WO2006112482A1

WO2006112482A1 - Prediction of prognosis of liver disease associated with hepatitis c virus infection

Info

Publication number: WO2006112482A1
Application number: PCT/JP2006/308233
Authority: WO
Inventors: Kyogo Itoh; Michio Sata; Shigeru Yutani; Nobukazu Komatsu; Akira Yamada
Original assignee: Green Peptide Co., Ltd.
Priority date: 2005-04-19
Filing date: 2006-04-19
Publication date: 2006-10-26
Also published as: TW200720656A; JP4615567B2; JPWO2006112482A1

Abstract

Disclosed is a method for the prediction of progress of a liver disease associated with HCV infection, the method comprising determining the antibody titer of an antibody against a peptide comprising an amino acid sequence composed of contiguous 8 or more amino acid residues containing amino acid Gly 32 of a hepatitis C virus core protein in the blood of a subject. Further, the antibody titer against NS5A-2132 peptide of hepatitis C virus and the antibody titer against C33 or C35 peptide of a hepatitis C virus core protein are also determined. Based on the combination of these results, it becomes possible to predict the prognosis with higher reliability. Also disclosed is a kit for use in the prediction of the progress of a liver disease associated with hepatitis C virus infection, the kit comprising a peptide which can be recognized by an anti-HCV antibody capable of binding to a peptide comprising an amino acid sequence composed of contiguous 8 or more amino acid residues containing amino acid Gly 32 of a hepatitis C virus core protein.

Description

Specification

Prediction of liver disease associated with hepatitis C virus infection

Technical field

[0001] The present invention relates to a prognostic prediction of liver disease associated with hepatitis C virus infection. More specifically, the present invention relates to liver disease associated with hepatitis C virus infection, from hepatitis to cirrhosis.

Furthermore, the present invention relates to a method for predicting progression to hepatocellular carcinoma and a kit for use in a powerful method.

Background art

[0002] Carriers of hepatitis C virus (HCV) are estimated to be 2 million in Japan and 200 million worldwide. In addition, many of these HCV-infected persons have symptoms of chronic hepatitis. Chronic hepatitis C, if left untreated, progresses to cirrhosis at an annual rate of 2-3%, and those patients further transition to liver cancer (hepatocellular carcinoma) at an annual rate of 5-7%.

[0003] As a method for detecting hepatitis C virus infection, a highly sensitive diagnostic method has become widespread. Screening methods include first-generation antibody testing using only the recombinant antigen (C100-3) corresponding to NS3,4 region of HCV, core region, NS3, NS4, NS5 (3rd generation only) Second- and third-generation antibody tests using recombinant antigens have been used. There is also a viral antigen gene identification method as a definitive method.

[0004] On the other hand, hepatitis C virus-related liver diseases can be diagnosed by examining liver function tests using various types of markers such as serum ALT and AST levels in patients, measurement of tumor markers AFP and PIVKA II, measurement of viral load, And methods of diagnosing the transition to fibrosis and canceration by biopsy are used. However, with these methods, it was difficult to predict the prognosis of hepatitis C virus-related liver disease. This is because, for example, because the viral load is high, the measured values of the above markers are not always parallel to the progression of liver lesions, such as not necessarily liver cancer!

[0005] As a new test method that can predict the prognosis, a test method that reflects the immune function of the virus elimination of the living body has been developed. The present inventors have previously found that antibodies against the C35 peptide of the HCV core protein are found in the plasma of patients with chronic hepatitis C. The antibody to this peptide was examined using a number of chronic hepatitis / cirrhosis / liver cancer patients (all with hepatitis c virus infection) and healthy human plasma. . As a result, the specificity of detection was 100% and the sensitivity was 93% or more, indicating that this is an extremely effective method for diagnosing hepatitis C virus. It was also revealed that this antibody is independent of the patient's pathology (hepatitis' cirrhosis / liver cancer) and virus variants (HCV—lb, 2a, 2b) (WO03Z025569).

[0006] Furthermore, IgG antibodies against the NS5A-2132 peptide, a non-structural protein different from the core protein described above, show high values in cured cases and chronic hepatitis in the early stages of HCV, but it decreases as the disease progresses. (WO2005Z028503). Since the serum levels of the above-mentioned anti-C35 peptide antibodies correlate with persistent infection, it is possible to diagnose HCV infection and determine prognosis, that is, predict the transition to liver cancer by monitoring antibody titers against these two peptides. It was shown that

[0007] Furthermore, for more reliable prognosis prediction, it is required to develop another marker that can be used for prognosis prediction in combination with these two types of peptides.

