Presentation Outline HCV Morphology History Structure of virus Epidemiology Genotypes of virus HCV-mutants (Quasi-species) Methodes for genotyping Conclusion Refrence 5
HCV MORPHOLOGY1-family Flaviviridae2-The hepatitis C virus (HCV) is a small 30-60 nm3-Icosahedral enveloped, 9.6 kb -single-stranded, positivesense RNA virus .4-The hepatitis C virus (HCV) is the major etiological agentof acute post-transfusional (1).5- Its genomic diversity and it is classified into 6 genotypesand 120 subtypes 6
History1-In the mid 1970s, Harvey J.Alter , Chief of the NationalInstitutes of health, and his research team demonstratedthat most post-transfusion hepatitis cases were not due tohepatitis A and B viruses.2-In April of 1989, Chiron discovery of the virus, re-namedhepatitis C virus (HCV), was published in two articles in thejournal Science. 7
The Iranian 1a and 3a strains indigenous toIran. Subtype 1a was frequent in South Iran(70%), while 3a was more prevalent in North-West Iran (83%), Patients infected by blood products had morefrequently subtype 1a (57%), while younger drug users had more frequentlysubtype 3a (54%). (13) 13
Genotypes of virusBased on genetic differences between HCVisolates, the hepatitis C virus species is classifiedinto six genotypes with several subtypes withineach genotype. Subtypes are further brokendown into quasispecies based on their geneticdiversity. (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
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 theentire genome spontaneous mutation rate estimated at 1.4 to 1.9 x10-3 mutations per nucleotide per year. 15
Clinical Importance of Hepatitis C Virus GenotypesA large number of studies showing that HCVgenotype is correlated with response to IFN-atherapy(5)Subtype 1b and type 4 infected patients respondpoorly (<8%) to IFN-a, subtype 1a shows amarkedly better response rate as compared withsubtype 1b (15-20%), while complete responsesare seen in over 30% of patients infected withsubtypes 2 or 3a 16
Genotyping techniquesA. Molecular biology based techniquesB. Serology based techniques
ELISArecombinant immunoblot assay RIBA FDA RIBA TRIBA RIBA intermediate RIBA ELISA RIBA ELISA HCV RNA HCV RNA intermediate RIBA NS3 core
Advantages of anti-HCV Antibody detection by ELISA1. Ease of automation2. Relative cost-effectiveness3. High sensitivity in screening4. The accuracy of third-generation test is verygood in high-prevalence populations.
Limitations of Serology1. The interval between HCV infection and detectionof 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-prevalencepopulations like blood donors.
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
Direct sequencingPCR fragments were cut out from theagarose gel and purified using a minicolumn system (MN Germany).For automated DNA sequencing, PCRamplified products were purified usingPCR purification kit
Disadvantagethis method is expensiveand time consuming andrequiresspecial equipment for sequencing, it hasbeen restricted to theresearch setting andconsidered impractical forlarge clinical studies
RFLP typing of the 5’ UTRThe amplified product obtained by using the primersRFLP analysis by using the restriction enzymes HaeIII, Hinf I and Bst NI (New England Biolabs, Beverly,USA)Size of the undigested amplicon is 256 bp. Theamplicon is digested with all the above threerestriction enzymes as indicated, and theelectrophoretype is compared with the predictedpatterns to determine the genotype.(Hae III and Mva I enzymes were originally used fortyping of the HCV isolates from India by Das etal.(1993)) 27
DisadvantageThe PCR-RFLP system couldnot distinguish all virussubtypes or some novelgenotypes discovered inThailand and Vietnam,(Mellor et al., 1996)It was quite expensive andtime consuming because itused many restrictionenzymes and also needed alarge amount of PCR product. 29
Real time PCRReal-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
Advantages Disadvantages1-Rapid 1- Requires high level of2-Less hands-on time technical skills3-Closed-tube system, reduced 2-High capital cost for the risk of PCR contamination instrumentation4-High sensitivity and specificity5-Reproducible6-Quantitative results with wide dynamic range
conclusionthe new HCV genotyping assay is highlysensitive,specific,reproducible and capable ofgenotyping reliably HCV RNA directly from clinicalsamples in routine diagnostic laboratory Genotypeis clinically important in determining potentialresponse to interferon-based therapy and therequired duration of such therapy. 34
Refrence1: Lindenbach B, Rice C (2005). "Unravelling hepatitis C virusreplication 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, Thiel3:H, Viazov S, Weiner A, Widell A (2005). "Consensus proposals fora 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 chronicviral hepatitis. N. Engl. J. Med. 336, 347-356. 35
Refrence6: 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, 200410)WHO Internet site, 200411)Hepatitis resource network12)Hatami H. Malekzadeh R. Emerging Hepatitis C, In : Emergingand 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.