Codelivery of DNA coding for the soluble form of CD86 results in the down-regulation of the immune response to DNA vaccines.

The costimulatory pathway that includes CD80, CD86, CD28, and CTLA-4 plays a key role in regulating T cell activation and tolerance and is a promising therapeutic target. We have studied the possibility of down-regulating the immune response to DNA vaccine by codelivery of a plasmid coding for the extracellular domains of CD86 (pDelta86). We found that DeltaCD86 was able to inhibit the engagement of FcCTLA-4 but not of FcCD28 to CD80 and CD86 expressed on COS cells. Coadministration of plasmid pDelta86 encoding for the extracellular domains of CD86 along with a plasmid encoding for the glycoprotein D (pgD) of herpes simplex virus-2 (a membrane-bound protein) by the im route in mice resulted in a strong inhibition of the cell-mediated immune response in the spleen and in draining lymph nodes. In addition, when pDelta86 was coadministered together with a plasmid encoding for the ovalbumin (pOVA) (a soluble protein), a strong inhibition of the cell-mediated immune response was observed in draining lymph nodes and only a partial inhibition was found in the spleen. Furthermore, only a partial down-regulation of the humoral immune response was observed. The mechanism involved could be a preferential engagement of DeltaCD86 to CTLA-4 leading to the transmission of a negative signal to T lymphocytes.

Oral transgene vaccination mediated by attenuated Salmonellae is an effective method to prevent Herpes simplex virus-2 induced disease in mice.

An attenuated strain of Salmonella typhimurium has been used as a carrier for oral genetic immunization. The eukaryotic expression vector pCMV containing the gene of the glycoprotein D (gD) of the herpes simplex virus 2 was used to transform Salmonellae. The oral immunization with the transformed salmonellae elicited a strong cellular immune response in both, the mucosal and systemic compartments (spleen, ileal lymph nodes and Peyer patches). The immune response mainly consisted in a dramatic activation of IFN-gamma-secreting cells. Twenty hours following the challenge with five lethal doses of virus, mRNA for IFN-gamma was observed in vaginal tissues from mice immunized with salmonella harboring the plasmid pgD but not in tissues from mice immunized by the intramuscular route with pgD. After an intravaginal challenge all immunized mice survived without developing symptoms. Furthermore, the immunization with Salmonella resulted in a more effective control of viral shedding than intramuscular immunization. We have unequivocally demonstrated by the introduction of an intron in the green fluorescent protein that the expression of the plasmid was due to the transcription of the protein by an eukaryotic nuclear process and not as a result of expression of the protein by the bacteria. Macrophages and dendritic cells were found expressing the protein in systemic and mucosal compartments of the immune system.

Modulation of the immune response to DNA vaccine by co-delivery of costimulatory molecules.

We have investigated methods for modulating immune responses, against herpes simplex virus (HSV), generated from DNA vaccination by co-delivery of genes encoding costimulatory molecules. A strong delayed-type hypersensitivity (DTH) reaction was induced in mice co-injected via the intradermal (i.d.) route with a eukaryotic expression plasmid encoding the CD80 molecule (pCD80) and a plasmid encoding the glycoprotein D of the HSV-2 (pgD). Furthermore, when spleen cells from these mice were cultured in the presence of inactivated HSV, a significant increase in the expression of interleukin-2 receptor (IL-2R) was observed in the CD4 subset compared with mice immunized only with pgD. Analysis of cytokine synthesis at the single-cell level indicated that CD80 genes induce a significant increase in the number of interferon-gamma (IFN-gamma)-, IL-2- and IL-4-secreting cells in the spleen. On the other hand, co-administration of the CD80 gene via the intramuscular (i.m.) route did not induce an increase in the cell-mediated immune response. When a plasmid carrying the CD86 gene (pCD86) was co-injected via the i.m. route with the pgD plasmid, a small decrease in the number of IFN-gamma-secreting cells was observed. This down-regulation of the immune response was also observed when eukaryotic expression cassettes for CD80 and for CD86 were co-administered with the pgD plasmid via the i.d. route. However, co-injection of pCD86 via the i.m. route produced a small increase in the number of IL-4-secreting cells. When immunized mice were challenged intravaginally with 100 plaque-forming units of virus, only co-injection of the CD80 gene by the i.d. route provoked an adjuvant effect compared with mice immunized with pgD alone. A reduction in the titres of HSV in vaginal washings was observed together with a decrease in the lesion score.