[0008] References cited in the present specification are as follows. All the contents described in these documents are cited herein as part of this specification.

Patent Document 1: WO03 / 025569

Patent Document 2: WO2005Z028503

Disclosure of the invention

Problems to be solved by the invention

[0009] An object of the present invention is to provide a novel marker that can be used for predicting the prognosis of liver disease associated with hepatitis C virus infection.

Means for solving the problem

[0010] The present inventors have found that the antibody titer against the HCV core protein 30-39 peptide increases in patients with hepatitis C virus infection as the liver lesion progresses, and thus completed the present invention.

[0011] The present invention measures the antibody titer of an antibody against a peptide consisting of eight or more consecutive amino acids including amino acid Gly32 of hepatitis C virus core protein in the blood of a subject. Provides a method for predicting the progression of liver disease associated with HCV infection. In the present specification, the HCV peptides are numbered sequentially from the N-terminal side to the amino acid sequence of the entire translation region encoded by the consensus sequence of the HCVlb virus genome. The amino acid sequence encoded by the consensus sequence is shown in FIG.

[0012] Anti-HCV antibody means an antibody capable of binding to a peptide derived from hepatitis C virus. Anti-HCV antibodies can be detected in the serum of patients infected with HCV. Liver disease associated with hepatitis C virus infection refers to liver disease caused by hepatitis C virus infection and includes, for example, hepatitis, cirrhosis and hepatocellular carcinoma. The progression of liver disease is typically progression from chronic hepatitis to cirrhosis, and further from cirrhosis to liver cancer.

[0013] More preferably, in the method of the present invention, the antibody titer against a peptide consisting of 8 or more consecutive amino acids including amino acid Arg2132 of non-structural protein NS5A of hepatitis C virus is measured in the blood of the subject. To do.

[0014] Preferably, in the method of the present invention, the antibody titer against a peptide having 8 or more consecutive amino acid strengths among Gly33 to Val48 amino acids of the hepatitis C virus core protein is also measured.

[0015] In another aspect, the present invention includes a peptide recognized by an anti-HCV antibody capable of binding to a peptide consisting of eight or more consecutive amino acids including amino acid Gly32 of hepatitis C virus core protein. A kit is provided to predict the progression of liver disease associated with hepatitis C virus infection.

[0016] Preferably, the kit of the present invention contains a peptide having a power of 8 or more consecutive amino acids including amino acid Gly32 of hepatitis C virus core protein. Particularly preferably, the kit of the present invention comprises a peptide selected from the group consisting of IVGGVYLLPR (SEQ ID NO: 1), VGGVYLLPRR (SEQ ID NO: 2) and GGV YLLPRRG (SEQ ID NO: 3).

[0017] Preferably, the kit of the present invention further comprises a peptide recognized by an antibody capable of binding to a peptide having a power of 8 or more consecutive amino acids including the amino acid Arg2132 of the nonstructural protein NS5A of hepatitis C virus. . Also preferably, the kit of the present invention further comprises a C type. It includes peptides that are recognized by antibodies that can bind to a peptide consisting of 8 or more consecutive amino acids among the amino acids from Gly33 to Val48 of the hepatitis virus core protein.

The invention's effect

[0018] According to the present invention, not only positive / negative of viral infection can be determined, but also the prognosis of diseases associated with hepatitis C virus infection can be predicted. The method of the present invention enables prediction of prognosis more accurately by combining with the conventional liver function test.

Brief Description of Drawings

[0019] FIG. 1 shows antibody titers against C30-39 peptide, NS5A-2132-2140 peptide, and C33-42 peptide in the serum of subjects.

[FIG. 2] FIG. 2 shows antibody titers against C30-39 peptide, C31-40 peptide, and C32-41 peptide in the serum of subjects.

FIG. 3 shows antibody titers against C33-42 peptide, C34-43 peptide, C35-44 peptide, C36-45 peptide and C37-46 peptide in the serum of subjects.

[Figure 4] Figure 4 shows the ratio of antibody titer against C30-39 peptide and antibody titer against C35-44 peptide in the serum of subjects.

