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Hepatitis A

Hepatitis A. Anders Widell Department of Medical Microbiology, Lund University, University Hospital , Malmö, Sweden. Hepatitis viruses - my minireview. Acute hepatiis A-E similar clinical picture; diagnostic testing a must

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Hepatitis A

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  1. Hepatitis A Anders Widell Department of Medical Microbiology, Lund University, University Hospital, Malmö, Sweden.

  2. Hepatitis viruses - my minireview • Acute hepatiis A-E similar clinical picture; diagnostic testing a must • 5 different viruses of which 4 are RNA viruses (HAV; HCV; HDV; HEV) and one DNA (HBV) • HAV, HEV non-enveloped, fecal-oral transmission – never chronic • HBV, HCV, HDV enveloped, parenteral transmission - can become chronic • Efficient vaccines against HAV, HBV (and therefore HDV) and against HEV – but not in sight for HCV • Antiviral treatment curative in 50-80% of HCV cases, less good for HBV – intense development

  3. HAV history • Hippocrates described benign transient jaundice • Was later thought to be mucus bile plug • Massive outbreaks among US soldiers during WWII initiated the search for etiology • Experiments at Willowbrook identified MS-1 • Further human experiment at Joliet prison • Feinstone identified the HAV particle by immune electron microscopy in 1973

  4. The HAV virus • Typical picornavirus by EM • Single stranded plus RNA, replicates via minus strand intermediate • Short guest play as ”enterovirus 72” in the 1980-ies, now instead the only member of the genus Hepatovirus

  5. HAV one of the more stable virues • T50,10 61°C for HAV compared to 43°C för polio • Survives 1 year in mineral water at r.t.

  6. HAV inactivation • HAV is however inactivated by: • Heat > 85°C instantaneously • Iodine 3 mg/l 15 min at r.t. • Chlorine 2 mg/l 30 min at r.t. • KMnO4 30 mg/l 15 min at r.t. • Glutaraldehyde 0,5% 3 min at r.t. • Formalin 1/4000 72 hours at r.t. (vaccine procedure) • Inactivation unreliable in the presence of proteins • HAV is NOT inactivated by ether, chloroform, lipid detergents, acid pH

  7. HAV - genomic organization Note that 3B (VPg) moves to the 5´-UTR where it serves as a protein RNA primer Martin & Lemon, Hepatology 2005

  8. HCV protein processing • Translation is initiated via the 5´-UTR Internal Ribosomal entry site (IRES) • A polyprotein is generated and cleaved cotranslationally, using the 3A protease • However, VP1/2A is cleaved by unknown host protease • The N terminal part of 2A is engaged in pentamer formation • The short VP4 is involved in assembling pentamers into capsids

  9. The HAV virus • No crystal structure yet, Cryo EM at best • Icosahedral virion with 60 copies of each VP0 (2,4), VP3, VP1, each presumably with wedge shaped structures (CHEF-BIGD of anti-parallel beta sheets) • No canyon • Neutralization sites on outer connecting loops, mostly on VP1 - N terminal • Several NS proteins generated intracellularly: protease, RNA-polymerase, VPg

  10. HAV structure by EM (left) and by Cryo EM (right) Martin & Lemon, Hepatology 2005

  11. Putative HAV receptor • HAV receptor on cells (Havcr-1) is a class I Transmembrane Immunoglobulin-like and Mucin like glycoprotein (TIM-1) with an extracellular domain containing an N-terminal cysteine-rich region • HAV binds to TIM-1 intestinal mucosa, migrates to the liver where replication occurs in the hepatocytes – mature virions are thereafter shed into the bile and excreted with feces • This receptor has however been disputed .

  12. HAV receptor and allergy • Havcr-1 is also associated with atopia and allergy; HAV exposed have much less allergy in many countries • TIM-1 present on CD4 and other cells, strong allergy regulator • TIM-1 coded by chromosome 5, shows polymorphism (deletions / insertions)

  13. HAV - In and out of the hepatocyte Martin & Lemon, Hepatology 2005

  14. Pathogenesis (1) • HAV only infects primates • (More species promiscuous in cell culture) • Probably short duration replication in intestinal crypts • Thereafter transport to and massive replication in the liver • Replication in the cytoplasm • HAV in cell culture – no cytopathic effect

  15. Pathogenesis (2) HAV in liver cells preceeds liver injury Inflammatory cells in the liver at injury T-cell caused liver damage CD8 cells in the liver in acute hepatitis A secrete INFg which recruits both innate/adaptive cells

  16. Pathogenesis (3) • Polycolonal IgM stimulation (compare EBV) • Like HCV, HAV inhibits interferon regulatory factor 3 (IRF-3) in the RIG-I signaling pathway • Also like HCV, HAV inhibits the TLR3 (dsRNA) signaling pathway • Unlike HCV, HAV does not influence on NF-kB • And HAV infection never becomes chronic

  17. Clinical presentation • Incubation time 2-6 weeks • Prodrome with nausea, fatigue, fever • Discoloured faeces, dark urine, jaundice • In children often no jaundice • Acute self limiting hepatitis in general • Acute fulminant hepatitis (about 1 per 1000), probably more common in HCV patients • Acute relapsing hepatitis over months

  18. Diagnosis • Clinical • Contact tracing • Anti-HAV IgM (acute hepatits A) • Only HAV IgG (natural immunity/vaccination • Virus RNA in serum/plasma and feces • Polyclonal IgM

  19. HAV epidemiology • Feco-oral transmission dominant • Raw seafood • Salad, fruit e.g. raspberries, fruit ice-cream • Associated with low socioeconomic status • Close contact (family) • MSM at high risk • Childhood infection under poor hygienic conditions • In childhood often subclinical without visible jaundice • Developing countries • Day care centres with immigrant children • Life long immunity • Anti-HAV prevalence rises with age

  20. Endemic - epidemic • Three global patterns South-North gradient • Endemic – early childhood exposure, in 95% little disease - strain conserved in region • Transient phase – people in richer population groups will pass childhood without exposure – others will not – clinical cases and small epidemics may occur – in those better off • Past endemic – no youngsters except immigrants have anti-HAV (and vaccinated). Anti-HAV rises sharply in the old

  21. Is hepatitis A virus also transmitted via blood ? • During incubation and acute hepatitis, HAV is also found in plasma by PCR • Viremia periods of weeks to months have been described • Rarely transfusion hepatitis A is reported • Hep A is common in IV drug users (waves) • Experimental infection via injection very efficient • Parenteral transmission can occur during the acute phase – but how often is it important?

