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Re: INfo on Genotypes, etc. Articles- Marty

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  • byteme
    These are some of the more acceptable articles on Genotype. But, I URGE anyone, take these with a grain of salt . They are studies from 1996 to 1999, which
    Message 1 of 1 , Apr 1, 2000
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      These are some of the more acceptable articles on Genotype. But, I URGE
      anyone, take these with a "grain of salt". They are studies from 1996 to
      1999, which usually means they are anywhere from 6 months to 2 years
      done prior to publication. They can offer some data, but, if you focus
      on these too much, you will either came out very happy or very
      depressed.

      Everyone is different, and so is their body chemistry, and today's
      knowledge, so don't take these are absolutes. They are just some
      articles I have, and, after wading through all the Medicaleeze and
      Scientificeeze, the conclusions are just a basis, or place to start. You
      may do much better, or not as well. Marty



      It is much easier to talk of the hepatitis C virus as if it is
      a single organism
      but in fact it is a range of viruses, similar enough to be
      called hepatitis C
      virus, yet different enough to be classified into subgroups.

      Viruses are microscopic and no person could ever see them with
      the
      naked eye. Indeed, HCV is so small that there's been no
      confirmed
      actual sighting of it using any type of microscope yet
      developed.

      Consequently, a better way to understand the terms HCV
      'genotypes'
      and 'subtypes' is to compare them to things that we can more
      readily
      relate to.

      Genotypes

      The group of birds we call 'raptors' (birds of prey) have
      evolved into
      different main types. Imagining raptors as being hepatitis C
      viruses, you
      could take one major raptor type, such as eagles, and imagine
      these as
      being one of HCV's main types (genotypes).

      Subtypes

      But eagles as a group are made up of different sub types such
      as the
      American Bald Eagle and Australia's Wedge Tailed Eagle and Sea

      Eagle. You could imagine each of these as being one of the HCV

      subtypes that make up an HCV genotype.

      Quasispecies

      Within each of above particular types of eagles, there are
      further
      differences. All Wedge Tailed Eagles, for example, differ from
      each
      other in regard to wing span, weight, colour, beak size, etc.
      Similarly,
      within a hepatitis C sub-type, individual viruses differ from
      each other
      ever so slightly. Such viral differences are not significant
      enough to form
      another sub-type but instead form what's known as
      quasi-species. It is
      believed that within an HCV sub-type, several million
      quasispecies may
      exist. Scientists predict that people who have hepatitis C,
      have billions of
      actual viruses circulating within their body. Although there
      may be one or
      two predominant sub-types, the infection as a whole is not a
      single entity
      and is composed of many different quasispecies.

      Classifications

      Biologists are generally not known for creativity when it
      comes to naming
      things - hence hepatitis C virus. The most commonly used
      classification
      of hepatitis C virus has HCV divided into the following
      genotypes (main
      types): 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11. As we've
      highlighted, HCV
      genotypes can be broken down into sub-types, some of which
      include:

      1a, 1b, 1c
      2a, 2b, 2c
      3a, 3b
      4a, 4b, 4c, 4d, 4e
      5a
      6a
      7a, 7b
      8a, 8b
      9a
      10a
      11a

      Genotype patterns

      It is believed that the hepatitis C virus has evolved over a
      period of
      several thousand years. This would explain the current general
      global
      patterns of genotypes and subtypes:

      1a - mostly found in North & South America; also common in
      Australia
      1b - mostly found in Europe and Asia.
      2a - is the most common genotype 2 in Japan and China.
      2b - is the most common genotype 2 in the US and Northern
      Europe.
      2c - the most common genotype 2 in Western and Southern
      Europe.
      3a - highly prevalent here in Australia (40% of cases) and
      South Asia.
      4a - highly prevalent in Egypt
      4c - highly prevalent in Central Africa
      5a - highly prevalent only in South Africa
      6a - restricted to Hong Kong, Macau and Vietnam
      7a and 7b - common in Thailand
      8a, 8b & 9a - prevalent in Vietnam
      10a & 11a - found in Indonesia

      It's believed that of the estimated 160,000 Australians with
      HCV,
      approx. 35% have subtype '1a', 15% have '1b', 7% have '2', 35%
      have
      '3' (mostly being 3a). The remaining people would have other
      genotypes.

      Genotype and treatment

      Current scientific belief is that factors such as duration of
      a person's
      HCV infection, their HCV viral load, age, grade of liver
      inflammation or
      stage of fibrosis may play an important role in determining
      response to
      interferon treatment. Recent studies have suggested that a
      person's HCV
      subtype (or subtypes) may influence their possible response to

      interferon, or interferon-ribavirin combination treatment.
      World-wide
      trials are being conducted which will soon shed more light on
      this belief.
      We'll publish any reports as they come to hand.



