2. C

3. 3

5. Presentation Outline
 HCV Morphology
 History
 Structure of virus
 Epidemiology
 Genotypes of virus
 HCV-mutants (Quasi-species)
 Methodes for genotyping
 Conclusion
 Refrence
5

6. HCV MORPHOLOGY
1-family Flaviviridae
2-The hepatitis C virus (HCV) is a small 30-60 nm
3-Icosahedral enveloped, 9.6 kb -single-stranded, positive
sense RNA virus .
4-The hepatitis C virus (HCV) is the major etiological agent
of acute post-transfusional (1).
5- Its genomic diversity and it is classified into 6 genotypes
and 120 subtypes
6

7. History
1-In the mid 1970s, Harvey J.Alter , Chief of the National
Institutes of health, and his research team demonstrated
that most post-transfusion hepatitis cases were not due to
hepatitis A and B viruses.
2-In April of 1989, Chiron discovery of the virus, re-named
hepatitis C virus (HCV), was published in two articles in the
journal Science.
7

8. The genomic organization of hepatitis C virus

9. The discovery of HCV types and subtypes. The total number of HCV types
(solld line) and subtypes (broken lme) is indicated by year.

10. Epidemiology

13. The Iranian 1a and 3a strains indigenous to
Iran.
Subtype 1a was frequent in South Iran
(70%), while 3a was more prevalent in North-
West Iran (83%),
Patients infected by blood products had more
frequently subtype 1a (57%),
while younger drug users had more frequently
subtype 3a (54%). (13)
13

14. Genotypes of virus
Based on genetic differences between HCV
isolates, the hepatitis C virus species is classified
into six genotypes with several subtypes within
each genotype. Subtypes are further broken
down into quasispecies based on their genetic
diversity. (1-2) a
gb a
e
c 6a
6b c
f cb b7
da
e 11
d
a 4 5 a
2 36 6a
1 6b
9c
b
c 9a 9b
c c
a a d b
l
a
10a 14

15. HCV-mutants (Quasi-species)
1-HVR–1 ANALYSIS N-terminal 27 residues (amino acid 384-411) of
E2 is highly variable and hence called Hypervariable Region or
HVR-I.
2-When mutant strains are eradicated, another strains will emerge.
3-Mutations help the virus to escape the immune response.
4-Quasi-species refers to the mutation that spontaneously
occur,without effecting a particular genotype.
5-Highly genetically diverse virus 13-18 mutations per year for the
entire genome spontaneous mutation rate estimated at 1.4 to 1.9 x
10-3 mutations per nucleotide per year.
15

16. Clinical Importance of Hepatitis C
Virus Genotypes
A large number of studies showing that HCV
genotype is correlated with response to IFN-a
therapy(5)
Subtype 1b and type 4 infected patients respond
poorly (<8%) to IFN-a, subtype 1a shows a
markedly better response rate as compared with
subtype 1b (15-20%), while complete responses
are seen in over 30% of patients infected with
subtypes 2 or 3a
16

17. Genotyping techniques
A. Molecular biology based techniques
B. Serology based techniques

18. ELISA
– ELISA
ELISA
ELISA

19. ELISA
recombinant immunoblot assay RIBA FDA
RIBA
TRIBA
RIBA
intermediate
RIBA ELISA
RIBA ELISA HCV RNA
HCV RNA intermediate
RIBA NS3 core

21. Advantages of anti-HCV Antibody detection by
ELISA
1. Ease of automation
2. Relative cost-effectiveness
3. High sensitivity in screening
4. The accuracy of third-generation test is very
good in high-prevalence populations.

22. Limitations of Serology
1. The interval between HCV infection and detection
of anti-HCV may be as long as 3 months and up to 6 months
in some cases,long after serum aminotransferase levels
have peaked.
2. Poor sensitivity in immunocompromised patients like post
liver-transplant patients and HIV positive patients.
3. An abundance of false-positives in low-prevalence
populations like blood donors.