  22. HAV vaccine HAV propagation in cell culture has generated: • Several formalin inactivated vaccines SKF, MSD • (Attenuated vaccine, only licensed in China) • SKF’s inactivated vaccine HAVRIX given in 2 doses 4 week apart gives seroconversion >99% • MSD’s vaccine protects after 21 days into an outbreak • HAV vaccines reduce use of immune globulin – use vaccine if exposure is less than 2 weeks ago • Global HAV childhood vaccination a goal

  23. HAV control • Hygiene, hygiene, hygiene • No sewage in seafood banks, avoid human manure in fruit-salad agriculture • For tourists “Peel it, cook it or forget it” • Post-exposition • Intramuscular immunoglobulin • Vaccination efficient with < 14 (-21) days of exposure • Long term immunity by vaccination 2 doses

  24. HAV genotyping and heterogeneity • Key classification paper by Robertson et al in 1992. • First target for sequence based typing: VP1/P2A • Nucleotide changes for HAV are mainly amino-acid silent, occurring at the 3rd base positions • Transitions (C/T) and (A/G) greatly outnumbered transversions (purines to pyrimidines and rev) • The VP3/VP1 and the entire V1 and the polymerase gene have also been proposed as alternative targets

  25. Viral heterogeneity (cont´d) • Six genotypes I-VI (Former type VII is now IIB) • I, II and III infect humans • Genotype difference 15%, subtype 7% • III both human and simian • IV, V and VI purely simian, so far found in single isolates • Human HAV has subtypes IA,IB, IIA, IB, IIIA, IIIB • Genotype I most commonly identified • Only one serotype of HAV

  26. GLOBAL DATA Relationship of 3582 HAV VP1/P2B sequences by Nainan et al at the CDC

  27. Local data over 20 years in Malmö, Sweden • Analysis of circulating HAV stains in a Swedish area (sporadic, epidemic, imported) during 20y. • The presence of HAV RNA in serum drawn during the acute phase of HAV infection permitted retrospective analysis on frozen sera. • Genetic information was then be linked epidemiological data. • Suggested transmission routes could be strengthened or rejected

  28. Local data over 20 years in Malmö, SwedenMethods • Two widely used nested PCRs done in parallel • One targeting the C-terminal part of the VP1 gene (Grinde et al) • One targeting the N-terminal (Araus-Ruiz et al) • Both applied on 598 on RNA from anti-HAV-IgM positive sera, collected from all known consecutive HAV cases in the County of Skåne from 1990 and onwards and stored in the Malmö biobank.

  29. Figure 1. Hepatitis A PCR, primers in VP3/VP1 and VP1/2A systems HA1 2170-2192 HAV6 2839-2857 HA7 3003-3024 HAV7 3357-3380 1st PCR VP3 VP1 2A 2208 3107 2nd PCR HAV8 2890-2914 HAV9 3264-3286 HA3 2618-2640 2167 2166-2191 • Arauz-Ruiz et al, 2001 (JMV) • Grinde et al, 1997 (JMV)

  30. Results (1) • In this biobank, maintained at -20oC, it was possible to amplify and obtain 460 N-terminal and 422 C-terminal VP1 HAV RNA sequences. • MOST CHANGES WERE TRANSITIONS • MOST CHANGES WERE AA SILENT • Clustering was very similar between the two systems • Bootstrap values were stronger in the N-terminal system which gave both longer products (392 analyzed bp versus 309 bp) and contained more diversity (figure 2)

  31. Dominant geno- and subtypes • The large majority of samples were genotype IA and IB. • The former contained one clonal outbreak of HAV among IVDUs in 1994-5, an outbreak that was preceded by circulation of similar strains in 1991-93. • This strain later migrated to IVDUs in Southern Norway, major outbreak

  32. Results (2) • Also we found closely strains in two related outbreaks (1996 and 2004) among homosexual men spread via a gay club in Copenhagen • The latter outbreak showed two discrete strain variants these strains also belong to related strains circulating among MSM in Norway, Sweden, UK and Holland)

  33. Genotype I • Excluding the larger IVDU, MSM and family outbreaks, similar numbers of IA and IB cases occurred • Imported cases often reflected region of endemic strain (e.g. Kosovo, etc) • No endemic “Swedish” strain • Most of the IB isolates which included several family outbreaks, were from the South-East Mediterranean region.

  34. Genotype IIIA, which had dominated HAV outbreaks among Swedish IVDUs in epidemics of 1979 and 1984-5, was now rare and only seen in import cases from Pakistan and Africa. And from Estonia Primer optimization? Four type II isolates found (African strains) Less common

  35. HAV summary • HAV a unique picornavirus in a separate genus (Hepatovirus), very stable, physically, genetically • Simple diagnostic test (IgM anti-HAV) • Epidemiology clarified, feco-oral transmission and childhood infection in developing countries • Replication in the liver, non-cytopathic • Liver damage via cellular innate and adaptive mechanisms • No chronicity - life long immunity • Vaccine now available - costs?

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