We evaluated the distribution of HCV genotypes in distinct geographic regions of the United States and determined the clinical characteristics of and response to interferon therapy in patients with one of several HCV genotypes. We used the classification system developed by Simmonds and colleagues [7] because it was recently adopted by consensus at the Second International Conference of HCV and Related Viruses (August 1994, San Diego, California). In this system, HCV genotypes are classified into six major genotypes (1 to 6, ordered according to when they were discovered) and 11 subtypes (1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b, 4a, 5a, and 6a).

Methods

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Serum Samples

We analyzed serum samples of 208 patients who were positive for antibody to HCV and had chronic liver disease. The samples were retrospectively obtained from four tertiary referral centers in the United States (59 consecutive samples from the Mayo Clinic, Rochester, Minnesota; 48 consecutive samples from the University of Vermont, Burlington, Vermont; 49 consecutive samples from the University of Miami, Miami, Florida; and 52 consecutive samples from the University of Washington Virology Laboratory, Seattle, Washington [this last center provided samples from Washington State, Idaho, Utah, Oregon, and California]). Twenty-nine patients were excluded from the study: Nineteen had no detectable products for DNA sequencing, and 10 had ambiguous sequencing results. The remaining 179 samples were the focus of this study. No clinical information was available on the patients whose samples were obtained from the University of Washington Virology Laboratory; thus, these samples were used only to study the geographic distribution of HCV genotypes. Data on interferon treatment were available for 78 patients from the Mayo Clinic and the University of Vermont. Samples from these two institutions were obtained from patients who had agreed to participate in trials of interferon treatment.

Reverse Transcriptase and Polymerase Chain Reaction

We selected the direct sequencing technique because it remains the gold standard and the only way to definitively identify all HCV genotypes and subtypes. Hepatitis C virus RNA was extracted from 100-µL aliquots of serum after the addition of 1 mL of RNAzol B solution (Biotecx Laboratories, Houston, Texas) (2 mol of guanidinium thiocyanate per L, 12.5 mol of sodium citrate per L, 0.25% N-laroylsarcosine, 0.05 mol of 2-mercaptoethanol per L, 100 mmol of sodium acetate per L, and 50% water-saturated phenol). After the addition of 100 µL of chloroform, samples were spun for 15 minutes at 14 000 g and the aqueous phase was extracted. Total RNA was precipitated by the addition of isopropanol and 2 µL of glycogen and incubation at 4 °C for 45 minutes. An RNA pellet was recovered by centrifugation at 14 000⁶ g, washed in 1 mL of 70% ethanol solution, dried, and resuspended in 10 µL of RNAase-free water (Promega, Madison, Wisconsin).

Ribonucleic acid was reverse-transcribed into complementary DNA by using reverse transcriptase and an antisense oligonucleotide primer (5'-CGCGGAATTCCTGGTCATAGCCTCCGTGAA-3') in the presence of reverse-transcriptase buffer (100 mmol of tris-HCl per L, 500 mmol of KCl per L, 1% Triton X-100, and a pH of 8.6 at 25 °C) (Promega) and 3.0 mmol of magnesium per L. Hepatitis C virus complementary DNA was amplified by polymerase chain reaction (PCR) in the presence of the sense oligonucleotide primer (5'-TGGGGATCCCGTATGATACCCGCTGCTTTGA-3'), PCR buffer (500 mmol of KCl per L, 100 mmol of tris-HCl per L, and a pH of 8.3) (Perkins-Elmer-Cetus, Norwalk, New Jersey), 2.0 mmol of magnesium per L, and Amplitaq DNA polymerase (Perkins-Elmer-Cetus). The PCR assay was done in a DNA thermal cycler for 50 cycles (94 °C for 1 minute, 58 °C for 1 minute, and 72 °C for 5 minutes). Products of the PCR assay were analyzed by gel electrophoresis in 3% agarose gel that was stained with ethidium bromide. The appearance of a band 401-base pair was considered a positive result. To avoid and monitor for possible contamination with exogenous sequences during extraction or amplification, extraction of nucleic acid and genomic amplification steps were done in separate laboratories. Ribonucleic acid samples from at least one negative and one positive sample were extracted, subjected to reverse transcription, and amplified in each batch of samples tested by PCR. No false-positive results were obtained in any of the negative controls.

