Page |2

Hepatitis C Virus (HCV)

Introduction:
Hepatitis is a disease characterized by inflammation of the liver, usually producing swelling and, in many cases, permanent damage to liver tissues. A number of different agents can cause hepatitis, including infectious diseases, chemical poisons, drugs and alcohol. Viral hepatitis refers to a set of at least six viruses that are known to cause hepatitis: hepatitis A (HAV), hepatitis B (HBV), hepatitis C (HCV), hepatitis D (HDV), hepatitis E (HEV), and hepatitis G (HGV). Recent scientific evidence also suggests the existence of other, as yet unidentified hepatitis viruses. The most common types of viral hepatitis are hepatitis A, B, and hepatitis C. Both hepatitis B and C can lead to serious, permanent liver damage, and in many cases, death. There are two primary types of viral hepatitis, food-borne and blood-borne hepatitis. The former, which is spread through contaminated food and water, does not cause chronic liver disease. By contrast, blood borne viral hepatitis may lead to long-term, persistent infections and chronic liver disease that has lethal consequences many years after infection.

The Hepatitis C Virus:

Hepatitis C is an RNA virus - which means that it mutates frequently. Once an infection has begun, hepatitis C creates different genetic variations of itself within the body of the host. The mutated forms are frequently different enough from their ancestors that the immune system cannot recognize them. Thus, even if the immune system begins to succeed against one variation, the mutant strains quickly take over and become new, predominant strains. As a result, the development of antibodies against HCV does not produce immunity against the disease like it does with most other viruses. More than 80% of the individuals infected with HCV will progress to a chronic form of the disease. There are six basic genotypes of HCV, with 15 recorded subtypes, which vary in prevalence in different regions of the world. Each of these major genotypes can differ significantly in their biological effects - in terms of replication, mutation rates, type and severity of liver damage, and detection and treatment options. However, these differences are not yet clearly understood. The 21 current variations in genotype, complicated by the constant mutation of the virus within infected individuals, represents a major challenge for the development of treatments and
Muhammad Ali Minhas HCV

Page |3

vaccines against HCV - and even for reliable detection of the virus. There is no guarantee that a treatment, test, or vaccine against one strain will be effective against all of them. Moreover, individuals cured of one strain will be prone to reinfection by any of the other strains.

Anatomy of the Hepatitis C Virus:

The structure of the hepatitis C virus is like that of most complex viruses - a core of genetic material (RNA), surrounded by a protective shell of protein, and further encased in a lipid (fatty) envelope of celluar material. However, the fact that the genetic information of the virus is stored in RNA, not DNA, has important consequences in the life cycle of the virus, and gives hepatitis C its dangerous ability to mutate. All organisms, with the exception of the RNA viruses, store their permanent information in DNA, using RNA only as a temporary messenger for information. DNA is quite a stable molecule, not particularly reactive with other molecules, and the processes which reproduce it make very few mistakes in the process of copying the molecule (between one in 1 million and 1 in 10 million). Most of these mistakes are normally corrected even when they do occur. This makes DNA an ideal format for the storage of information, for mutations (errors) only rarely occur, and most are not significant. RNA, by contrast, is a quite reactive molecule, capable of reacting even with itself under the correct conditions. It also makes frequent mistakes during copying - averaging one mistake per 10,000 nucleotides each time it is copied. These properties make RNA very poorly suited for the storage of information. However, these very propeties make RNA ideal for the storage of viral information. Once the immune system has learned to recognize an infecting virus and create antibodies against it (developed an immunity), it can quickly destroy it, so the virus can no longer use that host for reproduction. In order to reinfect a host - it must first change its nature enough that the immune system will no longer recognize it - in other words, it must mutate.

