Genotyping of hepatitis C virus-positive sera by means ofa line probe assay Indicated that <3% ofEuropean samples, but up to 30% of Gabonese sera, could not be classified as either la, lb, 2a, 2b, 3a, 3b, 4c, Sa, or 6a. Such samples were analyzed in the 5' untranslated region and in the nonstructural 5 (NS5) region. Classification based on phylogenetic analysis of the commonly used 222-bp-long NS5B region was possible for most but not all of the selected sera. Therefore, the core/envelope 1 region (579 bp) and a larger NS5B (340 bp) region were also analyzed. Only the phylogenetic analysis of the 340-bp NS5B region of these newly identified and published isolates provided unambiguous classification into types and subtypes. Furthermore, unequivocal evidence for four subtypes in type 2 and eight subtypes in type 4 was provided. A specific recognition sequence in the 5' untranslated region was observed for every newly identified subtype. Based on 1830 pair-wise comparisons in NS5B, isolates belonging to the same subtype showed evolutionary distances of <0.127 and isolates of the same type exhibited evolutionary distances of <0.328. These phylogenetic border distances can be conveniently used for classification of hepatitis C virus isolates into types and subtypes.Hepatitis C virus (HCV) is thought to be the causative agent of most non-A, non-B hepatitis cases. A very high number of HCV-infected patients develop chronic hepatitis, which often results in liver cirrhosis and occasionally progresses to hepatocellular carcinoma (1). DNA complements ofthe complete RNA genome of HCV have been cloned (2-5) and show an organization comparable to those of the genomes of pestiviruses and flaviviruses (6). Within the HCV genus, a high degree of sequence heterogeneity exists. Four groups of complete genomes have been reported. HCV-1 (3),

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Because of the enormous variability of hepatitis C virus (HCV), the development of reliable genotyping assays is a formidable challenge. The optimal genotyping region appears to be the 5 untranslated region (UR) because of high conservation within, but considerable variability between, genotypes. In this study, 21 probes dispersed over seven variable 5 UR areas were applied to a line probe assay (LiPA) and used to analyze 506 HCV-infected sera from different geographical regions representing a multitude of subtypes. At least 31 different reactivity patterns emerged, with 404 (80%) of 506 distributed over 11 prototype patterns, in general corresponding to subtypes 1a, 1b, 2a/2c, 2b, 3a, 5a, and 6a and several type 4 subtypes. Subtyping specificity ranged from 97% in Hong Kong to 90% in Europe but was only 11% in West Africa, while typing specificity was always 100% when samples from Vietnam were excluded. In a second evaluation, the subtype prediction by LiPA of 448 GenBank 5 UR HCV sequences was scored. Of the 58 theoretically predicted patterns, 321 sequences (72%) were covered by the 11 prototype patterns. We concluded that (i) the selected probes detected the corresponding signature motifs in the seven variable regions with 100% reliability; (ii) these motifs allowed correct type interpretation of samples collected worldwide, with the exclusion of Vietnam, Thailand, or Vietnamese patients residing in European hospitals; and (iii) subtyping specificities vary according to geographical region, with 11 prototype subtyping patterns identifying the majority of samples from Europe and the Americas. These results indicate that the LiPA is a reliable assay applicable to routine typing and subtyping of HCV specimens.

Between February 1991 and January 1992, elevated alanine aminotransferase (ALT) levels were observed in several hemodialysis patients in a dialysis center in Dendermonde, Belgium. By the end of 1992, 25 out of 68 patients had seroconverted for HCV antibodies. The HCV strains from 23 of these seroconverters were genotyped and classified as genotype 1b. Sequence analysis of the HCV Core region was carried out in 12 patients, 9 of whom were infected with a strain bearing a unique sequence motif as compared with the currently known HCV 1b strains. A new 5' UR/Core line probe assay was designed to screen for such variations. Twenty patients tested positively for this special sequence motif, while the other 3 showed the regular subtype 1b sequence. Phylogenetic analysis of the Core sequences further revealed that the latter three were neither related to the main special strain of the infection, nor to each other. These three strains could be traced to two patients already infected at the time of residence in other dialysis units and to one patient who already showed ALT elevations in 1989. Epidemiological studies revealed no traceable source for this outbreak. In conclusion, molecular analysis demonstrates nosocomial transmissions by a peculiar genotype 1b strain in a dialysis center. Three other genotype 1b strains were also present in the unit, but were not responsible for the outbreak.

A protein was isolated from rat C6 glioma‐conditioned medium and was biochemically characterized. The heparin‐binding protein has a native molecular mass of 55–75000Da, a molecular mass of 40–48000 Da under denaturing conditions, and a pI of 5.0–6.0. Based on the determined partial amino acid sequences, the full lenght cDNA encoding the rat and human proteins were cloned. The cDNA sequences identified the isolated rat and human protein as the homologue of a recently reported mouse osteoblast‐transforming‐growth‐factor‐β1‐inducible protein, encoded by the TSC‐36 gene [Shibanuma, M., Mashimo, J., Mita, A., Kuroki, T. & Nose, K. (1993) Eur. J. Biochem. 217, 13–19]. Analysis of the human, rat and mouse amino acid sequences indicates that these proteins are highly conserved (>92% sequence identity). Sequence similarities with follistatin and the follistatin‐like domain of agrin are revealed. The relationship with follistatin and agrin points to possible common functions for the cloned follistatin‐related proteins (FRP). The protein has no effect on the inhibitory action of transforming growth factor‐β1, on CCl‐64 cell growth.