Sequencing and Genotyping

Each fragment of the PCR product, which was approximately 401 base pairs long, was desalted before undergoing sequencing with a direct column-purification method (Wizard PCR Preps DNA Purification System, Promega). Automated sequencing was done by using a standard Sanger procedure, which involved the incorporation of fluorescein-labeled dideoxynucleotides and detection on an acrylamide gel (ABI model 373 A, Applied Biosystems, Hercules, California).

Nucleotide sequences were aligned and compiled with the previously reported sequences by using the Pileup program (Wisconsin Genetic Computer Group, Madison, Wisconsin)^[8]. Cluster analysis was done by using the unweighted-pair group mean average, which was included in the program. These methods allowed comparison of a 222-base pair fragment of DNA that was homologous to nucleotide positions 7975 to 8196 in the NS5 region of the prototype virus.

Collection of Epidemiologic Data

We studied the geographic distribution of the HCV genotypes identified in the blood samples. Data from all samples were combined to define the prevalence of the HCV genotypes in patients with chronic hepatitis C in the United States. When available, age, sex, risk factors for HCV acquisition, and liver histologic findings at the time of presentation were recorded for each patient. Risk factors for acquiring HCV included history of blood transfusion, history of injection drug use, and employment at a health care facility. Liver histologic findings were classified into three groups: mildly active hepatitis (portal inflammation without substantial hepatocyte necrosis), moderately active hepatitis (inflammation with hepatocyte necrosis), and liver cirrhosis. Accurate history of alcohol consumption was not available for many of these patients and thus was not included in the analysis. The investigator who did the genotyping was blinded to the clinical data of patients at the time of analysis. Liver biopsy specimens were independently interpreted at each center.

Pathogenicity of Hepatitis C Virus Genotypes

To study the possible differences in the pathogenicity of HCV genotypes, we divided patients into two groups: patients with mild hepatitis and patients with severe hepatitis. Mild hepatitis was defined as 1) pretreatment alanine aminotransferase levels that were less than three times the normal level and 2) no cirrhosis seen during examination of the liver biopsy specimen obtained before treatment. Severe hepatitis was defined as pretreatment alanine aminotransferase levels greater than three times the normal level or the presence of liver cirrhosis on pretreatment biopsy.

Response to Interferon

Seventy-eight patients received an average dose of 3 million U of interferon (interferon- or consensus interferon) for 6 months. Response to interferon was defined as the normalization of alanine aminotransferase levels at the end of therapy. Partial response to interferon (defined as decreased but not completely normal alanine aminotransferase levels) was considered to be a treatment failure. Sustained biochemical response was defined as a normal alanine aminotransferase level 6 months after the discontinuation of interferon treatment.

Statistical Analysis

We used the rank-sum and Kruskal-Wallis tests to compare continuous variables (such as age) between groups, and we used the Fisher exact test to assess associations in tabular data. Because few patients had genotype 2a, 3, or 4, all tests of association between genotype and other factors are based on data that were collapsed into four groups: genotype 1a, genotype 1b, genotypes 2a and 2b, and genotypes 3 and 4. Logistic regression was used to evaluate the association between response to interferon and the combined predictors of cirrhosis and genotype. We used the SAS statistical analysis package (SAS Institute, Cary, North Carolina) for all calculations.

Results

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Geographic Distribution of Hepatitis C Virus Genotypes

Hepatitis C virus genotype 1a was present in 104 of 179 (58%) patients with chronic HCV infection; genotype 1b was the second most common genotype encountered (38 of 179 patients [21%]). Genotype 2b was present in 23 patients (13%), and genotype 3a was present in 8 patients (5%). Four patients (2%) had HCV genotype 2a, and 2 (1%) had genotype 4a. Geographic region and distribution of genotypes were not significantly associated (P = 0.18). However, samples obtained from the western United States contained a slightly higher proportion of HCV genotype 1b. Samples obtained from the southeastern United States contained HCV genotype 4a, which was not seen in the other three regions (Table 1).

Epidemiology of Hepatitis C Virus Genotypes

The mean patient age varied significantly among the groups of patients with different HCV genotypes (P = 0.006; Kruskal-Wallis test). The major difference was seen between patients with genotype 1b (mean age ±SD, 49 ± 2.5 years) and patients with one of the other serotypes (40 ± 1 years) (P < 0.001; rank-sum test). Seventy-four percent of all patients were men (range, 62% [in the group with genotype 3a] to 100% [in the group with genotype 2a or 4a]) (Table 2).

