A reverse-hybridization assay, the line probe assay (LiPA), based on variations found in the 5' untranslated regions of the different hepatitis C virus (HCV) genotypes was developed, permitting simple and fast determination of four HCV genotypes and their subtypes. Using this assay, 61 PCR-positive Brazilian HCV sera were typed. Of the sera, 33% had a type 1 HCV infection, 38 % had type lb (related to HCV-J), 1.5 % had type 2a (related to HC-J6), 24.5 % had type 3 (related to E-bl and HCV-T), and 3 % of the sera were co-infected. This assay format was further evaluated using 13 sera from Belgium and the Netherlands, and all of these could be classified. Two pools of Japanese sera were classified as either type 2a or were co-infected with types lb and 2a, but no type 2b sequences were detected. Another eight PCR-positive sera were obtained from Burundi and Gabon. The sequence of the 5' untranslated region of these African viruses was strongly divergent from the three previously described types. Therefore, these isolates were tentatively classified as type 4. These and some of the other non-type 1 sera often demonstrated weaker reactivities than type 1 isolates in currently used second generation antibody confirmation assays.

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.

Upon prolonged treatment with various antiretroviral nucleoside analogs such as 3'-azido-3'-deoxythymidine, 2',3'-dideoxyinosine, 2',3'-dideoxycytidine, (-)- beta-L-2', 3'dideoxy-3'thiacytidine and 2',3'-didehydro-3'-deoxythymidine, selection of human immunodeficiency virus type 1 (HIV-1) strains with mutations in the reverse transcriptase (RT) gene has been reported. We designed a reverse hybridization line probe assay (LiPA) for the rapid and simultaneous characterization of the following variations in the RT gene: M41 or L41; T69, N69, A69, or D69; K70 or R70; L74 or V74; V75 or T75; M184, I184, or V184; T215, Y215, or F215; and K219, Q219, or E219. Nucleotide polymorphisms for codon L41 (TTG or CTG), T69 (ACT or ACA), V75 (GTA or GTG), T215 (ACC or ACT), and Y215 (TAC or TAT) could be detected. In addition to the codons mentioned above, several third-letter polymorphisms in the direct vicinity of the target codons (E40, E42, K43, K73, D76, Q182, Y183, D185, G213, F214, and L214) were found, and specific probes were selected. In total, 48 probes were designed and applied to the LiPA test strips and optimized with a well-characterized and representative reference panel. Plasma samples from 358 HIV-infected patients were analyzed with all 48 probes. The amino acid profiles could be deduced by LiPA hybridization in an average of 92.7% of the samples for each individual codon. When combined with changes in viral load and CD4+ T-cell count, this LiPA approach proved to be useful in studying genetic resistance in follow-up samples from antiretroviral agent-treated HIV-1-infected individuals.