Superiority of intramuscular route and full length glycoprotein D for DNA vaccination against herpes simplex 2. Enhancement of protection by the co-delivery of the GM-CSF gene.

Immunization with naked DNA has been analyzed in two critical variables: the site of injection and the cellular compartment to which the coded protein is directed. The gene for the full length of the glycoprotein D (gD) of HSV-2 under the control of the citomegalovirus (CMV) promoter was injected via the intradermal (i.d.) or the intramuscular (i.m.) routes in mice. Immunization in the quadricep muscle was superior to the intradermal immunization in the footpads. A stronger activation of IFN-gamma-secreting cells in the spleen and draining lymph nodes (DLN) was induced, resulting in a more efficient protection against an intravaginal challenge. In order to analyze the effect of the cellular localizations of the coded protein, the DNA for the truncated form of the gD (DeltagD) was injected via the i.m. route. Immunization with a vector encoding for DeltagD resulted in higher antibody levels in serum and vaginal washes than immunization with the gene for the full length gD. However, immunization with the DeltagD DNA elicited a much weaker cell-mediated immune response and was inferior to gD DNA in providing protection against a lethal intravaginal challenge with HSV. Co-injection of an expression cassette for the granulocyte-macrophage colony-stimulating factor (GM-CSF) increased both the humoral and cell-mediated immune response with both gD and DeltagD. A strong activation of IL-4-secreting cells was observed in the spleen and DLN together with an increase in the number of IFN-gamma-secreting cells. In addition, a reduction in the vaginal virus titers after an intravaginal challenge was observed in mice co-injected with the GM-CSF gene as compared to those immunized with pCDNAgD only.

Cohort effect of HCV infection in liver cirrhosis assessed by a 25 year study.

BACKGROUND: The role of HCV infection in the development of chronic liver disease is still unclear. OBJECTIVES: Assess the presence of HCV infection in patients with liver cirrhosis. STUDY DESIGN: 123 cases of cirrhotic liver randomly selected over a 25 years period (1969-1994) from the autopsy archives of the Pathology Department of the University of Trieste, Italy, were analyzed for the presence of HCV viral genome. METHODS: Total RNA was extracted from formalin-fixed paraffin-embedded tissues of the cirrhotic liver. Genotype analysis for HCV was performed after RT-PCR by dot-blot hybridization with the three major genotype-specific probes (G1, G2 and G3). RESULTS: The overall HCV genome frequency was 50.4% (62/123). The positivity was quite constant in the 1969-1979 period (35-38%), rose to 65% in 1984, peaked to 77% in 1989 (P<0.005 vs. the previous decade), and decreased to 50% in 1994. HCV genotype G1 was found in 89% of the 62 positive samples. The mean age of death of HCV-positive and HCV-negative patients was comparable (69+/-12 vs. 67+/-16 years, NS). CONCLUSIONS: These data show an increasing frequency of HCV infection in cirrhotic liver tissues from 1969 to 1994, which peaked in 1989. The genotype G1 was the almost uniquely associated with cirrhosis. These findings indicate that the HCV infection occurred around the late 1950s-early 1960s, thus supporting the hypothesis of a cohort effect. HCV infection seems not to alter the natural history of liver cirrhosis as indicated by the comparable age at death of HCV positive and HCV negative patients.

Influence of correct secondary and tertiary RNA folding on the binding of cellular factors to the HCV IRES.

Structural integrity of the hepatitus C virus (HCV) 5' UTR region that includes the internal ribosome entry site (IRES) element is known to be essential for efficient protein synthesis. The functional explanation for this observation has been provided by the recent evidence that binding of several cellular factors to the HCV IRES is dependent on the conservation of its secondary structure. In order to better define the relationship between IRES activity, protein binding and RNA folding of the HCV IRES, we have focused our attention on its major stem-loop region (domain III) and the binding of several cellular factors: two subunits of eukaryotic initiation factor eIF3 and ribosomal protein S9. Our results show that binding of eIF3 p170 and p116/p110 subunits is dependent on the ability of the domain III apical stem-loop region to fold in the correct secondary structure whilst secondary structure of hairpin IIId is important for the binding of S9 ribosomal protein. In addition, we show that binding of S9 ribosomal protein also depends on the disposition of domain III on the HCV 5' UTR, indicating the presence of necessary inter-domain interactions required for the binding of this protein (thus providing the first direct evidence that tertiary folding of the HCV RNA does affect protein binding).

Immunoreactivity to putative B-cell epitopes of hepatitis G virus polyprotein in viremic and nonviremic subjects.