23. Molecular techniques

24. Molecular Genotyping
(1) RFLP
(2) type specific amplification of the core gene
(3) type specific amplification of the NS5 gene
(4) serotyping using type specific peptides from the NS4 region
of the HCV polyprotein
(5) direct sequencing of the NS5 gene
(6) direct sequencing of the viral nucleic acid.(Zein et al., 1996).
(7) real-time polymerase chain reaction (PCR)
24

25. Direct sequencing
PCR fragments were cut out from the
agarose gel and purified using a mini
column system (MN Germany).
For automated DNA sequencing, PCR
amplified products were purified using
PCR purification kit

26. Disadvantage
this method is expensive
and time consuming and
requiresspecial equipment
for sequencing, it has
been restricted to the
research setting and
considered impractical for
large clinical studies

27. RFLP typing of the 5’ UTR
The amplified product obtained by using the primers
RFLP analysis by using the restriction enzymes Hae
III, Hinf I and Bst NI (New England Biolabs, Beverly,
USA)
Size of the undigested amplicon is 256 bp. The
amplicon is digested with all the above three
restriction enzymes as indicated, and the
electrophoretype is compared with the predicted
patterns to determine the genotype.
(Hae III and Mva I enzymes were originally used for
typing of the HCV isolates from India by Das et
al.(1993))
27

28. 28

29. Disadvantage
The PCR-RFLP system could
not distinguish all virus
subtypes or some novel
genotypes discovered in
Thailand and Vietnam,
(Mellor et al., 1996)
It was quite expensive and
time consuming because it
used many restriction
enzymes and also needed a
large amount of PCR product
.
29

30. TYPE SPECIFIC AMPLIFICATION

31. Real time PCR
Real-time PCR systems rely on the detection and
quantitation of a fluorescent reporter, the signal of which
increases in direct proportion to the amount of PCR
product. The real-time PCR instrument combines
thermocycling with fluorescence detection and
measures the amount of fluorescence after each
amplification cycle. Several fluorescent reporters have
been

33. Advantages Disadvantages
1-Rapid 1- Requires high level of
2-Less hands-on time technical skills
3-Closed-tube system, reduced 2-High capital cost for the
risk of PCR contamination
instrumentation
4-High sensitivity and specificity
5-Reproducible
6-Quantitative results with wide
dynamic range

34. conclusion
the new HCV genotyping assay is highly
sensitive,specific,reproducible and capable of
genotyping reliably HCV RNA directly from clinical
samples in routine diagnostic laboratory Genotype
is clinically important in determining potential
response to interferon-based therapy and the
required duration of such therapy.
34

35. Refrence
1: Lindenbach B, Rice C (2005). "Unravelling hepatitis C virus
replication from genome to function.". Nature 436 (7053): 933-8.
PMID 16107832.
2: Simmonds P, Bukh J, Combet C, Deléage G, Enomoto N,
Feinstone S, Halfon P, Inchauspé G, Kuiken C, Maertens G,
Mizokami M, Murphy D, Okamoto H, Pawlotsky J, Penin F, Sablon E,
Shin-I T, Stuyver L, Thiel
3:H, Viazov S, Weiner A, Widell A (2005). "Consensus proposals for
a unified system of nomenclature of hepatitis C virus genotypes.".
Hepatology 42 (4): 962-73. PMID 16149085.
4: The Lasker Foundation. Accessed 20 February 2008.
5:Hoofnagle, J. and Di Bisceglie, A. (1997) The treatment of chronic
viral hepatitis. N. Engl. J. Med. 336, 347-356.
35

36. Refrence
6: http://www.innogenetics.com/
7)East Mediterr Health J. 2000 Mar-May;6(2-3):372-7.Zali MR,
Mayumi M, Raoufi M, Nowroozi A.
8)J Med Virol. 2004 Oct;74(2):246-52.Samimi-Rad K, Nategh R,
Malekzadeh R, Norder H, Magnius L.
9)CDC Internet site, 2004
10)WHO Internet site, 2004
11)Hepatitis resource network
12)Hatami H. Malekzadeh R. Emerging Hepatitis C, In : Emerging
and reemerging infectious diseases and employee health, 1th ed.
2004.
13)J Med Virol. 2004 Oct;74(2):246-52.Samimi-Rad K, Nategh R,
Malekzadeh R, Norder H, Magnius L.