Hepatitis C Virus

Introduction

Family: Flaviviridae

Genus- Hepacivirus

• Hepatitis C is caused by infection with the hepatitis C virus.
• The hepatitis C virus is a single-stranded, enveloped, positive sense RNA virus.
• The infection usually begins in a gradual manner with symptoms like tiredness, loss of appetite, abdominal discomfort, nausea and vomiting, fever etc.
• Slowly it may progress to jaundice in few cases. Out of these cases, some get fully recover and the rest become chronic carriers of the infection.
• This virus can infect the person and may cause severe inflammation of the liver leading to severe liver damage and long-term complications.

The virus

• The hepatitis C virus is an envelope.
• It is single stranded RNA virus with a diameter of about 50 nm.
• The 5’ end of the gene codes for core and envelope proteins, followed by non-structural proteins.
• The rapid mutation in a hypervariable region of the genome coding for envelope proteins allows it to escape from immune surveillance.
• Most people infected with HCV develop chronic infection.
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• It belongs to the genus (Hepacivirus) within the Flaviviridae family.
• Hep C virus primarily affects humans but has been experimentally found to infect chimpanzees as well.
• Most hepatitis C infected persons which go on to develop chronic infection.

Genotypes of hepatitis C virus

• There are several genotypes of hepatitis C virus.
• Specifically, 11 genotypes with several distinct subtypes have been identified throughout the world.
• HCV has been classified into 6 genotypes (type 1-6) on the basis of phylogenetic analysis.
• Genotype 1-4 has a poorer prognosis and response to interferon therapy.
• The genotypes do not vary in their virulence of pathogenicity whereas; vary in their responsiveness to interferon/ribavirin combination therapy.
• There are about 100 different strains based on the gene sequence differences.
• Genotype 1 is generally associated with a poor response to interferon alone.
• Genotypes 2 and 3 are associated with more favorable responses.
• Genotypes 1-3 are found worldwide.

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Gene’s order

• HVR-1 represents the hypervariable region of envelope glycoproteins.
• HCV displays genomic diversity with different clades predominating in different parts of the world.
• The virus undergoes sequence variation during chronic infections.
• This complex viral population in a host is a host is referred to as “quasi- species”.
• Genetic diversity is not correlated with a difference in clinical disease although differences do exist in response to antiviral therapy according to viral genotypes.

Gene expression

• Like other positive SSRNA virus, HCV RNA genome is translated on entry into a host.
• HCV RNA is not capped, although most ribosome usually first recognizes the presence of a cap structure at 5’ end of an RNA before initiating translation.
• HCV has circumvented the cap requirement by using an internal ribosome entry site (IRES) at its 5’ end as a ribosome binding site (RBS) that directs internal initiation of RNA translation.
• The product of translation is a single large polyprotein, containing two protease domains capable of cleaving the polyprotein into a number of functional subunits.
• This protease is likely to be useful for the design of specific antiviral protease inhibitors.
• Once the HCV polyprotein is processed, viral nonstructural proteins participate in genome replication.
• RNA – dependent RNA polymerase first copies the positive RNA into negative RNA and then copies the negative RNA into more positive RNA.

Pathogenesis

• The liver disease caused by HCV is similar to those caused by HBV.
• HCV causes long-term consequences of chronic infection.
• The rate of progression of acute HCV to chronic infection is much higher than HBV.
• Liver cirrhosis is substantial following several decades of chronic HCV infection.
• HCV is associated with extrahepatic disease mixed cryoglobulinemia and immune complex disorder of the kidney and other sites.
• Antibodies complex to virion are deposited in affected tissues, triggering an inflammatory response that results in tissue damage.
• Factors promoting progression or severity of chronic hepatitis C include:

1. Increased alcohol intake
2. Age greater than 40 years at the time of infection,
3. HIV co- infection
4. Male gender and other co-infections (e.g. HBV infection).

Transmission

• HCV is a major cause of post- transfusion hepatitis, 0.5-1% adults are seropositive.post- transfusion hepatitis is also called non- A , non-B hepatitis.
• Route of transmission

Percutaneous

• Infecting drug use, clotting factors transfusion.
• Transplant from infected donor, therapeutic (contaminated equipment, unsafe infecting practice)

Occupational transmission

Perinatal transmission

Sexual transmission

Laboratory diagnosis

HCV detection is normally conducted in two phases a screening enzymes immune assay (EIA) detecting antibodies to HCV proteins followed by a confirmatory assay which employs either a more specific recombinant assay ((RIBA) antibody test or direct detection of HCV RNA.

Screening test

• The second generation screening test (EIA-2) detects antibodies to 3 HCV- encoded proteins: C22 (core protein), C33 nonstructural region 3 proteins (NS3), and core non-structural region 4 proteins (NS4).
• The EIA-2 has sensitivity of 92-95%
• The third generation EIA test (EIA-3) also detects a fourth HCV protein (NS5).
• EIA-3 has a sensitivity (97-99%) and specificity (>99%).
• A false negative can occur in immunocompromised populations, such as HIV-positive patients and in hemodialysis patients.

Confirmatory assay

• Once an initial screening test returns as positive, a confirmatory assay can be performed to rule out a false positive screen.
• Two methods are currently available:

1. RIBA

• RIBA also detects the C22, C33 and C100 proteins and is interpreted as:

Two or more antibodies present positive

One antibody presents Indeterminate

No antibodies present Negative

2. Molecular testing for HCV RNA

• Nucleic acid testing (NAT) detects actual viral presence
• NAT also allows earlier detection of acute HCV infection when antibodies are undetectable, but an active virus is present in the blood.
• RT-PCR, real-time PCR or branched chain DNA method (b DNA) are commonly used.
• HCV antibodies are detectable at least 4 weeks after infection. Antibodies detection helps to determine the time frame at which a patient is presenting.
• The HCV PCR should be repeated at week 4 and week 12 to look for spontaneous clearance of the virus. If HCV still persists at weeks after initial infection, strong consideration should be given to treat the infection at the early stage.

3.Antigen detection

• EIA for HCV Ag is commercially available
• The technique is similar to that of HCV RNA for early detection.
• Much easier to carry out

4. Genotyping

• Includes RNA sequencing, PCR hybridization.
• Genotypes 1 and 4 have a worse prognosis.

5. Viral load measurement

• Patients with high viral load are thought to have poorer prognosis
• Viral load measurement is used for monitoring response to interferon therapy.
• For viral load measurement, commercial test are available.

REFERENCE

Cheesbrough, M.Medical Laboratory Manual for Tropical Countries. Vol 2. ELBS London, 2007.

Tille, P.Diagnostic Microbiology.13th. Elsevier, 2014.

D Grenwood, Slack RCB, and Peutherer J.Medical Microbiology.Dunclude Livingstone: ELBS, 2001.