      Genotypes and Genetic Variation of Hepatitis C Virus
      by G. Maerterns & L. Stuyver,
      reviewed by Dr Greg Dore
      of the National Centre in HIV Epidemiology & Clinical
      Research.
      From The Hep C Review; Ed 23, December 1998; Paul Harvey

      Hepatology, March 1999, p. 897-903, Vol. 29, No. 3
      Relationship of the Genomic Complexity of Hepatitis C
      Virus With Liver Disease Severity and Response to
      Interferon in Patients With Chronic HCV Genotype 1b
      Infection

      Francesc-Xavier Lspez-Labrador1,*, Sergi Ampurdanhs1, Mireia
      Giminez-Barcons1, Magdalena Guilera1, Josep Costa2, Marma
      Teresa
      Jiminez de Anta2, Jose M. Sanchez-Tapias1, Juan Rodis1, and
      Juan-Carlos Saiz1

      From the 1Liver Unit, Department of Medecine, Institut
      d'Investigacions
      Biomhdiques August Pm i Sunyer (IDIBAPS); 2Microbiology
      Department, Hospital Clmnic, Facultat de Medicina, Universitat
      de
      Barcelona, Spain.

      ABSTRACT

      In patients with chronic hepatitis C, the influence of the
      genetic
      heterogeneity of the hepatitis C virus (HCV) on the
      progression of liver
      disease and on the responsiveness to interferon therapy is a
      matter of
      controversy. In this study we evaluated the genetic complexity
      of HCV
      by single-strand conformation polymorphism (SSCP) analysis of
      amplicons from the hypervariable region 1 (HVR1) in 168
      patients with
      chronic genotype 1b HCV infection, of whom 122 received a
      single
      course of interferon therapy (3 MU, three times weekly for 6
      months).
      No correlation was observed between the degree of genetic
      complexity
      of HCV (indicated by the number of bands in the SSCP assay)
      and
      patient age, serum alanine aminotransferase activity, or serum

      HCV-RNA concentration, measured by competitive polymerase
      chain
      reaction. HCV genomic complexity was not related to gender nor
      to the
      presumed source of infection. The number of SSCP bands
      detected in
      serum samples from patients with chronic hepatitis, either
      mild
      (8.1 1 3.9), moderate (8.0 1 3.3), or severe (9.2 1 3.3), and
      in patients
      with liver cirrhosis, either compensated (8.0 1 2.9),
      decompensated
      (6.3 1 2.9), or with superimposed hepatocellular carcinoma
      (9.5 1 2.9),
      was similar. The number of SSCP bands detected in patients
      with
      sustained response (7.5 1 3.9), transient response (8.3 1
      2.9), or no
      response (8.2 1 3.6) to interferon administration was similar
      as well.
      These observations suggest that the genetic complexity of
      hypervariable
      region (HVR1) of HCV, as estimated by SSCP analysis, is not
      related
      to the severity of liver injury nor to the type of response to
      interferon
      therapy. Thus, information offered by SSCP analysis of HVR1 of
      HCV
      in chronic HCV genotype 1b infection does not appear to be
      useful in
      the clinical management of these patients. (HEPATOLOGY
      1999;29:897-903.)
      (HEPATOLOGY 1999;29:897-903.)

      INTRODUCTION

      The hepatitis C virus (HCV) is an RNA virus that replicates
      with a high
      rate of mutation,1 which is particularly evident in the
      hypervariable region
      1 (HVR1) of the N-amino terminal region of the second envelope

      domain of the viral genome.2,3 Under the influence of
      environmental
      factors, continuous viral mutation gives raise to a mixed and
      changing
      population of mutants, which is known as quasispecies.4 As in
      infections
      with other RNA viruses, the quasispecies nature of HCV5 may
      have
      important biological implications concerning viral
      persistence,
      pathogenicity, and resistance to antiviral agents. However,
      attempts
      aimed to define the relationship between clinical aspects of
      chronic HCV
      infection and the genetic heterogeneity of the infecting virus
      have
      provided conflicting results.

      By sequence analysis of HVR1, greater nucleotide sequence
      diversity
      between HCV variants was shown in isolates from patients with
      more
      advanced liver disease,6 but this finding has not been
      confirmed by
      others.7 Studies based on single-strand conformational
      polymorphism
      (SSCP) analysis of HVR1 derived amplicons have also provided
      controversial data. In 1995, Koizumi et al.8 found that the
      viral
      populations were more heterogeneous in patients with hepatic
      cirrhosis
      or hepatocellular carcinoma than in those with chronic
      hepatitis, but other
      studies did not disclose a clear association between the
      degree of HCV
      quasispecies complexity and the histological severity of liver

      disease.7,9,10

      At present, the relationship between the degree of genomic
      complexity
      of HCV and the type of response to interferon therapy is
      unclear.
      Previous studies of a relatively small series of patients
      indicated that, in
      general, poor response to interferon was associated with high
      genomic
      complexity of HCV, whereas sustained response was associated
      with
      low genomic complexity.11-15 However, data on patients
      infected with
      genotype 1b are conflicting because the significant
      correlation between
      low genetic complexity of HCV and long-term response to
      interferon
      therapy found in some studies8,16 was not observed in
      others.15,17

      In the present study we investigated the genomic complexity of
      HCV by
      SSCP analysis of the HVR1 in a large series of patients with
      chronic
      HCV genotype 1b infection and chronic liver disease of
      variable
      severity, ranging from asymptomatic mild chronic hepatitis to
      decompensated cirrhosis and hepatocellular carcinoma. Because
      a
      substantial proportion of the patients received a course of
      interferon-,
      the relationship between genomic complexity of HCV and
      response to
      interferon was also investigated.