Blood transfusion in the absence of other risk factors was documented in 24% of patients with HCV genotype 1a, 27% of those with genotype 1b, 23% of those with genotype 2b, and 50% of those with genotype 3a. A history of illicit injection drug use was noted in 50% of patients with genotype 1a, 41% of those with genotype 1b, 41% of those with genotype 2b, 37% of those with genotype 3a, 2 of 3 patients with genotype 2a, and 1 of 2 patients with genotype 4a. Eight percent of patients with genotype 1a, 23% of patients with genotype 1b, and 12% of patients with genotype 2b were employed as health care providers and reported no history of blood transfusion or injection drug use. Twenty-two patients (18%) denied having any of the three risk factors for HCV acquisition: Thirteen of these patients had HCV genotype 1a, 2 had genotype 1b, 1 had genotype 2a, 4 had genotype 2b, 1 had genotype 3a, and 1 had genotype 4a.

Of the 120 patients who were known to have had liver histologic testing, 29% had liver cirrhosis at the time of presentation (range, 11% [in the group with genotype 2a or 2b] to 67% [in the group with genotype 3a, 3b, or 4a]). Thirty-one percent of patients with genotype 1a and 25% of patients with genotype 1b had cirrhosis (P = 0.03).

Association with Disease Activity

Clinical data required for studying the severity of liver disease in relation to HCV genotype (based on our definitions of mild and severe chronic hepatitis) were available from 63 patients with genotype 1a, 20 patients with genotype 1b, 2 patients with genotype 2a, 14 patients with genotype 2b, 6 patients with genotype 3a, and 2 patients with genotype 4a. Severe hepatitis was seen in 43 of 63 patients (68%) with genotype 1a; 16 of 20 patients (80%) with genotype 1b; 7 of 8 (87%) patients with genotype 3a, 3b, or 4a; and 6 of 16 patients (37%) with genotype 2a or 2b (P = 0.03) (Table 3).

Response to Interferon Therapy

Seventy-nine patients received interferon for 6 months. Forty-six interferon recipients had genotype 1a, 15 had genotype 1b, 1 had genotype 2a, 13 had genotype 2b, and 4 had genotype 3a. Neither age (P = 0.2) nor sex (P = 0.14) was significantly associated with response to therapy. Table 4 shows the association between response to interferon and cirrhosis and genotype. Both variables were significant in a multivariate logistic regression analysis (P = 0.03 for genotype, P = 0.02 for cirrhosis). The overall rates of response to interferon for persons with and without cirrhosis were 14% and 46%, respectively. The rates of response to interferon for persons with genotype 1a, 1b, or 3a were all between 25% and 28%; the rate in persons with genotype 2a or 2b was 71%.

Data on sustained biochemical response 6 months after discontinuation of interferon therapy were available from 67 patients, 8 of whom (12%) had a sustained response. Age, sex, cirrhosis, and genotype were not significantly associated with response to treatment. One of 23 (4%) patients with cirrhosis had a sustained response compared with 7 of 45 patients (16%) in whom cirrhosis was not seen on examination of a liver biopsy specimen (P > 0.2). Five of 39 (13%) patients with HCV genotype 1a, 1 of 14 (7%) patients with genotype 1b, and 2 of 12 (18%) patients with genotype 2a or 2b had a sustained response (P > 0.2).

Discussion

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After Houghton and colleagues [9] initially characterized the full HCV genome, several other HCV isolates from different parts of the world were identified and sequenced [10-15]. Regions that encode the putative envelope proteins (E1, E2/NS1) are the most variable [16], and the 5' untranslated region is the most conserved [17]. Comparison of known genomic RNA sequences of HCV has led to the identification of many distinct genotypes that may diverge by as much as 33% over the entire length of the viral genome [18]. We used the Simmonds system for classifying HCV genotypes [7]. The other commonly used classification system, developed by Okamoto and colleagues [19], includes types I, II, III, IV, V, and VI, which correspond to genotypes 1a, 1b, 2a, 2b, 3a, and 3b of the Simmonds system.

We studied the distribution of HCV genotypes in patients with chronic HCV infection from four geographic regions in the United States. The distribution of genotypes was similar in all four areas with two exceptions: the finding that the two samples containing genotype 4a were both obtained from Florida and the finding that genotype 1b was slightly more common among the samples obtained from the western United States. Genotype 4a was originally described in patients from the Middle East [20]. The presence of this rare genotype in Florida may reflect the high rate of immigration to that state. The fact that HCV genotype 1b is the most common genotype among Japanese patients with chronic hepatitis C may explain the higher prevalence of this genotype in patients from the western United States. However, because the association was not statistically significant (P = 0.18), these small imbalances may have been caused by chance. Our results indicate that genotypes 1a and 1b may be found in most persons with HCV infection in the United States (in our study, 80% of the samples contained one of these genotypes). Genotypes 2b and 3a were present in 12% and 5% of samples, respectively. All samples were obtained from tertiary care centers and therefore may not represent all subpopulations in the United States.

