3. Presentation Outline
HCV MORPHOLOGY&CHARACTER
History
Structure of virus
Epidemiology
Genotypes of virus
Mutants
Treatment
Methodes for genotyping
Conclusion
Refrence
3
4. HCV MORPHOLOGY&CHARACTER
1-The hepatitis C virus (HCV) is a small - nm
2-enveloped, 9.6 kb -single-stranded, positive sense RNA virus
3-family Flaviviridae.
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. hepatitis C virus (HCV)
30-60 nm
4
5. 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.
5
6. A Brief History of genotyping
-1
RFLP PCR HCV
Davidson Mcomish
5'-UTR HCV Leiven stuyver -2
.
NS56 Chayama -3
HCV -4
Lipa Leiven stuyer -5
. HCV
core Holland -6
PCR -7
Holland
schorter Bullock -8
HCV
6
7. The discovery of HCV types and subtypes. The total number of HCV types
(solld line) and subtypes (broken lme) is indicated by year.
13. 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. The
preponderance and distribution of HCV genotypes
varies globally. (1-2)
13
14. HCV-mutants
(Quasi-species)
HCV mutates as it replicates inside the human body.
Mutation occurs in the hypervariable region of the
envelope gene.
When mutant strains are eradicated, another strains
will emerge.
Mutations help the virus to escape the immune
response.
Quasi-species refers to the mutation that spontaneously
occur, without effecting a particular genotype.
14
15. Laboratory Diagnosis
• HCV antibody - generally used to diagnose hepatitis
C infection. Not useful in the acute phase as it takes
at least 4 weeks after infection before antibody
appears.
• HCV-RNA - various techniques are available e.g.
PCR and branched DNA. May be used to diagnose
HCV infection in the acute phase. However, its main
use is in monitoring the response to antiviral therapy.
• HCV-antigen - an EIA for HCV antigen is available.
It is used in the same capacity as HCV-RNA tests
but is much easier to carry out.
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 of the HCV is currently
done by this method
(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) InnoLipa (Line probe assay) using the specific probes
(6) direct sequencing of the NS5 gene
(7) direct sequencing of the viral nucleic acid.
(Zein et al., 1996).
17
18. 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
19. 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
20. 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))
20
22. 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 .
22
24. 5`UTR
The 5’ UTR of HCV RNA is the most
highly conserved portion of the genome
and thus has been used in most
laboratories to develop sensitive detection
assays for HCV RNA.
24
25. LIPA
A particular kind of reverse hybridization
assays, called line probe assays (LiPA), were
developed at Innogenetics which allow an easy and
fast determination of multiple genetic variations, as
has been documented for HCV genotyping
INNO-LiPA assay was the easiest and least time
consuming method to perform
25
26. Advantages
A commercial DNA hybridization method called the line probe assay relies
on genotype-specific probes based on 5 noncoding region (5NCR) to
hybridize to a product amplified from a clinical sample (Stuyver et
al., 1993).
However, most of these techniques are time consuming, laborintensive,
expensive, confined to research or reference laboratories and not feasible for
routine genotyping (White et al., 2000; Nolte et al., 2003).
A simple, inexpensive, and accurate genotyping method is needed
(Smith et al., 1995), especially for countries like Pakistan, where HCV poses
a significant public health problem and there is a very limited feasibility to
diagnose infection at the genotypic level and detect infection with mixed
genotypes.
26
27.
28. LiPA Technology, Step 3: Conjugate Binding
Protein conjugate
marker line
Streptavidin/alkaline
phosphatase conjugate binds to
biotin at the terminus of the
surface-bound target
DNA-probe
Nitrocellulose strip
29. 4/28/2012 Free Template from www.brainybetty.com 29
30. 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. Genotypes 1 and
4 are less responsive to interferon-based treatment
than are the other genotypes (2, 3, 5 and 6) (1-2)
30
31. 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
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.
3:Paul Elias. " The
Associated Press, 27 February 20
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.
31
32. 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.