      PATIENTS AND METHODS

      Patients. One hundred sixty-eight patients with chronic liver
      disease
      related to chronic infection with HCV genotype 1b were
      included in the
      study. All patients were seropositive for anti-HCV antibodies
      by a
      third-generation enzyme-linked immunosorbent assay (ELISA)
      technique
      (HCV ELISA 3.0, Ortho Diagnostic Systems, Raritan, NJ) and had

      detectable HCV-RNA in serum by nested PCR of the 5'NCR.18 No
      patient had previously received antiviral or immunosuppressive
      therapy,
      nor did any patient have any other potential cause of chronic
      liver
      disease, such as current hepatitis B virus infection (positive
      test for
      hepatitis B surface antigen), excessive alcohol intake (40 g
      per day for
      women and 80 g per day for men), or autoimmune or metabolic
      disorders affecting the liver. Informed consent was obtained
      in every
      case, and the Ethics Committee of our Institution approved the
      study.

      The study population included 131 consecutive outpatients with

      asymptomatic or minimally symptomatic chronic hepatitis C who
      were
      referred for antiviral therapy between January 1995 and June
      1996, and
      37 randomly selected patients with advanced liver disease who
      were
      hospitalized during the same period of time. Among the former,
      the
      histological severity of chronic hepatitis was defined as mild
      in 41 cases,
      moderate in 45, and severe in 45, according to recently
      proposed
      criteria.19 Among the latter, 17 patients had histological
      evidence of
      cirrhosis and were classified as having compensated cirrhosis.
      Of the
      37 patients with advanced liver disease, 17 were admitted for
      treatment
      of major complications of hepatic cirrhosis, such as ascites,
      hepatic
      encephalopathy, or variceal bleeding, and were classified as
      decompensated cirrhosis; the remaining 20 had evidence of
      hepatic
      cirrhosis with superimposed hepatocellular carcinoma and were
      admitted
      for diagnostic or therapeutic purposes. The diagnosis of
      hepatocellular
      carcinoma was based on ultrasonographic or computed tomography

      findings and was confirmed by cytological or histopathological

      examination of liver specimens in all the cases.

      Of the 131 patients with uncomplicated chronic hepatitis, 122
      received
      one full course of interferon therapy, consisting of 3 MU
      injections of
      recombinant interferon-,-2b, three times a week for 6 months,
      and
      were followed for at least 6 months on no therapy. The
      severity of liver
      injury disclosed by pretreatment liver biopsy in treated
      patients was
      considered as mild in 41 cases, as moderate in 41, and as
      severe in
      40, of whom 17 had cirrhosis. The response to treatment was
      evaluated
      by biochemical (ALT) and virological (HCV-RNA in serum)
      parameters. The detection limit of the PCR technique used for
      qualitative
      detection of HCV-RNA was 100 copies per mL. Sustained response

      was defined by the presence of normal ALT and undetectable
      HCV-RNA at the end of the treatment period and at the end of
      post-treatment follow-up; transient response was defined by
      the
      presence of normal ALT and undetectable HCV-RNA at the end of
      treatment, followed by either biochemical or virological
      relapse on
      post-treatment follow-up; and no response was defined by
      elevated ALT,
      and or detectable HCV-RNA at the end of treatment.

      HCV-RNA Extraction and Complementary DNA Synthesis. Serum
      samples were
      collected immediately before the start of interferon therapy
      or at the time of liver
      biopsy in patients with chronic hepatitis or compensated
      cirrhosis and during
      hospitalization in patients with decompensated cirrhosis or
      with hepatocellular
      carcinoma. Sera was aliquoted 2 hours after venipuncture and
      stored at 700
      C. HCV-RNA was extracted from 180 5L of serum by the
      acid-guanidinium
      isothiocyanate phenol-chloroform method.20 Extracted RNA was
      resuspended in
      505L of diethylpyrocarbonate-treated water. cDNA synthesis was
      carried out with
      300 units of Moloney Murine Leukemia Virus reverse
      transcriptase (MMLV,
      GIBCO-BRL, Gaithersburg, MD) for 1 hour at 370C, using 5 5L of
      resuspended
      RNA, random hexanucleotides (Boehringer Mannheim, Mannheim,
      Germany), and
      20 units of ribonuclease inhibitor (RNAsin, Promega, Madison,
      WI) in a final
      volume of 25 5L.

      HCV Genotyping and HCV-RNA Quantitation. HCV genotype was
      determined
      by restriction fragment length polymorphism analysis of the
      5'NCR as previously
      described.21,22

      HCV-RNA concentration in serum was measured by an
      in-house-developed
      competitive PCR method as previously described.23 This method
      had a dynamic
      range between 6 W 103 and 6 W 107 copies per mL and measured
      values correlated
      well with measurements obtained by Amplicor HCV Monitor test
      (Roche
      Diagnostic Systems, Inc., Branchburg, NJ) in serum specimens
      infected with HCV
      genotype 1b.