Muhammad Ali Minhas

HCV

Page |4

Hepatitis C virus. Structure of the viral capsid is clearly visible The unstable nature of the RNA molecule provides this mutagenic factor, allowing the Hepatitis C virus to develop new genetic variations of itself. As discussed earlier, the mutated forms are frequently different enough from their ancestors that the immune system cannot recognize them, so if the immune system begins to succeed against one variation, the mutant strains quickly take over and become new, predominant strains. Because each surviving virus reproduces itself thousands of times, mutations in the RNA sequence occur frequently, allowing it to evolve faster than any other type of living organism. This evolution is known as antigenic drift. Mutations occur randomly across the entire length of the viral RNA, and so of course most are not beneficial, producing viruses which lack a needed protein or are otherwise disadvantaged. However, because of the enormous number of offspring produced by each virus, even a high rate of mutation does not threaten the survival of the virus - and when advantageous mutations do occur, they are rapidly selected for and reproduced.

Hepatitis C, as an RNA virus, has a powerful reproductive strategy. Because it stores its information in a "sense" strand of RNA, the viral RNA itself can be directly read by the host cell's ribosomes, functioning like the normal mRNA present in the cell. The virus thus needs no special abilities of its own - it uses the cell's own ribosomes to produce everything it needs for its takeover of the cell's processes and reproduction. This means hepatitis C requires only a small amount of RNA to encode its core information, and thus has lots of room

Muhammad Ali Minhas

HCV

Page |5

for genetic variation within the non-essential portions of its RNA. This also gives it fewer common characteristics that can be readily identified by the immune system - or, for that matter, exploited by scientists working to create a treatment.

Genome of HCV:
Hepatitis C virus has a positive sense single-stranded RNA genome. The genome consists of a single open reading frame that is 9600 nucleotide bases long. This single open reading frame is translated to produce a single protein product, which is then further processed to produce smaller active proteins. At the 5' and 3' ends of the RNA are the UTR, which are not translated into proteins but are important to translation and replication of the viral RNA. The 5' UTR has a ribosome binding site (IRES - Internal ribosome entry site) that starts the translation of a very long protein containing about 3,000 amino acids. This large pre-protein is later cut by cellular and viral proteases into the 10 smaller proteins that allow viral replication within the host cell, or assemble into the mature viral particles.

Muhammad Ali Minhas

HCV

Page |6

Structural proteins made by the hepatitis C virus include Core protein, E1 and E2; nonstructural proteins include NS2, NS3, NS4, NS4A, NS4B, NS5, NS5A, and NS5B.

The Life Cycle of Hepatitis C:

1) The hepatitis C virus must attach to and infect liver cells in order to carry out its life cycle and reproduce - this is why it is associated with liver disease. While little is known about the exact natural processes of hepatitis C, like other viruses, it must complete eight key steps to carry out its life cycle: 2) The virus locates and attaches itself to a liver cell. Hepatitis C uses particular proteins present on its protective lipid coat to attach to a receptor site (a recognizable structure on the surface of the liver cell). 3) The virus's protein core penetrates the plasma membrane and enters the cell. To accomplish this, hepatitis C utlilizes its protective lipid (fatty) coat, merging its lipid coat with the cells outer membrane (the coat is in fact composed of a fragment of another liver cell's plasma membrane). Once the lipid coat has successfully fused to the plasma membrane, the membrane engulfs the virus - and the viral core is inside the cell. 4) The protein coat dissolves to release the viral RNA in the cell. This may be accomplished during penetration of the cell membrane (it is broken open when it is released into the cytoplasm), or special enzymes present in liver cells may be used to dissolve the casing. 5) The viral RNA then coopts the cell's ribosomes, and begins the production of materials necessary for viral reproduction. Because hepatitis C stores its information in a "sense" strand of RNA, the viral RNA itself can be directly read by the host cell's ribosomes, functioning like the normal mRNA present in the cell. As it begins producing the materials coded in its RNA, the virus also probably shuts down most of the normal functions of the cell, conserving its energy for the production of viral material, 6) although it occasionally appears that hepatitis C will stimulate the cell to reproduce (presumably to create more cells that can produce viruses), which is why hepatitis C is often associated with liver cancer. The viral RNA first synthesizes the RNA transcriptase it will need for reproduction. 7) Once there is adequate RNA transcriptase, the viral RNA creates an antisense version (the paired opposite) of itself as a template for the creation of new viral RNA. The viral RNA is now copied hundreds or thousands of times, making the genetic material for new viruses. Some of this new RNA will contain mutations. 8) Viral RNA then directs the production of protein-based Capsomeres (the building blocks for the virus's protective protein coat). Ribosomes create the proteins and release them for use. 9) The completed Capsomeres assemble around the new viral RNA into new viral particles. The Capsomeres are designed to attract each other and fit together in a certain way. When enough Capsomeres are brought together, they self-assemble to form a spherical shell, called a capsid that fully encapsulates the virus's RNA. The completed particle is called a nucleocapsid.