The hepatitis G virus (HGV) polyprotein was scanned by computer-aided prediction of antigenicity to search for B-cell epitopes. Four polypeptide sequences, V37D (amino acids [aa] 1685 to 1721), V36S (aa 2102 to 2137), P37R (aa 2156 to 2192), and C40P (aa 2280 to 2319), were identified and synthesized for use in immunoassays. Antibodies to these peptides were searched for in a panel of 239 serum samples, which were also tested for anti-E2 antibodies and HGV RNA. Furthermore, the course of HGV markers was studied prospectively in four patients who had been transfused with HGV RNA-positive blood. There was a negative association between immunoreactivity to V37D and P37R and presence of HGV RNA (2 of 53 and 1 of 53, respectively; P < 0.05); none of the subjects with dual antibody positivity was HGV RNA positive. Anti-V37D and anti-P37R antibodies compared favorably with anti-E2 antibodies as markers of recovery from HGV infection. These results might be useful for the development of new, more sensitive diagnostic assays.

Prediction of successful outcome in a randomised controlled trial of the long-term efficacy of interferon alpha treatment for chronic hepatitis C.

To evaluate the efficacy of a 12-month course of recombinant interferon alpha (IFN-alpha2b), and to assess predictive factors of successful response to IFN therapy in chronic active hepatitis C (HCV CAH), 242 patients with histologically proven HCV CAH were assigned randomly to two groups, one treated with IFN-alpha2b (3 MU three times weekly, intramuscularly), the other untreated. To determine the efficacy of IFN-alpha2b 12 months after therapy, a second liver biopsy was carried out on 100 treated patients and 27 untreated patients. The biochemical, virological, and serological response of patients followed up for at least 50 months after treatment was also evaluated to confirm the efficacy of IFN-alpha2b. The genotypes of infecting HCV, anti-HCV core IgM, and HCV-RNA concentrations were also analysed and the predictors of response determined by univariate and multivariate analyses. Response was defined in terms of the normalisation of aminotransferase activities and the disappearance of HCV-RNA. The overall long-term response was 39.4%. Anti-HCV core IgM levels were significantly lower in long-term responders. Patients with increased levels of IgM anti HCV core (>3.8 sample/cut-off), infected with genotype 1b were nonresponders. Liver histology improved significantly in patients with long-term response. Multivariate analysis identified three independent predictors of the likelihood of long-term response to IFN therapy: age younger than 40 years, basal anti-HCV core IgM levels < or = 3.8, and genotypes other than 1b. These data indicate that the treatment with IFN-alpha2b used in this randomised controlled trial is effective in HCV CAH. Anti-HCV core IgM was the strongest predictor of long-term response in the present study.

IgM antibody response to the hepatitis C virus core protein in intravenous drug users.

To verify whether a solid-phase enzyme immunoassay for serum IgM antibodies to the hepatitis C virus (HCV) core protein (IgM anti-HCVcore) might be proposed as a surrogate test for serum HCV RNA, we studied 86 anti-HCV antibody-positive intravenous drug users. Serum HCV RNA was demonstrated by RT-PCR with primers derived from the 5' non-coding and the core region. IgM anti-HCVcore antibodies were found in 62/86 (72%) subjects; circulating HCV RNA was detected by the 5' noncoding assay in 53/86 samples (62%) and by the core region assay in 35/86 samples (41%). IgM anti-HCVcore reactivity was associated with core HCV RNA seropositivity (p < 0.05) but not with 5' noncoding HCV RNA seropositivity (p = NS). Patients infected by HCV type 1a were more-often positive for IgM anti-HCVcore (p < 0.05) and for core HCV RNA (p = 0.005) than patients infected by other HCV genotypes. IgM anti-HCVcore reactivity was significantly more common in subjects positive for core HCV RNA (p < 0.005) and in subjects aged > 30 years (p < 0.05). In conclusion, the IgM anti-HCVcore assay frequently tests positive in intravenous drug users, particularly when infected by HCV 1a, but is not a surrogate of testing for serum HCV RNA.

The neutralizing antibody response against a conserved region of human immunodeficiency virus type 1 gp41 (amino acid residues 731-752) is uniquely directed against a conformational epitope.