      Amplification of the HVR1 by Nested PCR. The HVR1 of HCV was
      amplified by
      a nested PCR procedure with genotype 1b-specific primers
      derived from the E2
      region. Primary PCR was performed with 5 5L of cDNA in a 25 5L
      final volume
      with primers HV1 (sense, nt. 1290-1312, 5'-CGC ATG GCT TGG GAT
      ATG ATG
      AT-3') and HV2 (antisense, nt. 2001-2023, 5'-GTG AAC CCA GTG
      CTG TTC ATC
      CA-3'). One microliter of the primary PCR reaction was used as
      template in a
      secondary PCR round with primers HV3 (sense, nt.1431-1453,
      5'-ATG GTG GGT
      ACC TGG GCT AAG GT-3') and HV6 (antisense, nt. 1598-1621,
      5'-AGG GAA TTC
      CTG TTG ATG TGC CA-3') in a 50 5L final reaction volume.
      Nucleotide positions
      are according to Okamoto et al.1 Secondary PCR produced a
      fragment of 190 base
      pairs (bp) of the E2 gene including the HVR1. Cycling
      conditions for both primary
      and secondary amplifications were as follows: 940C for 5
      minutes (940C for
      30 seconds, 550C for 30 seconds, 720C for 1 minute) W 35
      cycles; 720C 5 minutes.
      Final concentrations of reagents used were: MgCl2 1.5 mmol/L,
      dNTP 200 5mol/L,
      primers 0.25 5mol/L, and Taq DNA polymerase (GIBCO-BRL,
      Gaithersburgh, MD)
      0.125 units/25 5L reaction volume. To avoid PCR contamination,
      Kwok and
      Higuchi general guidelines were strictly observed.24 Four
      negative and two
      positive controls were included in each round of
      amplification. Amplified products
      were identified in 1.5% agarose gels after ethidium bromide
      staining.

      SSCP Analysis of the HCV Genomic Complexity in the HVR1. To
      avoid
      underestimation of HVR1 variants present in samples from
      patients with low
      viremia levels, approximately the same amount of the 190 bp
      HVR1 amplified DNA
      products from each sample was subjected to SSCP analysis. The
      amount of DNA
      in amplified HVR1 PCR products was estimated by comparison
      with DNA
      standards of known concentration after electrophoresis in
      agarose gels. About
      30 ng of double-stranded HVR1 secondary PCR products were
      denatured with
      10 5L of SSCP loading buffer (95% deionized formamide, 0.01%
      bromophenol blue,
      20 mmol/L ethylenediaminetetraacetic acid) at 950C for 10
      minutes and quickly
      cooled in an ice-water bath during 10 minutes. Samples were
      immediately loaded
      into 12% nondenaturing polyacrylamide minigels (Mini-protean
      II system, BioRad,
      Richmond, VA) and electrophoresed during 4 hours at 100 volts
      at room
      temperature. Bands were visualized on the gel by silver
      staining25 and the number
      of SSCP bands was determined by scanning the gels by using the
      Bioimage
      Whole-Band analyzer software, version 2.51 (Bioimage System
      Co, Ann Arbor,
      MI).

      To assess the reproducibility of the assay, a
      well-characterized sample with a
      specific SSCP pattern was used as a control in each
      experiment. One
      nondenatured and one denatured control was included in each
      SSCP gel. Only
      gels with no bands in the nondenatured control and the
      appropriate number of
      bands in the denatured control were considered. In addition,
      testing under the
      same conditions was carried out in 50 cDNA samples from
      different serum
      samples.

      To assess the specificity of the assay, SSCP bands were
      excised from the gel,
      reamplified as described above, purified, and directly
      sequenced by using the
      Thermosequenase Dye Terminator kit (Amersham, Cleveland, OH)
      in a 310 DNA
      sequencer (Applied Biosystems, Westerstad, Germany).

      The sensitivity of the assay was determined as follows:
      reciprocal amounts of two
      selected samples that had a similar concentration of cDNA, but
      displayed a
      different SSCP pattern, were serially mixed to obtain an
      equimolar amount of total
      cDNA in each mixture. Then, nested PCR of the HVR1 and SSCP
      analysis were
      performed in the mixed cDNA samples. The experiment was
      repeated twice to
      check the reproducibility of the technique.

      To further evaluate the sensitivity of the SSCP assay to
      detect minor variants,
      amplified cDNA from representative samples was cloned into TA
      cloning plasmid
      pGEM T (Promega, Madison, WI). Competent XL-1 cells were
      transformed with
      recombinant plasmids and 10 to 20 randomly selected clones
      from each isolate
      were sequenced as described above.

      Statistical Analysis. The median number of SSCP bands observed
      in samples
      under study was used to define the viral complexity in the
      HVR1 as low or high.
      Qualitative data were analyzed by using the 2 test or the
      Fisher's exact test when
      necessary. Quantitative values were compared by using the
      Student's t test, the
      Kruskal Wallis test or the analysis of the variance when
      necessary. P values lower
      than 0.05 were considered significant. All statistical
      calculations were performed
      by using the SPSS for Windows, version 6.0 software package.

      RESULTS

      Sensitivity, Specificity, and Reproducibility of the PCR-SSCP
      Assay. To assess
      the sensitivity of the PCR-SSCP assay, mixtures containing
      reciprocal amounts of
      two samples with a different SSCP pattern were analyzed (Fig.
      1). Variants
      representing 5% to 10% of the whole viral population present
      in the mixtures were
      identified as individual bands after silver staining.



