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. 209-2011)

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 quasi-species. 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 MethodsELISA Experiment

Materials used:

Recombinant HCV antigens: NS3, NS4, NS5 at a concentration of 10 ug/ml each Patient X’s 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 °C2. The next day reaction was blocked with 2% BSA solution in PBS for 1 hour and then washed twice with

PBS-Tween3. 100ul of serum was added and the solution was incubated for one hour4. Wells was washed with PBS-tween 5 times5. Mouse anti-human HRP was added and then wells were incubated for 1 hour6. Wells were washed with PBS-tween 5 times7. DAB substrate was added8. After 15 minutes reaction was stopped with 20ul of H2SO4 and read at 450nm

Figure 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 – 5’NCR (GenBank Accession # M67463), to amplify cloned HCV cDNA for preparation of HCV 5’-NCR RNA control. Primers had following sequence: 5_-FAM-GCGAGCCACCGGAATTGCCAGGACGACCGCTCGC- DABCYL-3, and position: 166-187. (Yang, et al., 2002)

Reverse-trancriptase reactionObtained 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 RNA2. Mixture was heated at 70°C for 5 minutes and then chilled on ice3. Reverse transcriptase mix was added4. Mixture was heated at 25°C for 5 minutes to anneal the primer5. First-strand synthesis reaction was carried (42°C for 60 minutes) 6. Reverse transcriptase was inactivated by heating the mixture at 90° for 5

minutes

RT-PCRPolimerase Chain Reaction was performed twice using Perkin-Elmer ABI Prism 7700 Detection System. Conditions were as follows: 95°C for 10 minutes, 45 cycles in 95°C for 15s, 60°C for 60s. Each PCR reaction used 2µl of cDNA added to 48µl 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 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

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 PCRViral genotype at the start of illness: 1Anti-HAV: negativeHBsAg: negativeAntinuclear antibody: negativeAnti-mitochondrial antibody: negative

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, Schäffer 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.