Amino acids 731-752 (731PRGPDRPEGIEEEGGERDRDRS752) of the transmembrane glycoprotein gp41 of human immunodeficiency virus type 1 (HIV-1) are conserved in most virus isolates and are controversially reported to be implicated in virus neutralization. The humoral response in infected patients against this region is poor and humans immunized with gp160 show high levels of antibodies against the peptide but poor neutralization titres. Nonetheless, several groups have succeeded in obtaining neutralizing antibodies against this sequence using different antigen-presenting systems. In order to identify the sequence(s) against which the neutralizing response was generated, we used the flock house virus (FHV) antigen-presenting system to analyse neutralizing antisera from mice immunized with a cowpea mosaic virus (CPMV) chimera expressing the 731-752 sequence. Data show that the neutralizing response is uniquely directed against a conformational epitope mapping to the ERDRD portion of this sequence, although the major antibody response, which is non-linear, and is not neutralizing, is against an IEEE epitope. These results provide an explanation for the controversy regarding the immunogenicity of this region of gp41 and suggest that this conformational epitope, in the absence of the non-neutralizing epitope, should be considered for a subunit vaccine. In addition, this study highlights the usefulness of antigen-presenting systems that preserve epitope conformation in the investigation of immune responses.

Factors associated with serum HCV RNA positivity in anti-HCV antibody positive intravenous drug users.

Serum hepatitis C virus (HCV) RNA, HCV genotypes and liver function tests were evaluated in a series of 189 unselected, consecutive anti-HCV positive intravenous drug users (IVDUs). Serum HCV RNA was detected in 106/189 patients. Abnormal liver function tests were associated with alcohol abuse, but not with the presence of serum HCV RNA. Among 109 patients retested after a mean follow-up of 21 months, 41 were intermittently serum HCV RNA positive. Patients persistently negative had more commonly a past history of acute hepatitis. A history of prostitution and/or a pattern of abuse involving >30 injections per week were related to infection by genotype 3a. In conclusion, serum HCV RNA is either transiently or persistently detectable in most anti-HCV positive IVDUs, but bears no association with abnormal liver biochemistry. Infection by HCV-3a is more common in IVDUs with more deviant life styles. In those cases where serum HCV RNA is found repeatedly negative, HCV infection may have been cleared, possibly through an episode of acute hepatitis.

Localization of the different hepatitis C virus core gene products expressed in COS-1 cells.

In this study we have investigated the subcellular localization in transfected COS-1 cells of the two major forms of the hepatitis C virus core protein: the immature protein of 191 residues (p21) and its proteolytically cleaved product of 173 residues (p19). In this study, and unlike previous investigations, we have been able to distinguish separately the localization of p21 from p19. This was achieved by the addition of a C-terminal HSV Tag to the p21 full coding sequence, and exploiting the fact that it is subsequently lost in the p19 product. In order to obtain an accurate localization of both p21 and p19 we used a mouse anti-HSV Tag MAb together with a human anti-core MAb (B12.F8) to perform double immunofluorescence studies. The results have shown that p21 is always localized around the nuclear envelope. On the other hand, p19 can be found diffused in the cytoplasm to different degrees. These in vivo results reinforce the proposed links between the regulated processing of the hepatitis C virus core protein and the possibility that this may contribute towards the regulation of its diverse biological functions.

Functional analysis of the interaction between HCV 5'UTR and putative subunits of eukaryotic translation initiation factor eIF3.

Translation initiation in Hepatitis C Virus is controlled by the presence of an internal ribosome entry site element (IRES) principally located in its 5' untranslated region (UTR). Mutation/deletion analyses have shown that the integrity of this structure is essential for initiation of cap-independent protein synthesis. We have developed a strategy to swap the position of the two major domains (II and III) on the 5'UTR sequence. The aim was to further characterize this mechanism by preserving domain-specific interactions but possibly losing contacts that require any interdomain geometry. The expression of dicistronic mRNAs containing these different UTRs showed that the positioning of the different domains on the 5'UTR is essential for efficient IRES functioning. We then used these mutants to identify cellular factors implicated in IRES activity. Using UV crosslinking assays we found that domain III makes direct contact with two proteins (p170/p120) which can be associated with efficient IRES activity. In particular, we have mapped the binding sites of these proteins and shown that p120 binds to the apical loop segment of domain III, whilst p170 binds in the stem portion, independently of domain III position or context. Finally, we provide evidence showing that p170 and p120 represent two subunits of eukaryotic initiation factor eIF3: p170 and p116/p110.

High prevalence of infection with hepatitis G virus in patients with hepatic and extrahepatic malignancies.