      To View Larger Image
      Fig. 1. Estimation of the
      sensitivity of the PCR-SSCP
      assay. Lane 1, molecular weight
      marker. Reciprocal amounts of
      cDNA from two isolates (A and
      B) producing a different SSCP
      pattern were mixed, amplified by
      PCR, and submitted to SSCP
      analysis (lanes 2 to 14). Bands in

      the upper and lower parts of the
      gel correspond to HCV variants
      present in isolate A and in
      isolate
      B, respectively. Variants
      representing at least 5% of the
      total viral population present in
      the
      mixtures produced visible bands in

      the gel (arrows).


      Analysis of denatured controls showed that the number of bands
      and the SSCP
      pattern were identical in repeated experiments, whereas no
      bands were observed
      following SSCP of nondenatured controls.

      Direct sequencing of reamplified bands excised from the gel
      was performed to
      confirm the accuracy of the analysis. Three to four nucleotide
      changes were
      observed between two different SSCP bands excised from the
      same isolate when
      compared with the consensus sequence (Fig. 2).





      To View Larger Image

      Click on Graphic
      Fig. 2. (A) PCR-SSCP analysis
      of cDNA from isolate A (see Fig.
      1). (B) Nucleotide sequences
      obtained after excision from the
      gel of the two bands. The
      sequences obtained from each
      band differed in three to four
      nucleotides from the consensus
      sequence. Dashes represent
      nucleotide identity with the
      consensus sequence. Lane 1,
      molecular weight marker. R = A
      or G.


      Cloning experiments were carried out by using amplified DNA
      from samples with
      low SSCP complexity and from samples with high SSCP complexity
      (the definitions
      of SSCP complexity are given below). Sequence analysis of 10
      to 20 clones from
      each isolate showed that the number of individual clones
      differing from the
      uncloned sequence in a few nucleotide positions was higher in
      samples with high
      than in those with low SSCP complexity (data not shown).

      To assess the reproducibility of the method, 50 samples were
      retested from the
      cDNA synthesis step. The same numbers of bands and SSCP
      patterns were
      observed in all retested samples (data not shown).

      HCV Genomic Complexity in the HVR1 and Disease Severity.
      Nested PCR of
      the HVR1 generated a single DNA band of the expected size in
      all the cases. The
      number of bands detected by SSCP analysis in the whole series
      ranged from 2 to
      20.

      The basal features of patients and the results of the SSCP
      analysis are summarized
      in Table 1. The number of SSCP bands observed in individual
      cases did not
      correlate with patient's age (r = 0.09, P = .2), serum ALT
      activity
      (r = 0.11, P = .14), or HCV-RNA concentration in serum (r =
      0.10, P = .3). No
      differences related to sex were found (8.03 1 3.3 in men, 8.56
      1 3.7 in women, P =
      .3). The genomic complexity of HCV did not appear to be
      related to the presumed
      route of HCV transmission because the number of SSCP bands was
      similar in
      patients with history of blood transfusion (8.7 1 3.7), in
      intravenous drug abusers
      (8.2 1 3.1), or in those with an unknown source of exposure
      (7.9 1 3.2) (P = 0.4).

      To View This
      Table
      Table 1. Basal Features and Complexity of
      the
      HVR1, as Determined by SSCP Analysis, in
      Patients With Genotype 1b HCV Chronic
      Infection According to the Severity of the
      Underlying Liver Disease


      The degree of HVR1 quasispecies complexity in the 114 patients
      with chronic
      hepatitis (8.3 1 3.5) and in the 54 with liver cirrhosis (8.0
      1 3.1) was similar (P = 0.6).
      Among patients with chronic hepatitis, no significant
      difference concerning
      quasispecies complexity between patients with mild, moderate,
      or severe hepatitis
      was observed. Similarly, there was no significant difference
      in the number of
      observed SSCP bands among patients with compensated or
      decompensated
      cirrhosis, or with cirrhosis and hepatocellular carcinoma
      (Table 1 and Fig. 3).


      To View Larger Image

      Click on Graphic
      Fig. 3. Relationship between
      HCV HVR1 complexity, as
      determined by the number of
      bands produced by SSCP
      analysis, and the histological and

      clinical severity of liver disease
      in
      patients with chronic HCV
      infection.


      To further analyze the possible meaning of quasispecies
      complexity, patients were
      separated into two groups with low or high genomic complexity
      according to the
      median value of the number of SSCP bands observed in the whole
      series. Low
      viral complexity ( 8 SSCP bands) was recorded in 103 patients
      and high viral
      complexity (> 8 SSCP bands) in 65. The proportion of patients
      with low or high
      viral complexity was similar when they were grouped according
      to clinical or
      histopathological severity of liver disease (Table 1).

