HEPATITIS TITLE

Hepatitis C is an infectious flavivirus disease, caused by HCV - member of the family of

positive viruses with a single stranded RNA. It has been identified in 1989, by
recombinant technology. HCV genome encodes a gene which produces a single-strand
chain which consists of nearly 3000 amino acids. (Coleman and Tsongalis, 2009, p. 2092011)

Fig1. Simplified diagram of the structure of Hepatitis C virus. (GrahamColm, 2008)

HCV can be present in six different genotypes (numbered from 1 to 6). Genotype 1 is the
most common one, followed by the genotype 2 and 3. Genotypes 4, 5 and 6 mainly occur
only in Africa and
Asia.
In the United States and the most of European countries genotype 1 is found in
approximately 75% of HCV infected (Cichy Zabojca WZW C, 2010). Hepatitis Central
(Hepatitis Central, 2011) states that even though patients are only infected with one
genotype, each genotype is actually a mixture of closely-related viruses called quasispecies. These quasi-species have the ability to mutate very quickly and become
immune to current treatments, which explains why chronic Hepatitis C is so difficult to
treat.
Hepatitis C can be called the silent killer infection is often asymptomatic or the
symptoms are uncharacteristic. Only 20% of patients develop easily diagnosed
symptoms. The disease can remain silent for decades, whilst effectively destroying the
liver of the infected person. In the 20-30 years of chronic HCV infection, around third of
the patients develops cirrhosis of the liver.

Fig2. Simplified diagram presenting the pathogenesis of Hepatitis C virus

Hepatitis C is a disease of a global footprint. Currently hepatitis accounts for three-fourth
of all the cases of liver disease in the world.(Trustees of Dartmouth College, 2012) Figure
3 presents most important facts and statistics on magnitude of HCV problem around the
world:

Fig3. Chart presenting magnitude of Hepatitis C problem worldwide.

Infection

Hepatitis C is easily spreadable by blood-to-blood contact. Very common ways of

infection are:

Sharing personal hygiene items

Sharing needles to drug injections
Using unsterilized piercing and tattoo equipment
Sexual transmission does not seem to be very
common, as well as mother-baby transmission
over the pregnancy and birth.
Treatment

Current therapies for HCV infection allow recovery in an increasing number of patients.
Pharmacological Treatment is necessary and is determined individually for each patient,
based on diagnosis of the virus genotype. The standard treatment of chronic hepatitis are
subcutaneous injections of interferon which fight infections within the body, including
viruses. It is supported with intake of the antiviral agent ribavirin.

Response to the treatment varies between different genotypes of hepatitis:

For viruses of the genotype 1,4,5 and 6, standard therapy lasts around 48 weeks
Therapy of genotype 2 and 3 viruses usually lasts up to 24 weeks
(Cichy Zabojca WZW C, 2010)

Materials and Methods

ELISA Experiment
Materials used:

Recombinant HCV antigens: NS3, NS4, NS5 at a concentration of 10 ug/ml

each
Patient Xs serum
Positive and negative control sera
Mouse anti-human immunoglobulin conjugated with horseradish
peroxidase and 3,3-diaminobenzidine tetrahydrochloride substrate (DAB)
Phosphate buffered saline (PBS)
PBS-tween (0.5%)
Stopping reagent here H2SO4
Microtiter plates
Microtiter plate reader equipped with 405 nm filter
Procedure

1. Recobinant HCV antigens was added on the wells and incubated (covered)
overnight at around 4 C
2. The next day reaction was blocked with 2% BSA solution in PBS for 1 hour and
then washed twice with PBS-Tween
3. 100ul of serum was added and the solution was incubated for one hour
4. Wells was washed with PBS-tween 5 times
5. Mouse anti-human HRP was added and then wells were incubated for 1 hour
6. Wells were washed with PBS-tween 5 times
7. DAB substrate was added
8. After 15 minutes reaction was stopped with 20ul of H2SO4 and read at 450nm