Muhammad Ali Minhas

HCV

Page |7

10) The newly formed viruses travel to the inside portion of the plasma membrane and attach to it, creating a bud. The plasma membrane encircles the virus and then releases it - providing the virus with its protective lipid coat, which it will later use to attach to another liver cell. This process of budding and release of new viruses continues for hours at the cell surface until the cell dies from exhaustion. Each surviving virus - those which are not destroyed by the immune system or other environmental factors - can produce hundreds or thousands of offspring. Over time, this endless cycle of reproduction results in significant damage to the liver, as millions upon millions of cells are destroyed by viral reproduction or by the immune system's attacks on infected cells.

Muhammad Ali Minhas

HCV

Page |8

Cellular immune responses against hepatitis C virus

HCV, like other viruses, induces multiple immune effector responses, but this review focuses primarily on T lymphocytes. There is good evidence that both CD4+ and CD8+ T lymphocytes play a major role in determining outcome after acute infection and therefore in the long term. This comes from the following observations.

Clearance of acute infection in both man and in chimpanzee models is accompanied by strong CD4+ and CD8+ T cell responses against numerous HCV derived antigens. The evidence was obtained first for CD4+ T cell responses and initially some specific epitopes were highlighted as potentially protective. Although these do appear to be targeted, this is not exclusive and responses to other gene products are also seen. The strength of the CD8+ T cell response against one epitope when measured using a tetramer, may be up to 8% of the total CD8+ T cells, and can include responses to at least 8 separate epitopes. By ELISpot analysis, the CD4+ T cell responses appear to be of a similar magnitude.

Figure: Interactions of HCV proteins with different effectors of the immune response. The effects of HCV proteins on the different components of the innate and specific immunity are summarized.

Muhammad Ali Minhas

HCV

Page |9

The timing of these responses appears to correlate with resolution of viraemia in those cases where virus is cleared. The level of activation of HCV-specific T cell responses (assessed by CD38 expression) correlates with the degree of liver inflammation analysed by blood ALT levels. There is an association between possession of specific HLA genes (DRB1*1101 and/or DQ1*0301) and spontaneous clearance of virus. This strongly suggests that selection of particular epitopes is associated with better initial control of viraemia. Those bearing HLA DQ1*0301 (which is in tight linkage disequilibrium with DRB1*1101) were found to be more likely to possess significant HCV-specific CD4+ T cell responses, further evidence that the responses in these individuals are more robust.

So much for successful responses which are in fact the exception. The mechanism for viral persistence, i.e. failure of T cell responses in the majority of patients, is not yet clear. Studies of those who go on to develop persistent infection have highlighted the weak CD4+ T cell responses, although it is not clear yet whether persistence of virus causes attenuation of T cell responses or vice versa. Reemergence of CD4+ T cell responses upon clearance of virus with interferonalpha/ribavirin therapy suggests the latter , i.e. suppression of T cells by virus may be important.

Muhammad Ali Minhas

HCV

P a g e | 10

The picture with regards to CD8+ T cell responses is even less clear. CD8+ T cell responses have been observed in the acute phase of infection in those who fail to clear virus at levels of 1 3% of CD8+ lymphocytes against 12 separate T cell epitopes. Whether these are the main epitopes targeted in these individuals and how, overall, the responses differ in magnitude between clearers and nonclearers is not known. It appears that failure to clear virus is not due to failure to mount any CTL response whatsoever, although, like CD4+ T cell responses, these may be poorly maintained in the face of ongoing viraemia. The overall quality of the response may differ in terms of magnitude or breadth or, importantly, peptide selection. It is this latter issue that forms the focus of this review.

