Hepatitis A

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Hepatitis A

• Anders Widell
• Department of Medical Microbiology, Lund
  University, University Hospital, Malmö, Sweden.

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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

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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

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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

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HAV one of the more stable virues

• T50,10 61C for HAV compared to 43C för polio
• Survives 1 year in mineral water at r.t.

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HAV inactivation

• HAV is however inactivated by
• Heat gt 85C 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

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

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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

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

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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)

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

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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

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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

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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

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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

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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

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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

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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?

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HAV vaccine

• HAV propagation in cell culture has generated
• Several formalin inactivated vaccines SKF, MSD
• (Attenuated vaccine, only licensed in China)
• SKFs inactivated vaccine HAVRIX given in 2
  doses 4 week apart gives seroconversion gt99
• MSDs 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

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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 lt 14 (-21) days of
  exposure
• Long term immunity by vaccination 2 doses

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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

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Viral heterogeneity (contd)

• 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

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

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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.
  

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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)

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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

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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)

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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.

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Less common

• 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)

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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?