      HCV Genomic Complexity in the HVR1 and Response to Interferon
      Treatment.
      The basal features and the results of SSCP analysis in
      relation to the observed
      response to interferon therapy in treated patients are shown
      in Table 2. HCV-RNA
      serum concentration was significantly lower in sustained
      responders than in
      transient responders or in non responders. (P = .04).
      Differences between these
      three groups of patients concerning demographic,
      epidemiological, or biochemical
      features were not observed. The number of SSCP bands detected
      in sustained
      responders, transient responders, and nonresponders was
      similar (Fig. 4). No
      significant difference was observed in the proportion of
      patients with high or low
      complexity of HVR1 in relation to the type of response to
      interferon therapy.

      View This
      Table
      Table 2. Basal Features and Complexity of the

      HVR1, as Determined by SSCP Analysis, in
      Patients With Genotype 1b HCV Chronic Infection

      According to the Response to a Course of
      Interferon Therapy




      To View Larger Image

      Click on Graphic
      Fig. 4. Relationship between
      HCV HVR1 complexity, as
      determined by the number of
      bands produced by SSCP
      analysis, and the type of response

      to one course of interferon-
      therapy in patients with chronic
      hepatitis C.


      DISCUSSION

      Host and virus-related factors may influence the natural
      outcome and the
      responsiveness to interferon therapy of patients with chronic
      hepatitis C. Several
      studies have shown that HCV genotype and viremia are related
      to the
      effectiveness of interferon therapy in these patients,26-28
      but the relationship of
      these factors with the severity of the underlying liver
      disease is still unclear.

      The long-term outcome of HCV infection and its responsiveness
      to interferon
      therapy may also be related to the quasispecies nature of HCV,
      a
      well-characterized feature of this virus with important
      biological implications.3,5 In
      infections caused by many RNA viruses, targets for
      neutralizing antibodies and
      for cytotoxic T cells often include epitopes that are located
      in hypervariable
      regions with high capacity to change their aminoacid sequence.
      These changes
      can provide a better adaptation (fitness) to environmental
      factors.4 Rapid
      selection of pre-existent, or emergence of new variants, may
      confer important
      biological properties to the virus, including escape from
      immune clearance, viral
      persistence, and resistance to antiviral agents.4

      In HCV infection, HVR1 escape mutants seem to play a major
      role in the
      establishment of persistent infection3 and in the development
      of resistance to
      interferon therapy.11-13,29 Sequence analysis of cDNA clones
      derived from PCR
      products from individual patients has provided important
      information on the
      genetic variability of HCV HVR1.6,7,11,12,17,30,31 However,
      taking into account the
      quasispecies nature of HCV and the marked heterogeneity of
      patients with
      chronic HCV infection, sequence analysis of a large number of
      clones would be
      necessary for accurate assessment of the role of viral
      heterogeneity in the
      pathogenesis of the disease and its response to therapy, but
      this methodology
      cannot easily be applied to studies involving a large number
      of patients. This
      limitation can partially be solved by using alternative
      indirect methods, such as
      SSCP analysis of amplified DNA products.32,33 By using this
      approach, the
      degree of complexity of the sequences present in a viral
      population is proportional
      to the number of bands visible after gel electrophoresis of
      DNA, and enables a
      rough estimation of the quasispecies variants present in a
      given sample. Under
      the conditions used in the present study, and in agreement
      with previous
      observations on the genetic variability of the HCV-NS5A
      region,34 SSCP analysis
      allowed detection of quasispecies variants representing as
      little as 5% to 10% of
      the whole viral population. Similar results have previously
      been reported by
      others.29,35

      The possible association between the HVR1 complexity of HCV
      and the severity
      of liver disease in chronically infected patients is a
      conflicting issue. A positive
      correlation between the degree of HCV genomic complexity and
      the severity of
      liver injury has been found in some studies that focused on
      the hypervariable
      region6,8 or on the core region of HCV,36 but this finding has
      not been confirmed
      by others, either by cloning and sequence analysis7 or by SSCP
      analysis9,10 of
      the HVR1 of HCV. However, data from these studies can not be
      directly compared
      because of differences in sample size and composition,
      diversity of HCV
      genotypes, unequal diagnostic criteria, and no standardization
      of laboratory
      methodology. Differences in sensitivity of SSCP analysis may
      be a crucial point
      because the number of viral variants in samples from patients
      with low level of
      viremia may be underestimated by this technique.10

      To overcome these problems, the current study focused on a
      large and
      heterogeneous group of patients that included a reasonably
      high number of
      subjects with well-defined liver disease at different stages
      of clinical and
      histopathological severity. All patients were infected with
      the same genotype, and
      approximately the same amount of amplified PCR product was
      analyzed by using a
      sensitive and reproducible SSCP technique. In our study, HVR1
      genomic
      complexity was unrelated to patient age and sex, presumed
      source of infection,
      ALT serum level, or HCV viremia. Furthermore, no significant
      differences in the
      number of SSCP bands were observed between patients with
      different degrees of
      liver injury, ranging from asymptomatic mild chronic hepatitis
      to decompensated
      cirrhosis and hepatocellular carcinoma. Similar results were
      obtained when
      patients infected with isolates with low or high HVR1
      complexity were compared.
      These observations suggest that the width of the mutant
      spectra of HCV
      quasispecies, estimated by SSCP analysis, does not appear to
      be related to degree
      of liver damage.