Clinical features of HCV

After inoculation, unlike Hepatitis A and B, the acute illness caused by HCV is not well documented. This is partly because it is genuinely milder, and possibly it is poorly recognized by physicians, and also those in the current western risk groups may not present to hospital. This is unfortunate, as it now appears that early intervention is of benefit. When it has been documented, or in animal models, the following features are apparent:

The peak of viraemia may take several weeks to arise. The liver inflammation/liver enzyme level in the blood (usually measured as ALT or AST) does not parallel the viral load consistent with the idea that at this stage much of the liver damage is not caused directly by the virus. Resolution of the viraemia (accompanied by cellular immune responses) is associated with liver inflammation and in some but not all cases, clinical jaundice. The level of ALT may be 5002000 IU/l, compared with HBV where it may be 5 or 10 times higher. After this period, viraemia either persists or the individual, in about 15% of cases, becomes RNA negative in the blood. There may be a state of instability where virus may become undetectable in blood temporarily and then reappears. If viraemia is established, the level of viraemia does not correlate with progression of disease, unlike HIV. Disease progression is measured by the development of liver inflammation, as assessed by blood ALT and histological indices of lymphocytic infiltration and also by creeping fibrosis. The extent of these vary widely between individuals, and apart from a few factors such as alcohol and coinfection (for example with HIV), the basis of this variation is not understood.

Muhammad Ali Minhas

HCV

P a g e | 11

In those where disease has progressed, therapy (in the form of interferon-alpha and ribavirin) may lead to long-term clearance of virus from blood, with accompanying improvement in liver histology. The effects of the drugs are not entirely understood, and it is likely that in addition to antiviral activity they influence the immune response, both directly, and indirectly through lowering viral load.

Diagnosis and Testing

How and when is HCV diagnosed?
HCV infection is not infrequently diagnosed as a complete surprise and may cause considerable upset for patient and those around the patient including doctor. The disease can have a broad spectrum of presentations such as:

Major symptoms (swelling of legs and abdomen, confusion)and findings (abnormal laboratory tests (blood) or imagines (Ultrasound, CT scan). HCV can be diagnosed in a completely asymptomatic individual during health or life insurance check focused test result after someone's history indicated risk factor's (sometimes decades ago!) Asymptomatic individual may becomes more symptomatic once knowing that they carry HCV virus. This may due to prior denial of symptoms or occur against the background of increased anxiety about the diagnosis. As part of work-up for a variety of symptoms and findings, often - but not necessarily including abnormal liver tests: o Fatigue o Jaundice (relatively rare) o Fluid retention in abdomen or legs o Skin and joint complaints o Red blood cells in urine o Abnormal looking liver and/or spleen on ultrasound or CT o Liver tumor on imaging o Mental status changes, disturbance of sleeping pattern

Testing for HCV

1) HCV antibodies:
All HIV-infected patients should be tested for HCV infection with the HCV antibody test. Patients with risk factors for HCV infection should be retested at regular intervals. In HIVInfected patients, the HCV antibody test result sometimes is falsely negative; therefore, if HCV infection is suspected (e.g., because of a history of high-risk behavior, unexplained elevated

Muhammad Ali Minhas

HCV

P a g e | 12

ALT, or evidence of cirrhosis), the HCV RNA should be tested even if the HCV antibody test result is negative. A false-negative HCV RNA result is very unlikely in chronic infection.

2) HCV RNA:
All patients who test positive for HCV antibody should have HCV RNA testing performed. As noted above, if patients have negative results on HCV antibody tests but persistently abnormal transaminases or suspected acute or chronic infection, HCV RNA testing should be performed. The definition of chronic HCV infection is the presence of HCV RNA 6 months after the estimated time of infection. If a patient is HCV antibody positive but HCV RNA negative, the patient has cleared the HCV and does not have chronic HCV infection.

There are quantitative RNA tests and qualitative RNA tests. Although both types of RNA tests are highly sensitive and specific, the qualitative tests can detect lower levels of viremia than the quantitative tests. The choice of RNA test can be important. The quantitative RNA tests will be reported as a value, with a measured number of international units per milliliter (IU/mL). Quantitative tests are useful for determining the prognosis of HCV treatment and then monitoring while on HCV treatment. Qualitative RNA tests will be reported as a present or absent value, but without a numerical value. They are useful for serial testing during suspected acute infection and for determining whether spontaneous viral clearance has occurred, a sustained virological response has occurred during treatment, or a relapse has occurred after treatment.

