. Analysis of 5'NCR sequences from a large number of isolates, including newly obtained sequences from Pakistan, Zaïre and Scotland, produced separate groupings of Asian, African and European/North American variants. These groupings are associated with specific polymorphisms in the 5'NCR, many of which were covariant and consistent with a proposed secondary structure for this region. The relatively low level of amino acid sequence variation observed between these geographically and phylogenetically defined groups of variants suggests that they are unlikely to display significant biological differences.
Asunto(s)
Flaviviridae/genética , Variación Genética , Secuencia de Bases , ADN Viral/química , ADN Viral/genética , Femenino , Flaviviridae/clasificación , Genoma Viral , Hepatitis Viral Humana/virología , Humanos , Intrones , Masculino , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Filogenia , ARN ViralRESUMEN
A comparison of two precipitation methods of IgY from normal hen eggs was made. For method I the precipitation agent is represented by PEG 6000, and for method II by organic solvents. The comparative study of some parameters (protein concentration, ovalbumin content, presence of non-specific inhibitors, PAA-gel electrophoresis) shows that method I is more efficient and more convenient than method II. Using this method, we isolated and characterized IgY preparations from hens immunized with circulating influenza virus strains: A/Singapore/6/86 (H1N1), A/Shanghai/11/87 (H3N2) B/Beijing/1/87 and B/Yamagata/16/88 These viral IgY antibody preparations are homogeneous, lacking anti-host cell antibodies and non-specific inhibitors. Their NI titres and HI titres are higher than those found in the sera of immunized birds.