[FIG. 5] FIG. 5 shows the numbering of the amino acid sequences of all translation regions encoded by the consensus sequence of the HCVlb virus genome, sequentially from the N-terminal side. The amino acid sequence encoded by the consensus sequence is shown. In the figure, the C30-39 peptide and the NS 5A-2132-2140 peptide are underlined.

[FIG. 6] FIGS. 6-1 to 6-16 show the homology of the amino acid sequences of various isolates of HCVlb.

BEST MODE FOR CARRYING OUT THE INVENTION

[0020] The present invention relates to HCV infection, comprising measuring the antibody titer of an antibody against a peptide consisting of 8 or more consecutive amino acids including amino acid Gly32 of hepatitis C virus core protein in the blood of a subject. Methods for predicting the progression of associated liver disease are provided.

[0021] Liver disease associated with hepatitis C virus infection means liver disease caused by hepatitis C virus infection, and includes, for example, hepatitis, cirrhosis and hepatocellular carcinoma. The progression of liver disease typically progresses from chronic hepatitis to cirrhosis, and further from cirrhosis to liver cancer. Line.

[0022] The antibody titer in the blood of a subject can be measured using an antigen-antibody reaction well known in the art. For example, when a typical ELISA method is used, the measurement can be performed as follows. The antigen peptide is bound to a conventional ELISA plate such as 96well, and the plate is appropriately blocked to prevent nonspecific adsorption. Next, the serum prepared by the subject's blood force is diluted as appropriate and added to each well of the plate, and reacted for a predetermined time. After washing the plate to remove unbound components, add an antibody that can bind to a human antibody (eg, a rabbit anti-human antibody). If it is desired to detect IgG, γ-chain specific anti-HgG can be used. After reacting for the specified time, the plate is washed and detectably labeled antibody (eg anti-rabbit IgG) is added. Labeling can be performed by methods known to those skilled in the art using enzymes, fluorescent dyes, chemiluminescent substances, piotin, radiation compounds and the like. After reacting the plate for a specified time, the label is detected by adding an appropriate substrate and measuring the decrease in substrate or increase in product, or by measuring fluorescence, luminescence, or radioactivity. In this way, the amount of antibody against a specific peptide in the serum of a subject can be measured.

[0023] Among such methods for detecting an antibody by antigen-antibody reaction, one of highly sensitive methods is xMAP technology. This is a flowmetry measurement method developed by Luminex using a fluorescent microbead array system. Microbeads to which peptides are bound are brought into contact with serum, and then a fluorescently labeled secondary antibody is bound to the flow. Measure fluorescence intensity by measurement. Details of antibody titer measurement using this method are shown in the examples below.

[0024] In addition, when the antibody titer is measured in a hospital laboratory or the like, an immunochromatography method can be used. In this method, a portion in which the antigen (or antibody) is linearly distributed is made on the test paper, and the complex of the antibody in the sample and the antigen labeled with the colored particles moves on the test paper. Qualitative analysis is based on the presence or absence of colored lines that appear by intensive capture by antigens (or antibodies). Using this method, results can be obtained in a short time (within 20 to 30 minutes) with simple equipment.

[0025] The peptide used for detection in the present invention is based on the known gene sequence of hepatitis C virus, and may be 8 or more consecutive amino acids including the amino acid Gly32 of the core protein. Can be obtained from the above. Particularly preferred for the present invention! / ヽ peptides are C30 peptide: IVGGVYLLPR (SEQ ID NO: 1), C31 peptide: VGGVY LLPRR (SEQ ID NO: 2) and C32 peptide: GGVYLLPRRG (SEQ ID NO: 3). These peptides only need to have the above sequence to the extent that the effects of the present invention are exhibited, and may contain additional sequences. A person skilled in the art would have the N-terminal side and the C-terminal side to obtain a desired effect as an antigen peptide for detecting an antibody, that is, to be able to specifically bind to the target antibody. Naturally, it is possible to understand how much sequence is allowed on each side. Therefore, for example, an amino acid sequence convenient for expressing a peptide as a fusion protein and an amino acid sequence convenient for peptide production and purification are added to the N-terminal side and the Z- or C-terminal side of the peptide according to the present invention. May be. In addition, the peptide according to the present invention may be chemically modified or a polymer or sugar chain may be added as long as the specificity of binding to the antibody is not lost. Further, as is well known in the art, there is a sequence norelation between various strains of HCV and various isolates of the same strain. Those skilled in the art can also select peptide sequences corresponding to these variation sequences in accordance with the teachings of the present invention, and peptides having such sequences are also within the scope of the present invention. As examples of sequence variations, the homology of the amino acid sequences of various isolates of HCVlb is shown in Figures 6-1 to 6-16.