3) Genotyping:
The HCV genotype is the strongest predictor of response to HCV treatment and also is a critical determinant of the dosage and duration of treatment. HCV genotyping should be performed once for all patients with detectable HCV RNA; it does not need to be repeated.

Muhammad Ali Minhas

HCV

P a g e | 13

4) Alanine aminotransferase:
Monitoring of ALT can be useful to assess acute infection, chronic liver inflammation, and response to HCV treatment. However, ALT does not always correlate with the degree of fibrosis and in addition, ALT can be persistently normal in 25% of HCV patients, including patients with cirrhosis or advanced liver disease. Small fluctuations in ALT usually are not clinically significant in HCV, though trends can be significant during or following HCV treatment.

Additional tests:
Check complete blood cell count with platelet count, albumin, total bilirubin, and prothrombin time. Test all patients for hepatitis B (HBsAg, anti-HBsAb, and anti-HBcAb). Patients with a negative HBsAg and negative anti-HBsAb result should be vaccinated against HBV. Test for hepatitis A virus (HAV) antibodies (total). All patients with a negative HAV antibody result should be vaccinated against HAV.

5) Imaging
Ultrasonography can be performed to screen for cirrhosis or focal hepatic masses. Computed tomography (CT), magnetic resonance imaging (MRI), and single-photon emission computed tomography (SPECT) are more expensive and generally are reserved for further evaluation of liver masses detected by ultrasound.

6) Liver biopsy
Liver biopsy is used to define the degree of inflammation (the grade) and degree of fibrosis (the stage) to determine the need for HCV treatment. Unless there is clear evidence of cirrhosis, laboratory tests and radiology studies are unable to quantitate the degree of fibrosis in the liver. Liver biopsy carries some risk, primarily from bleeding (the risk of significant bleeding or fatality is approximately 1/10,000). Patients with severe thrombocytopenia or coagulopathy should not undergo liver biopsy. Fibrosis is scored from 0 to 4, with 0 indicating no fibrosis and 4 indicating cirrhosis. Biopsy can be useful in making management decisions for some HCV patients, for example when determining whether to treat a patient, particularly those with genotype 1 virus (see below). If the biopsy reveals only mild-to-moderate fibrosis, it may be preferable to defer treatment and monitor the patient. Conversely, if the biopsy reveals more advanced fibrosis, treatment should be considered more urgently. With genotype 2 or 3 patients, some providers consider biopsy to be unnecessary because treatment outcomes are sufficiently high that findings from a biopsy would not necessarily change the management strategy. For HIV/HCV-coinfected patients, a biopsy may be particularly useful in determining the stage of disease and in planning whether or when to initiate HCV treatment, as the course of liver disease may accelerate. Overall, deciding

Muhammad Ali Minhas

HCV

P a g e | 14

whether to conduct a biopsy largely is a matter of individual choice. It is not a requirement for treatment of any patient, but may be useful for helping the provider and patient make a decision about whether or when to undergo treatment. Test results to detect, diagnose, and monitor HCV include: Anti-HCV Negative Positive Positive Negative Need to do Negative HCV RIBA HCV RNA, Qualitative HCV Infection No infection or, rarely, insufficient antibody No infection; likely a false positive Likely no infection, past infection, or HCV viral load low Past infection or HCV viral load low Current infection No infection, past infection, or HCV viral load low

Positive Positive or Weak or Indeterminate Indeterminate or Positive

Positive Not done or Positive Indeterminate

Negative Positive Negative

Basics of diagnostic tests

1. HCV ELISA
An enzyme-linked immunosorbent assay (ELISA) was developed by using a synthetic polypeptide (SP) whose sequence was derived from the structural region of hepatitis C virus (HCV). Since the introduction in 1990 of HCV antibody screening of blood donations, the incidence of this infection in transfusion recipients has been significantly reduced. The first generation of HCV ELISAs showed limited sensitivity and specificity and was produced using recombinant proteins complementary to the NS4 region of the HCV genome. Second generation tests, which included recombinant/synthetic