      Recent data have suggested that HVR1 quasispecies complexity
      may also be
      related to the response to interferon therapy. By using SSCP
      analysis of the HVR1
      of HCV, several studies that included a relatively small
      number of patients
      infected with a variety of HCV genotypes have shown that the
      degree of viral
      complexity was higher in isolates from nonsustained responders
      than in those
      from sustained responders to interferon therapy.9,10,13,14 In
      these studies,
      however, only a small proportion of the patients infected with
      HVC genotype 1b
      developed a sustained response to interferon, and this
      circumstance makes it
      difficult to evaluate the role of viral complexity in response
      to interferon in
      genotype 1b infected patients. Data from studies that focused
      on genotype 1b
      patients are rather conflicting: a good correspondence between
      low genetic
      complexity and sustained response to interferon was found in
      studies from
      Japan8,16 but not in European patients in whom a relationship
      between viral
      complexity and therapeutic response has been observed in
      genotype 3a, but not
      in genotype 1b infected patients.15 The reasons for these
      discrepancies are
      unclear, but they may be related to differences in patients
      selection, laboratory
      methodology or therapeutic schedules.

      In the current study, a large number of genotype 1b infected
      patients who
      received an identical course of interferon therapy were
      studied. The number of
      visualized SSCP bands was similar in long-term responders, in
      transient
      responders, and in nonresponders. These data suggest that HVR1
      complexity, as
      evaluated by an accurate SSCP procedure, is not related to the
      efficacy of
      interferon therapy. Therefore, SSCP analysis of HVR1 does not
      appear to be of
      value as prognostic indicator of response to conventional
      interferon therapy in
      patients with chronic hepatitis caused by genotype 1b HCV.

      Because the number of HVR1 variants does not appear to be
      associated with liver
      disease severity or interferon responsiveness in HCV genotype
      1b infection,
      important questions concerning the pathogenic role of
      virus-related factors
      remain unanswered. In the viral quasispecies model, the
      imposition of either
      preexisting or emerging mutants with a marked degree of
      divergence with respect
      to the global viral population may represent an adaptive
      advantage for the virus.4
      That is, under some circumstances, certain HCV quasispecies
      variants may be
      more pathogenic or virulent than others,37 and a single
      variant bearing drug
      resistance would survive to the drug more efficiently than
      multiple variants
      lacking this specific biological advantage.34 Such particular
      variants could not be
      recognized by SSCP and variants present in a very low amount
      might not be
      detected. Recently, Polyak et al.,38 by using the heteroduplex
      tracking assay have
      suggested that viral diversity in the HVR1, i.e., the genetic
      divergence between
      variants, but not viral complexity (the number of quasispecies
      variants) may be a
      key factor determining the type of response to interferon.
      However, it is also
      possible that mutations in the HCV genome conferring different
      pathogenicity,
      virulence, or drug-resistance would be located in genomic
      regions other than
      HVR1. If so, variations in the HVR1 would merely reflect the
      high capacity of this
      region to accept mutations under different environmental
      circumstances.

      In conclusion, we have found that the genomic complexity in
      the HVR1 region of
      HCV genotype 1b is not related to the clinical or
      histopathological severity of liver
      disease nor to the type of response to interferon therapy when
      analyzed by SSCP.
      Thus, as we recently suggested,34 the long-term outcome of
      liver disease and the
      effectiveness of interferon therapy might be related to
      qualitative, rather than to
      quantitative features of viral quasispecies variants present
      in these patients.

      Acknowledgment

      The authors thank Antoni Lloreng for technical assistance and
      Lloreng Quints for
      help in the statistical analysis.

      Abbreviations

      Abbreviations: HCV, hepatitis C virus; HVR1, hypervariable
      region 1; SSCP,
      single-strand conformational polymorphism; PCR, polymerase
      chain reaction;
      5'NCR, 5' noncoding region; ALT, alanine aminotransferase;
      cDNA,
      complementary DNA

      FootNotes

      Received May 17, 1998; accepted October 26, 1998. Present
      address of F.-X.L.-L.:
      Division of Gastroenterology, Stanford University School of
      Medicine, Veterans
      Administration Medical Center, Palo Alto, CA.

      Supported in part by grant SAF 97/0103 from the Comisisn
      Interministerial de
      Ciencia y Tecnologma (CICYT). F.-X. Lspez-Labrador and S.
      Ampurdanhs were
      supported by Fundacis Privada Clmnic and M. Giminez-Barcons by
      Institut
      d'Investigacions Biomhdiques August Pm i Sunyer (IDIBAPS).

      Address reprint requests to: Dr. Josi M. Sanchez-Tapias, Liver
      Unit, Hospital
      Clmnic, Villarroel 170, 08036 Barcelona, Spain. E-mail:
      sanchez@...; fax:
      34-93-4515272.