Figu
re 4
Procedure was repeated 6, 9, 36, 48 and 64 months after initial diagnosis which allowed
to obtain results shown in Figure 4.
Design of primer
Primers for PCR were designed based on HCV sequence 5NCR (GenBank Accession #
M67463), to amplify cloned HCV cDNA for preparation of HCV 5-NCR RNA control.
Primers had following sequence: 5_-FAM-GCGAGCCACCGGAATTGCCAGGACGACCGCTCGCDABCYL-3, and position: 166-187. (Yang, et al., 2002)
Reverse-trancriptase reaction
Obtained RNA came useful in creating a cDNA template using the Reverse Transcription
System (Promega, Madison, WI). Process can be described in a few simple steps:
1. Primer was added to RNA
2. Mixture was heated at 70C for 5 minutes and then chilled on ice
3. Reverse transcriptase mix was added
4. Mixture was heated at 25C for 5 minutes to anneal the primer
5. First-strand synthesis reaction was carried (42C for 60 minutes)
6. Reverse transcriptase was inactivated by heating the mixture at 90 for 5
minutes
RT-PCR
Polimerase Chain Reaction was performed twice using Perkin-Elmer ABI Prism 7700
Detection System. Conditions were as follows: 95C for 10 minutes, 45 cycles in 95C for
15s, 60C for 60s. Each PCR reaction used 2l of cDNA added to 48l of PCR Mastermix (
taq polymerase, molecular beacon, water, buffer, MgCl2, dNTP, forward and reverse
primers). 5-carboxy-X-rhodamine, added to the buffer worked as the reference day which
normalized the rections.

BLAST
According to Altschul (1997) BLAST is an algorithm for fast sequence database searching.
In compared sequences, the program finds pairs of segments length W (W = 3 for protein
sequence, W = 6 for DNA sequences) for which the result of the comparison (score) is
higher than a specified threshold value [T].It is a widely used tool for searching

protein and DNA databases for sequence similarities. It can help in analysing
unidentified genes, using genes which functions are already known.
RESULTS

Table 1: Lab investigations of the patient X at the

time of his first visit to his physician.
Total Bilirubin: 3 mg/dl (normal: 0.3 to 1.9 mg/dL)
ALT: 100 U/L (normal: 7 to 56 units per liter)
Anti HCV antibody titer: 1/80 dilution (cut-off 1/20 dilution)
HCV RNA: 10^5 RNA IU/ml (cut-off 200 IU/ml)
by SmartCycler II Real-time PCR
Viral genotype at the start of illness: 1
Anti-HAV: negative
HBsAg: negative
Antinuclear antibody: negative
Anti-mitochondrial antibody: negative

Table one contains results of lab investigations of the patient X at the time of his
first visit to his physician. It can be concluded that:

Patient suffers from jaundice, as a level of bilirubin (3 mg/dl) is higher

than normal.
The increase in ALT can be a result of liver disease - here hepatitis
(regardless of etiology)
Viral genotype at the start of illness determined that patient suffered
from genotype 1 HCV
Lack of HAV Antibodies- hepatitis A was not detected
Negative outcome of HBsAg test patient did not suffer from Hepatitis
B
Negative results of Antinuclear antibody as well as Anti-mitochondrial
antibody showed that patient was free from autoimmune disease

Figure 6Liver sample of Patient X. Chronic inflammatory cells and lipid deposition
together with thick
fibrous connective tissue are symptoms of progressing fibrosis. Absence of
hepatic artery and portal vein.

Reference list
Trustees of Dartmouth College (2012) Hepatitis C World Prevalence. Available
At: http://www.epidemic.org/thefacts/theepidemic/worldPrevalence/ (Accessed:
17 December 2012)

GrahamColm (2008) Simplified diagram of the structure of Hepatitis C virus

[Online]. Available at: http://en.wikipedia.org/wiki/Hepatitis_C_virus (Accessed: 17
December 2010).
Altschul SF, Madden TL, Schffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ.
Nucleic Acids Res. 1997 Sep 1;25(17):3389-402. Review.
Coleman, W.B, Tsongalis G.J. (2009) Understanding Molecular Pathogenesis: The
Biological Basis of Human Disease and Implications for improved Treatment of
Human Disease, in Coleman, W.B, Tsongalis G.J. Molecular Pathology. Academic
Press, pp. 209
Cichy Zabojca WZW C (2010) Available at: http://www.wzwc.pl/wzwc/czymjest
(Accessed: 10 March 2010)
Hepatitis Central (2011) Hepatitis C Genotypes. Available at:
http://www.hepatitis-central.com/hepatitis-c/hepatitis-c-genotypes.html
(Accessed 22 November 2012)
Yang JH, Lai JP, Douglas SD, Metzger D, Zhu XH, Ho WZ. Real-time RT-PCR for
quantitation of hepatitis C virus RNA. J Virol Methods. 2002, 102:119-28.