BACKGROUND/AIMS: The pathogenic role of hepatitis G virus, the recently discovered blood-borne agent, is controversial. Our aim was to ascertain the prevalence of hepatitis G virus infection in hepatic and in extrahepatic malignancies. METHODS: We studied 166 Italian patients (112 male, 54 female, mean age 61.8+/-9.3, mean+/-SD, range 34-85). One hundred and eighteen had cirrhosis, which was complicated by hepatocellular carcinoma in 66 cases. Forty-eight patients had extra-hepatic malignancies. Circulating HGV RNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) of both the nonstructural-3 and 5'noncoding regions of the hepatitis G virus genome. Antibodies to the E2 protein of hepatitis G virus were detected by means of an enzyme-linked immunosorbent assay. RESULTS: Ongoing HGV infection was detected in 30/66 (46%) patients with hepatocellular carcinoma, 12/52 (23%) patients with cirrhosis, and 14/48 (29%) patients with extrahepatic malignancies (p<0.05). Evidence of exposure to hepatitis G virus (detection of either HGV RNA or anti-E2 antibodies) was found in 46% of patients with cirrhosis, 66% of patients with hepatocellular carcinoma, and 39% of patients with extrahepatic malignancies. Serum HGV RNA positivity was associated with a hematocrit value < or = 0.35 and with history of exposure to blood products (p<0.005). CONCLUSIONS: Ongoing hepatitis G virus infection is detected at a very high rate in patients with hepatocellular carcinoma, but is also fairly common in extrahepatic malignancies. Hepatitis G virus infection in these patients is likely to originate from exposure to blood products, and to persist because of deficient immune surveillance.

In vivo translational efficiency of different hepatitis C virus 5'-UTRs.

Initiation of translation in hepatitis C virus (HCV) is dependent on the presence of an internal ribosome entry site (IRES) contained in its 341-nt-long 5'-untranslated region (UTR). This region is very conserved among different isolates and has been used to classify HCV isolates in six different genotypes. These genotypes differ in nucleotide sequence that generally preserve the IRES structure. However, the small differences seen may be biologically and clinically significant as the HCV strains seem to differ from each other in several important ways, such as the behaviour of the viral infection and the response to interferon therapy. Therefore, differences in translational initiation efficiency amongst the various genotypes could provide an explanation for these phenomena. Using a bicistronic expression system we have compared the in vivo translational ability of the three most common European genotypes of HCV (1, 2, and 3). The results show that genotype 3 is less able than 1 and 2 to promote translation initiation. In addition, by site-directed mutagenesis of the sequence of the IRES domain III apical stem loop structure, we have shown that the conservation of the primary nucleotide sequence and not only the structure, is important for the conservation of IRES function.

Endogenous interferon-alpha concentration and outcome of interferon treatment in patients with chronic hepatitis C.

To verify its value with regard to the outcome of therapy in chronic hepatitis C, serum interferon-alpha (IFN) was measured by ELISA in 70 patients (43 male, 27 female) with chronic hepatitis C, treated with IFN 9 MU/week subcutaneously for up to one year. Serum IFN was detectable in 49/70 patients, 16 of whom had IFN values >125 pg/ml. Only 1/22 patients who maintained a sustained response six months after the end of treatment had pretreatment serum IFN > 125 pg/ml, in comparison to 15/48 patients who did not respond or who relapsed (chi2 6.1, P < 0.02). At multivariate analysis the predictive value of serum IFN was independent of age, sex, presence of cirrhosis, infection by genotype 1b (improvement chi2 7.1, P < 0.01). In patients with chronic hepatitis C, measurement of serum IFN at baseline might be useful for the selection of patients with higher probability of long-term response.

A neutralizing monoclonal antibody previously mapped exclusively on human immunodeficiency virus type 1 gp41 recognizes an epitope in p17 sharing the core sequence IEEE.

We report here that a human immunodeficiency virus type 1 (HIV-1)-specific neutralizing monoclonal antibody (MAb 1575) mapped to the conserved putative intracellular region from amino acid residues 735 to 752 (735-752 region) of gp41 also recognizes a region in an extracellular portion of p17. Both epitopes have a core recognition sequence (IEEE) in a nonhomologous context. The IEEE motif found in HIV-1 p17 is located in a region known as HGP-30 (residues 86 to 115) which has been previously associated with virus neutralization, cytotoxic T lymphocyte activity, and mother-to-child transmission. An analysis of available gp41 and p17 sequences demonstrates that in these regions both IEEE sequences are highly conserved in different HIV-1 clades. The presence of the IEEE epitope in p17 allows us to explain some unexpected neutralizing characteristics of MAb 1575. In addition, the gp41 735-752 region has been previously reported both in intra- and extracellular locations. Our results suggest that the extracellular location was the result of cross-reactivity with p17.