Among the 11 HCV genotypes, we found no statistically significant differences in the distribution of the sex of the patients, the mode of virus transmission, or liver histologic findings at presentation. However, patients with HCV genotype 1b were significantly older than patients with other genotypes. A recent study by Pol and colleagues [21] showed that the relative prevalence of genotype 1b was significantly higher in patients who were infected in the 1960s than in those who were infected in the 1980s. These data may indicate a change in the epidemiology of HCV genotypes over time, which could explain the older mean age of patients with genotype 1b. We did not explore the possible association between specific HCV genotypes and sexual or vertical transmission; this association may need to be considered in future studies.

Previous reports [22] have suggested that HCV genotype 1b may be associated with more severe liver disease and a more aggressive course of infection. We also found that most patients with genotype 1a or 1b had severe hepatitis and that most patients with genotype 2a or 2b had mild hepatitis. These data are further supported by our previous findings that genotype 1b is more commonly seen in patients with decompensated liver disease that requires liver transplantation and is associated with a higher incidence of and more severe post-transplantation hepatitis [23, 24]. The mechanism by which genotype 1b is more pathogenic in the host has not yet been determined. Preliminary data have shown that genotypes 1a and 1b are associated with levels of viremia in the host that are significantly higher than those associated with genotype 2b [25]. These data may explain some of the differences in pathogenicity among genotypes.

Much effort has been expended in defining factors that may play a role in predicting long-term responses to interferon therapy [26]. Interferon dose [27], duration of treatment [28], viral RNA level [29], and liver histologic findings [30] may all help to predict response. Recent studies of patients with chronic HCV infection who live in countries other than the United States have suggested that some HCV genotypes are more likely than others to respond to interferon therapy. Chayama and colleagues [31] assessed the susceptibility of different HCV genotypes to interferon in 69 patients with chronic HCV infection. Partial or complete short-term responses to interferon occurred in 55% of patients with genotype 1b and in 89% of patients with other genotypes combined (primarily genotypes 2a and 2b). In a similar study, Takada and coworkers [32] found significantly lower rates of response to interferon in patients with HCV genotype 1b than in patients with other genotypes. Our findings in patients from the United States agree with those of the above studies. Patients with genotype 1b had the lowest rate of complete response after 6 months of interferon therapy and the lowest rate of sustained response, whereas genotype 2b was associated with the most responders. These results may partly explain the higher rates of long-term response to interferon reported in Europe, where genotypes 2a and 2b are more prevalent than they are in the United States. We also found that patients without liver cirrhosis had a higher rate of response to interferon than did patients with cirrhosis, regardless of genotype. Therefore, HCV genotype and liver histologic findings may be independent variables in the determination of rate of response to interferon in patients with chronic HCV infection. Causse and colleagues [27] found that younger age and female sex were associated with better response to interferon. We found no such correlations in our patients, and the association may need to be clarified in future studies.

We conclude that HCV genotype 1a and, to a lesser extent, genotype 1b are the most common HCV genotypes in the United States. In contrast to genotypes 2a and 2b, genotypes 1a and 1b seemed to be associated with more severe liver disease and a lower rate of response to interferon therapy.

Appendix

The following are members of the Collaborative Study Group: Mayo Clinic, Rochester, Minnesota: Terry M. Therneau, PhD, John B. Gross Jr., MD, John J. Poterucha, MD, and Andria A. Gossard, RN; University of Miami, Miami, Florida: Lennox J. Jeffers, MD, and Eugene R. Schiff, MD.

From the Mayo Clinic, Rochester, Minnesota; the University of Miami, Miami, Florida; and the University of Vermont, Burlington, Vermont.

Dr. Rakela: Division of Transplantation Medicine, 301 Lhormer Building, 200 Lothrop Street, Pittsburgh, PA 15213-2582.

Dr. Krawitt: University of Vermont, One South Prospect Street, Given Medical Building, Burlington, VT 05405.

Dr. Reddy: University of Miami, 1600 NW 10th Avenue, Miami, FL 33101.