Muhammad Ali Minhas

HCV

P a g e | 15

antigens from the Core and nonstructural regions NS3 and NS4 resulted in a remarked improvement in sensitivity and specificity. Clinical studies show that significant amount of HCV infected individuals develop antibodies to NS5 non-structural protein of the virus. For this, the third generation tests include antigens from the NS5 region of the viral genome in addition to NS3, NS4 and the Core. The third generation tests have improved sensitivity and have shorten the antibody detection window period to 70 days. Principle: This anti-HCV employs solid phase, indirect ELISA method for detection of antibodies to HCV in two step incubation procedure. Polystyrene microwell strips are pre-coated with recombinant, highly immunoreactive antigens corresponding to the core and the non-structural regions of HCV (third generation HCV ELISA). During the first incubation step, anti-HCV specific antibodies, if present, will be bound to the solid phase pre-coated HCV antigens. The wells are washed to remove unbound serum proteins, and rabbit anti-human IgG antibodies (antiIgG) conjugated to the enzyme horseradish peroxidase (HRP-Conjugate) are added. During the second incubation step, these HRP-conjugated antibodies will be bound to any antigen-antibody (IgG) complexes previously formed and the unbound HRP-conjugate is then removed by washing. Chromogen solutions containing Tetramethylbenzidine (TMB) and urea peroxide are added to the wells and in presence of the antigen-antibody-anti-IgG (HRP) immunocomplex, the colorless Chromogens are hydrolyzed by the bound HRP conjugate to a blue-colored product. The blue color turns yellow after stopping the reaction with sulfuric acid. The amount of color intensity can be measured and it is proportional to the amount of antibody captured in the wells, and to the amount of antibody in the sample respectively. Wells containing samples negative for anti-HCV remain colorless.

Ag(p) + Ab(s) Ag(p)+ Incubation1 30min

[Ag(p)Ab(s) + ENZ] +ENZ]

[Ag(p)Ab(s)ENZ] [Ag(p) ]

blue yellow no color Coloring 15min

(+) (-) results

[Ag(p)

Incubation 2 30min

Immobilized Complex

Ag(p)pre-coated HCV antigens(core, NS3/4,NS5); Ab(s)HCV antibodies in sample (IgG); ENZHRP conjugated rabbit anti-human IgG.

Muhammad Ali Minhas

HCV

P a g e | 16

2. HCV PCR
HCV RNA is first separated from the sample. There are different methods available for doing so. When viral RNA is separated, its cDNA is formed by a process called Reverse Transcription (RT) by a reverse transcriptase enzyme. Then this DNA fragment is amplified by PCR.

The reverse transcription (RT) and polymerase chain reaction (PCR) amplification steps are performed sequentially in a single tube. First, genomic HCV RNA is reverse transcribed into complementary DNA (cDNA) using HCV-specific primers. Next, the mixture is heated to activate the DNA polymerase for the PCR amplification step and simultaneously inactivate the reverse transcriptase. Portions of the 5' UTR and core regions of the HCV genome are co-amplified from the cDNA using two pairs of biotinylated primers to produce two distinct biotinylated DNA fragments of 240 and 270 base pairs, representing the 5' UTR and core HCV regions respectively. The nucleotide sequence of the primers has been optimized to yield comparable amplification of six HCV genotypes. PCR can be used for qualitative as well as quantitative study of HCV.

The Future

HCV will continue to put a tremendous burden on future health care expenditures. Many patient have already often irreversible damage. An enormous number of infected patients, many of them as yet unidentified and who contracted blood through blood products or past iv drug abuse are at risk of developing cirrhosis with all its complications including hepatocelluar carcinoma. Once advanced cirrhosis with complications, viral eradication (still) very difficult, often impossible or associated with detrimental complications. Continued contamination / infection through needle transmission & illicit drug use. A fraction of all patients will be eligible for liver transplantation: this is beyond the reach of most worldwide and if available: the organ shortage is tremendous. ********************

Muhammad Ali Minhas

HCV