Improved reactivity of hepatitis C virus core protein epitopes in a conformational antigen-presenting system.

Recent studies have identified several epitopes in the N-terminal portion of the nucleocapsid protein which are predominantly recognized by sera of patients infected with hepatitis C virus (HCV). The characterization of the sequences recognized by theses antibodies and the evaluation of their reactivities have been performed mainly with synthetic peptides. However, synthetic peptides are notoriously unreliable as antigens when the immune response is directed against conformational epitopes. In order to improve the detection of antibody responses in HCV-infected patients, we have evaluated the reactivities of three immunodominant regions of the HCV core protein (residues 1 to 20, 21 to 40, and 32 to 46) displayed in a conformation-specific manner on the surface of the Flock House virus (FHV) capsid protein. The results obtained with these proteins in the analysis of 94 serum samples positive by anti-HCV enzyme-linked immunosorbent assay where then compared with those obtained with the corresponding synthetic peptides. The sequence most reactive both with the peptide and with the FHV protein was the region from residues 1 to 20, confirming the low conformational requirements for the display of these residues. On the other hand, the already reported conformational nature of residues 32 to 46 is in keeping with its observed high reactivity when displayed by the FHV recombinant protein and with the low reactivity displayed by its corresponding synthetic peptide. Finally, the high reactivity observed for the chimeric protein displaying the region from residues 21 to 40, as opposed to the results obtained with the synthetic peptide, also suggests that this sequence contains one or more conformational epitopes whose structures cannot be mimicked correctly with synthetic peptides.

V3 loop core region serotyping of HIV-1 infected patients using the FHV epitope presenting system.

We have reported recently a new epitope presenting system based on the Flock House Virus (FHV) capsid protein. The HIV-1 V3 loop core sequence IGPGRAF was inserted in different sites of this carrier molecule. Immunoreactivity experiments and molecular modelling consistently showed that the most reactive recombinant protein displayed the IGPGRAF sequence in a conformation which is most similar to that of a V3 loop reference structure. The same insertion site was then used to display the V3 loop apex sequences of six different HIV-1 isolates. Sera from 32 HIV-1 infected patients were examined for their reactivity to our chimeric proteins and the results were compared with those obtained using synthetic V3 loop peptides. The data obtained were confirmed by nested PCR amplification and direct sequencing of the patient's V3 loops. The results showed that the V3 loop serotyping using the FHV hybrid proteins, was more specific than that obtained using synthetic peptides. This system will therefore be a useful tool for the correct evaluation of the immune response against different V3 loop core sequences.

Discordant results from hepatitis C virus genotyping by procedures based on amplification of different genomic regions.

We compared the results of genotyping hepatitis C virus (HCV) either by PCR amplification of the core region or by hybridization of PCR-amplified products of the 5' untranslated region (5'UTR assay). Serum samples from 144 Italian anti-HCV-positive patients (106 drug abusers and 38 patients with chronic viral liver disease but no history of drug abuse) were studied. The original core region assay described by Okamoto et al. (H. Y. Okamoto, Y. Sugiyama, S. Okada, K. Kurai, Y. Akahane, Y. Sugai, T. Tanaka, K. Sato, F. Tsuda, Y. Miyakawa, and M. Mayumi, J. Gen. Virol. 73:673-679, 1992) allowed genotyping of 75 of 144 samples. A modified version of Widell et al. (A. Widell, S. Shev, S. Månsson, Y.-Y. Zhang, U. Foberg, G. Norkrans, A. Frydén, O. Weiland, J. Kurkus, and E. Nordenfelt, J. Med. Virol. 44:272-279, 1994) allowed genotyping of 11 of 79 samples (50 of 79 samples remained unclassified by the method of Okamoto et al. In contrast, all 144 samples were genotyped by the 5'UTR assay. Forty-six of 75 (61 percent) of the samples genotyped by the method of Okamoto et al. and 10 of 11 (91 percent) of the samples genotyped by the method of Widell et al. had results consistent with those obtained by the 5'UTR assay. According to the results of direct sequencing, the method of Okamoto et al. erroneously classified seven samples as having mixed infections. In conclusion, HCV genotyping seems more reliable when it is performed by the 5'UTR assay than by either of two core region assays. The major advantage provided by the 5'UTR assay is a much lower proportion of negative or indeterminate results in younger patients with histories of drug abuse or infection by genotypes other than HCV type 1.