[0026] More preferably, in the method of the present invention, a peptide consisting of 8 or more consecutive amino acids containing the amino acid Arg2132 of the non-structural protein NS5A of hepatitis C virus, such as NS5A— The antibody titer against 2132 peptide: RYAPACKPL (SEQ ID NO: 4) is measured. Antibodies to this peptide show high levels in cases of HCV infection-related liver disease and chronic hepatitis in the early stages of HCV, but are known to decrease as the disease progresses! Therefore, by combining with the above-described method of the present invention, a more reliable prognosis can be predicted.

[0027] Preferably, in the method of the present invention, a peptide comprising 8 or more consecutive amino acids of Gly33 to Val48 of hepatitis C virus core protein in the blood of the subject, for example, C33 Peptide: GVYLLPRRGP (SEQ ID NO: 5) , C34 peptide: VYLLPRRGPR (SEQ ID NO: 6) or C35 peptide: YLLPRRGPR L (SEQ ID NO: 7). Antibodies to these peptides are high in the plasma of patients with chronic hepatitis, cirrhosis and liver cancer associated with HCV infection, and are found in the plasma of patients with chronic hepatitis associated with hepatitis B virus. It has been found that it is not allowed. That is, the presence of antibodies against these peptides in serum correlates with persistent viral infection. Therefore, when combined with the above-described method of the present invention and when combined with measurement of antibody titer against NS5A-2132 peptide, a more reliable prediction of prognosis, that is, prediction of transition to liver cancer is possible. It becomes.

[0028] Peptides used as antigens in the method of the present invention can be obtained by a conventional chemical synthesis method or a gene recombination technique using a host transformed to express a base sequence encoding a target amino acid sequence. Obtainable.

[0029] When a target peptide is produced by a chemical synthesis method, it can be produced by a method commonly used in normal peptide chemistry. For example, using a peptide synthesizer, It can be synthesized by a solid phase synthesis method. The crude peptides thus obtained can be purified by purification methods commonly used in protein chemistry, such as salting out, ultrafiltration, reverse phase chromatography, ion exchange chromatography, -Teak It can be purified by a method such as mouth-matography.

[0030] On the other hand, when a peptide is produced by genetic recombination technology, a DNA fragment encoding the target amino acid sequence is incorporated into an appropriate expression vector, and microorganisms and animal cells are used using this expression vector. The desired peptide can be obtained by culturing the resulting transformant after transformation. As expression vectors that can be used, plasmids, virus vectors, and the like known in the art can be used. In order to facilitate the expression and purification of peptides, techniques for expressing peptides as fusion proteins are also well known in the art.

[0031] As a method for transforming host cells using an expression vector, a conventional method such as a calcium chloride method, a calcium phosphate coprecipitation method, a DEAE dextran method, a lipofectin method, or an electopore position method may be used. it can. Purification of the resulting peptide Can be performed from the cell extract or culture supernatant recovered from the cultured medium by the purification method described above.

[0032] The contents of all patents and references explicitly cited herein are hereby incorporated by reference as part of the present specification. In addition, the contents described in the specification and drawings of Japanese Patent Application No. 2005-121293, which is the application on which the priority of this application is based, are cited herein as a part of this specification.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example

[0034] Antibody titers against the following peptides were measured using sera of HCV-infected and healthy individuals with various liver diseases:

C30- 39: IVGGVYLLPR (SEQ ID NO: 1)

C31 -40: VGGVYLLPRR (SEQ ID NO: 2)

C32—41: GGVYLLPRRG (SEQ ID NO: 3)

NS5A-2132-2140: RYAPACKPL (SEQ ID NO: 4)

C33-42: GVYLLPRRGP (SEQ ID NO: 5)

C34-43: VYLLPRRGPR (SEQ ID NO: 6)

C35—44: YLLPRRGPRL (SEQ ID NO: 7)

C36-45: LLPRRGPRLG (SEQ ID NO: 8)

C37— 46: LPRRGPRLGV (SEQ ID NO: 9)

[0035] Specimens used were a group of patients diagnosed with liver disease (chronic hepatitis (CH) 21 cases, cirrhosis (LC) 21 cases, liver cancer (HCC) 27 cases), and healthy subjects (HD) 20 cases. It was. Peptides were purchased from Thermo with a purity of over 90%.

