Abbas morovvati


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Abbas morovvati

  1. 1. HCV Genotyping methods Abbas Morovvati
  2. 2. Presentation Outline HCV MORPHOLOGY&CHARACTER History Structure of virus Epidemiology Genotypes of virus Mutants Treatment Methodes for genotyping Conclusion Refrence 3
  3. 3. HCV MORPHOLOGY&CHARACTER1-The hepatitis C virus (HCV) is a small - nm2-enveloped, 9.6 kb -single-stranded, positive sense RNA virus3-family Flaviviridae.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 genotypes and 120subtypes. hepatitis C virus (HCV) 30-60 nm 4
  4. 4. History1-In the mid 1970s, Harvey J.Alter , Chief of the National Institutes ofhealth, and his research team demonstrated that most post-transfusionhepatitis 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
  5. 5. 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 -6PCR -7 Holland schorter Bullock -8 HCV 6
  6. 6. The discovery of HCV types and subtypes. The total number of HCV types(solld line) and subtypes (broken lme) is indicated by year.
  7. 7. The genomic organization of hepatitis C virus
  8. 8. Epidemiology
  9. 9. HCV- Genotypes Genotype Geographical Location1,2 & 3 Worldwide 4 Middle East & Africa 5 South Africa 6 South East Asia 11
  10. 10. 12
  11. 11. Genotypes of virusBased on genetic differences between HCVisolates, the hepatitis C virus species is classified intosix genotypes with several subtypes within eachgenotype. Subtypes are further broken down intoquasispecies based on their genetic diversity. Thepreponderance and distribution of HCV genotypesvaries globally. (1-2) 13
  12. 12. 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
  13. 13. 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.
  14. 14. 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 a markedlybetter 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
  15. 15. 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 fromthe 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
  16. 16. Direct sequencingPCR fragments were cut out from the agarosegel and purified using a mini column system(MN Germany).For automated DNA sequencing, PCRamplified products were purified using PCRpurification kit
  17. 17. Disadvantagethis method is expensive andtime consuming andrequiresspecial equipmentfor sequencing, it has beenrestricted to the researchsetting and consideredimpractical for largeclinical studies
  18. 18. 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 EnglandBiolabs, Beverly, USA)Size of the undigested amplicon is 256 bp. The amplicon isdigested with all the above three restriction enzymes asindicated, and the electrophoretype is compared with thepredicted patterns to determine the genotype.(Hae III and Mva I enzymes were originally used fortyping of the HCV isolates from India by Das et al.(1993)) 20
  19. 19. 21
  20. 20. DisadvantageThe PCR-RFLP system couldnot distinguish all virus subtypesor some novel genotypesdiscovered in Thailand andVietnam,(Mellor et al., 1996)•it was quite expensive and timeconsuming because it used manyrestriction enzymes and alsoneeded a large amount of PCRproduct . 22
  21. 21. Line probe assay(6)
  22. 22. 5`UTRThe 5’ UTR of HCV RNA is the mosthighly conserved portion of the genomeand thus has been used in mostlaboratories to develop sensitive detectionassays for HCV RNA. 24
  23. 23. LIPAA particular kind of reverse hybridizationassays, called line probe assays (LiPA), weredeveloped at Innogenetics which allow an easy andfast determination of multiple genetic variations, ashas been documented for HCV genotypingINNO-LiPA assay was the easiest and least timeconsuming method to perform 25
  24. 24. AdvantagesA commercial DNA hybridization method called the line probe assay relieson genotype-specific probes based on 5 noncoding region (5NCR) tohybridize to a product amplified from a clinical sample (Stuyver etal., 1993).However, most of these techniques are time consuming, laborintensive,expensive, confined to research or reference laboratories and not feasible forroutine 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 posesa significant public health problem and there is a very limited feasibility todiagnose infection at the genotypic level and detect infection with mixedgenotypes. 26
  25. 25. LiPA Technology, Step 3: Conjugate Binding Protein conjugatemarker line Streptavidin/alkaline phosphatase conjugate binds to biotin at the terminus of the surface-bound target DNA-probe Nitrocellulose strip
  26. 26. 4/28/2012 Free Template from 29
  27. 27. 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. Genotypes 1 and4 are less responsive to interferon-based treatmentthan are the other genotypes (2, 3, 5 and 6) (1-2) 30
  28. 28. Refrence1: Lindenbach B, Rice C (2005). "Unravelling hepatitis C virus replicationfrom genome to function.". Nature 436 (7053): 933-8. PMID 16107832.2: Simmonds P, Bukh J, Combet C, Deléage G, Enomoto N, FeinstoneS, Halfon P, Inchauspé G, Kuiken C, Maertens G, Mizokami M, MurphyD, Okamoto H, Pawlotsky J, Penin F, Sablon E, Shin-I T, Stuyver L, ThielH, Viazov S, Weiner A, Widell A (2005). "Consensus proposals for aunified system of nomenclature of hepatitis C virus genotypes.". Hepatology42 (4): 962-73. PMID 16149085.3:Paul Elias. " TheAssociated Press, 27 February 204: , TheLasker 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. 31
  29. 29. Refrence6: 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, NateghR, 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 : Emerging andreemerging infectious diseases and employee health, 1th ed. 2004.13)J Med Virol. 2004 Oct;74(2):246-52.Samimi-Rad K, NateghR, Malekzadeh R, Norder H, Magnius L.