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      Analysis of the Community Liver Health Program Data Base

      12-15-96

      Quarterly Results Analysis of the Community Liver Health Program Data
      Base 12-15-96 Introduction: At the
      time of this analysis (12-15-96), 3522 chronic HCV patients have been
      enrolled into the Community Liver
      Health Program. This is an increase of over 100% since the last
      quarterly analysis. None of these patients have
      completed a course of interferon treatment. Thus, the only analysis
      that will be presented in this quarterly
      analysis will be those baseline characteristics specified in the Data
      Analysis Plan (see Data Analysis Plan for
      more detail). I. Analysis of the Distribution of baseline HCV RNA
      concentrations. The distribution of the
      baseline HCV RNA concentrations for 3310 anti-HCV (ELISA) positive
      patients who are participating in the
      Community Liver Health is shown in Figure 1. HCV RNA was assessed using
      the SUPERQUANT. test. As
      illustrated by Figure 1, the cut-off for the lower 25th percentile of
      this population (low titer) was 810,000
      copies/mL. The median for this population was 3,200,000 copies/mL with
      the distribution of the median titer
      ranging from >810,000 copies/mL to 5,000,000 copies/mL. Finally, the
      cut-off for the upper 25th percentile of
      this population (high titer) was >5,000,000 copies/mL.



      The distribution of HCV RNA did not significantly change from the last
      quarterly analysis. More specifically
      analysis of the 1510 patients for the September, 1996 was revealed the
      cut-off for the lower 25th percentile of
      this population was 820,000 copies/mL. The median for this population
      was 3,200,000 copies/mL and the
      cut-off for the upper 25th percentile of this population "high titer"
      was >5,000,000 copies/mL. II. Analysis of
      the Genotype Distribution HCV genotype for 3538 anti-HCV (ELISA)
      positive patients participating in the
      Community Liver Health program was assessed using the Inno-lipa assay.
      The distribution of the HCV
      genotype for this population is shown in Table 1.

      Table 1: Distribution of HCV Genotype in the Community Liver Health
      Program Patients

      Genotype
      Count
      Probability
      1
      137
      3.9%
      1/2b
      0
      0.0%
      1/3a
      1
      0.0%
      1/4a
      1
      0.0%
      1a
      1403
      39.7%
      1a/1b
      119
      3.4%
      1a/2
      2
      0.1%
      1a/2b
      2
      0.1%
      1a/4e
      3
      0.1%
      1b
      1055
      29.8%
      1b/2b
      3
      0.1%
      1b/4a
      3
      0.1%
      2
      6
      0.2%
      2a/1
      2
      0.1%
      2a
      133
      3.8%
      2a/2b
      13
      0.4%
      2a/4e
      3
      0.1%
      2b
      351
      9.9%
      3a
      256
      7.2%
      3b
      1
      0.0%
      3c
      1
      0.0%
      4
      11
      0.3%
      4a
      1
      0.0%
      4c/4d
      19
      0.5%
      4f
      1
      0.0%
      4h
      1
      0.0%
      5a
      1
      0.0%
      6a
      8
      0.2%
      Total
      3538


      In order to simplify the analysis, the genotype classifications were
      collapsed into their respective higher order
      categories. For example HCV genotype 1, 1a and 1 b were combined into
      the category 1. The distribution of
      HCV genotype by collapsed category is shown in Figure 2. As illustrated
      by the figure, the population is
      dominated by HCV genotype 1 patients (73%). The next most frequently
      observed HCV genotype was
      genotype 2 followed by genotypes 3, and mixed. The genotype
      distribution seen in this update of the CLHP
      analysis is not different than the distribution seen in the last
      analysis in that both analyses demonstrated that the
      population of chronic HCV patients has a higher distribution of
      genotype 1 patients than has previously been
      reported for patients participating in clinical trials. The reason for
      the discrepancy is unknown at this time.


      In order to discern if there are any regional difference in HCV
      genotype distribution in the United States, the
      distribution for this population was examine by individual State (Table
      2). As seen by Table 2, there are no
      regional difference with respect to HCV genotype distribution in the
      United States.

      Table 2 HCV Genotype by State73.5%

      1
      2
      3
      4
      5
      6
      Mixed
      N
      AL
      73.5%
      14.3%
      8.1%
      0.0%
      0.0%
      0.0%
      4.0%
      223
      AR
      62.7%
      17.9%
      10.4%
      0.0%
      0.0%
      0.0%
      9.0%
      67
      AZ
      68.8%
      12.5%
      12.5%
      0.0%
      0.0%
      1.6%
      4.7%
      64
      CA
      67.7%
      15.9%
      10.6%
      0.9%
      0.4%
      1.8%
      2.7%
      226
      CO
      69.0%
      20.2%
      8.3%
      0.0%
      0.0%
      0.0%
      2.4%
      84
      CT
      70.8%
      16.7%
      4.2%
      0.0%
      0.0%
      0.0%
      8.3%
      24
      DC
      86.4%
      4.5%
      4.5%
      2.3%
      0.0%
      0.0%
      2.3%
      44
      FL
      77.2%
      13.7%
      6.0%
      0.4%
      0.0%
      0.4%
      2.5%
      285
      GA
      75.2%
      13.8%
      4.6%
      0.9%
      0.0%
      0.0%
      5.5%
      109
      HI
      73.0%
      14.3%
      6.3%
      0.0%
      0.0%
      1.6%
      4.8%
      63
      IL
      64.7%
      29.4%
      5.9%
      0.0%
      0.0%
      0.0%
      0.0%
      17
      IN
      75.8%
      13.6%
      6.1%
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