Hepatitis C virus (HCV) isolates from a cohort of 315 patients from the Benelux countries (Belgium, The Netherlands, Luxembourg) were genotyped by means of reverse hybridization Inno-LiPA (line probe assay). Genotypes la, lb, 2a, 2b, 3a, 4a and 5a were detected. From the cohort, isolates representing all types and those showing an aberrant LiPA pattern were further analysed by sequencing parts of the 5' UTR, core (nt 1 to 326; aa residues 1 to 108) and core/E1 (nt 477 to 924; aa residues 159 to 308) regions. Molecular evolutionary analysis of the core and core/E1 regions allowed discrimination between known and additional subtypes, especially within types 2 and 4. The core region is not suitable for classification of new subtypes because of the relatively high level of conservation. The core/E 1 region displays a higher level of sequence variation and allows much more distinct discrimination between subtypes. Genotypes 2 and 4 are particularly heterogeneous, with at least 7 and 10 subtypes, respectively. In contrast to previous reports from Europe, HCV isolates from the cohort constituted a highly heterogeneous population of virus variants, especially within genotypes 2 and 4.
The RNAs of hepatitis C virus (HCV) isolates from 62 patients with chronic HCV infection were analyzed by direct sequencing of the 5' untranslated region. Two important sequence motifs were recognized: one between positions-170 and-155 and the other between positions-132 and-117. These motifs are partly complementary. All three previously published genotypes were observed; 34 (55%) isolates were classified as type 1 (including prototype [from the United States] and HCV-BK [from Japan] sequences), 11 (18%) were classified as type 2 (including HC-J6 and HC-J8), and 12 (19%o) were classified as type 3 (including EB1); one patient was infected with genotypes 1 and 2. Four (6%) isolates showed aberrant sequences and were therefore provisionally classified as genotype 4. These results indicate the significance of sequence variation among the 5' untranslated regions of different HCV genotypes and indicate that this region could possibly be used for consistent genotyping of HCV isolates.
In 24 patients with hepatitic C virus (HCV) infection who participated in a randomized trial with alpha 2B interferon, HCV RNA analysis by the polymerase chain reaction with two separate primer sets was performed at weeks 0, 4, and 24 and during a follow-up period of 6 to 9 months. Prior to therapy all patients were HCV RNA positive. During therapy, HCV RNA decreased to an undetectable level (<1 chimpanzee infectious dose per ml) in nine patients at week 4. After week 4, no additional cases of HCV RNA disappearance (<1 chimpanzee infectious dose per ml) were observed. During follow-up, HCV RNA could not be detected in four of the six patients with a sustained alanine aminotransferase response. These results suggest the probable predictive value of HCV RNA levels for detecting the failure of therapy in an early stage of HCV infection.In recent years, several randomized controlled studies were performed with alpha interferon (IFN) for the treatment of non-A, non-B hepatitis (NANBH) (9,10,13,16,19,20). The effect of IFN was evaluated by measuring the alanine aminotransferase (ALT) level in serum, which reflects the activity of liver disease.Recently, the etiological agent for NANBH has been identified (6, 12). The causative agent is now known as hepatitis C virus (HCV). The HCV genome is a positivestranded RNA molecule of about 9,400 nucleotides. Sequence homology among the known HCV strains is about 80% (3, 7, 14, 17, 21).In the study described here, 24 patients were investigated for the presence of HCV RNA during and after IFN therapy, to determine directly the effect of IFN treatment on viremia. HCV RNA analysis by the polymerase chain reaction was performed by using primer sets from the highly conserved noncoding (NC) region and a conserved sequence from nonstructural region 5 (NS5).Patients between 18 and 70 years of age with elevated ALT levels (22 times the upper limit of normal), a biopsyproven chronic NANBH, antibodies to HCV determined by a second-generation enzyme immunosorbent assay (Abbott, North Chicago, Ill.) and a confirmatory assay RIBA IV (Ortho, Raritan, N.J.), and no recent history of hepatitis B virus, hepatitis A virus, cytomegalovirus, or Epstein-Barr virus infection were included in the study. All patients gave informed consent prior to participation in the study. Patients were treated in a randomized controlled trial with either a standard scheme (12 patients) consisting of 3 MU of recombinant IFN-a2B (Intron A; Schering Plough, Kenilworth, N.J.) three times a week for 24 weeks or an experimental scheme (12 patients). In the experimental scheme, therapy was started with 6 MU of recombinant IFN-ot2B three times a week for at least 8 weeks. Therapy was stopped at week 12 if ALT levels remained elevated. If the ALT level normalized, therapy was continued with 3 MU of recombinant IFN-ot2B three times a week for 8 weeks; this was followed * Corresponding author. 595by treatment with 1 MU of recombinant IFN-ox2B three times a week until a normal ALT level was accompanied by an undetectable HCV RN...
Two rapid genotyping methods for hepatitis C virus (HCV), the line probe assay (Inno-LiPA) and the subtype-specific core amplification system [Okamoto et al., (1992b) Journal of General Virology 73:673-679], were applied to 58 HCV isolates which were typed as type 1 (n = 37) and type 2 (n = 21) by sequence analysis of the 5' untranslated region (5'UTR). The line probe assay targets the 5'UTR and recognized 12 subtype 1a, 25 subtype 1b, 18 subtype 2a, 2 subtype 2b and 1 subtype 2d in accordance with sequence analysis of this region. Subtype-specific core amplification revealed 7 discrepancies among the 37 type 1 isolates when compared to LiPA. A different subtype was observed in 3 isolates (1a versus 1b), 2 isolates remained untyped and 2 isolates showed a coinfection of subtype 1a and 1b. The first 5 discrepancies were confirmed by sequence analysis of the core region whereas the coinfection could not be confirmed. Of the 21 type 2 isolates only one could be typed by subtype-specific core amplification. HCV RNA was detected in all 21 cases after the general first round of polymerase chain reaction (PCR). Direct sequencing of the core region indicated sequence variation as a source of failure. It is concluded that LiPA results are conclusive for typing of HCV. However, LiPA is hampered occasionally for subtyping by lack of subtype-specific sequence variation in 5'UTR. Subtyping results by subtype-specific core amplification were accurate. However, it seems that this assay is not suitable for the identification of genotype 2 isolates that circulate in patients living in Western Europe.
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