[0036] Preparation of peptide coupled beads

The following operations were performed at room temperature (25 ° C) unless otherwise specified. The filter plate was washed with 0.05% Tween—20ZPBS. Place 100 / zl of carboxy beads (Luminex) into each well of the filter plate and suck at some point, and wash with 0.1 M MES (2-morpholinoethanesulfonic acid) buffer (pH 7.0). 0.1M MES buffer of 1 Solution (pH 7.0) was added. EDC (Nethyl N,-(3 dimethylaminopropyl) -force rubodiimide hydrochloride) (lOmgZmlZO. 1M MES buffer (pH 7.0)) was added to each of the tubes one by one.

[0037] Each peptide was dissolved in dimethyl sulfoxide at 10 mg / ml to obtain a peptide solution.

In each well of a separate plate, a mixture of 60 1 0.1 M MES buffer (pH 7.0) and 151 peptide solution was prepared and 50 1 each was added to each well of the filter plate.

[0038] After reacting for 20 minutes at a certain place, EDC (lmg / ml / 0.1M MES buffer (pH 7.0)) was added at a rate of 10 / zl of each well and allowed to react for 20 minutes in the dark. Furthermore, EDC (lmgZmlZ 0.1M MES buffer (pH 7.0)) was added to each well 101 and allowed to react for 20 minutes in the dark. After aspirating the filter plate, 1M Tris-HCl buffer (pH 7.0) was added to each well 1001 and allowed to react for 15 minutes at room temperature in the dark. Next, each bead of each tool was washed with 0.05% Tween-20ZPBS, and collected with a storage solution prepared by adding 0.05% sodium chloride to Block Ace (registered trademark).

[0039] Pile peptide pile body I

Serum that also obtained subject strength was diluted 100-fold with 10% Block Ace (registered trademark) ZPBS. The filter plate was washed with 0.05% Tween-20ZPBS, and the previously prepared peptide-bound beads were added with 51 Zwell. The beads were washed with 0.05% Tween-20ZPBS, and diluted serum was covered with 100 1Z wells and reacted at room temperature for 2 hours. The beads were washed with 0.05% Tween-20ZPBS, added with 100 μlZ piotylated goat anti-human IgG (γ) antibody, and reacted at room temperature for 1 hour. After the beads were washed with 0.05% Tween—20ZPBS, streptavidin R-phycoerythrin conjugate (SAPE) was covered with 100 μl well and reacted at room temperature for 30 minutes. Next, the fluorescence intensity was measured using the Luminex fluorescence flowmetry system.

[0040] Each sample was measured using at least 50 beads, and the median fluorescence intensity (Median) indicated by each bead was used as the antibody titer. The results are shown in Fig. 1 and Fig. 2.

[0041] Figure 1 shows C30—39: IVGGVYLLPR (SEQ ID NO: 1), NS5A—2132-2140: RYAP ACKPL (SEQ ID NO: 4), and C33—42: GVYLLPRRGP ( The result of having plotted the antibody titer with respect to sequence number 5) according to the disease progression degree is shown. It can be seen that the antibody titer against C30-39 increases with disease progression (CH → LC → HCC), whereas the antibody titer against NS5A-2132-2140 decreases with disease progression. Furthermore, the antibody titer against C33-42 is high in HCV-infected individuals regardless of disease progression, but is low in healthy donors. In other words, it was shown that by measuring antibodies against these three peptides, it is possible to diagnose hepatitis C virus infection and determine the prognosis, that is, to predict the transition from chronic hepatitis to cirrhosis and liver cancer.

[0042] Figure 2 shows antibody titers against C30—39: IVGGVYLLPR (SEQ ID NO: 1), C31—40: VGGVYLLPRR (SEQ ID NO: 2), and C32—41: GGVYLLPRRG (SEQ ID NO: 3) in the serum of subjects. The results plotted according to the degree of disease progression are shown. Antibody titers against C31 and C32 were shown to increase with disease progression, as did antibody titers against C30.

[0043] Figure 3 shows C33—42: GVYLLPRRGP (SEQ ID NO: 5), C34—4 3: VYLLPRRGPR (SEQ ID NO: 6), C35—44: YLLPRRGPRL (SEQ ID NO: 7), C36 45: The result of having plotted the antibody titer with respect to LLPRRGPRLG (sequence number 8) and C37-46: LPRRGPRLGV (sequence number 9) according to the disease progression degree is shown. Antibody titers against any of these peptides are high in HCV-infected individuals regardless of disease progression, but are low in healthy donors. In other words, by combining the antibody titers against these peptides with the antibody titers against the C30, C31 or C32 peptides described above, a more accurate prognosis of HCV infection-related liver disease can be predicted.

[0044] As already known, antibody titers against C35-44 are high in HCV-infected individuals regardless of disease progression, but low in healthy HCV-uninfected donors. On the other hand, as shown in Fig. 1, the antibody titer against C30-39 increases with disease progression. Therefore, the ratio of the antibody titer against C30-39: IVGGVYLLPR (SEQ ID NO: 1) and the antibody titer against C35-44: YLLPRRGPRL (SEQ ID NO: 7) in the serum of the subjects was calculated. As a result, as shown in Fig. 4, using the C30ZC35 value, the correlation between disease progression and antibody titer was shown more clearly. The value of C30ZC35 is similar between HCC patients and healthy donors, but the antibody titer against C35 is significantly lower in healthy donors. CC patients and healthy donors can be easily distinguished. In other words, by combining the antibody titer against C30 peptide and the antibody titer against C35 peptide, a more accurate prognosis of HCV infection-related liver disease can be predicted.

Industrial applicability

The present invention is useful for diagnosis of liver diseases associated with hepatitis C virus infection and prediction of prognosis.

Claims

The scope of the claims

[1] Liver disease associated with HCV infection, including measuring the antibody titer of antibodies against 8 or more consecutive peptides containing amino acid Gly32 of hepatitis C virus core protein in the blood of the subject How to predict the progression of the.

[2] In the subject's blood !, hepatitis C virus nonstructural protein NS5A amino acid Arg

The method according to claim 1, further comprising measuring an antibody titer against a peptide having 8 or more consecutive amino acids including 2132.

[3] The method according to claim 1 or 2, further comprising measuring an antibody titer against a peptide having 8 or more consecutive amino acids from Gly33 to Val48 of hepatitis C virus core protein.

4. The method according to claim 13, wherein the progression of liver disease is progression from chronic hepatitis to cirrhosis.

5. The method according to claim 13, wherein the progression of liver disease is progression to chronic hepatitis or liver cirrhosis liver cancer.

[6] A kit for predicting progression of liver disease associated with hepatitis C virus infection, comprising a peptide recognized by an anti-HCV antibody, wherein the anti-HCV antibody is an amino acid of hepatitis C virus score protein A kit characterized in that it can bind to a peptide having 8 or more consecutive amino acids including Gly32.

[7] A kit for predicting the progression of liver disease associated with HCV infection, comprising a peptide consisting of 8 or more consecutive amino acids, including amino acid Gly32 of hepatitis C virus core protein

[8] The kit according to claim 6 or 7, wherein the peptide is selected from the group consisting of IVGGVYLLPR (SEQ ID NO: 1), VGGVYLLPRR (SEQ ID NO: 2) and GGVYLLPRRG (SEQ ID NO: 3).

[9] The hepatitis C virus non-structural protein NS5A further comprises a peptide recognized by an antibody capable of binding to a peptide having more than 8 amino acids, including amino acid Arg2132, The kit according to 1.

[10] Eight consecutive amino acids from Gly33 to Val48 of hepatitis C virus core protein The kit according to any one of claims 6 to 9, further comprising a peptide recognized by an antibody capable of binding to the peptide having the above amino acid strength.

[11] The kit according to any one of claims 6 to 10, wherein the progression of liver disease is progression of chronic hepatitis to cirrhosis.

[12] The kit according to any one of claims 6 to 10, wherein the progression of liver disease is progression to chronic hepatitis or liver cirrhosis liver cancer.