Hepatitis A

Hepatitis AHepatitis A

Anders WidellAnders Widell Department of Medical Microbiology, Lund Department of Medical Microbiology, Lund

University, University HospitalUniversity, University Hospital, , Malmö, Malmö, SwedenSweden..

Hepatitis viruses - my minireviewHepatitis 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

HAV historyHAV 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

The HAV virusThe 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

HAV one of the more stable HAV one of the more stable viruesvirues

• T50,10 61°C for HAV compared to 43°C för polio

• Survives 1 year in mineral water at r.t.

HAV inactivationHAV 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

HAV - genomic organizationHAV - genomic organization

Note that 3B (VPg) moves to the 5´-UTR where it serves as a protein RNA primer

Martin & Lemon, Hepatology 2005

HCV protein processingHCV 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

The HAV virusThe 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

HAV structure by EM (left) and by Cryo EM (right)


Putative HAV receptorPutative 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

.

HAV receptor and allergyHAV 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)

HAV - In and out of the hepatocyte


Pathogenesis (1)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


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 INFwhich recruits both innate/adaptive cells


• 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

Clinical presentationClinical 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

DiagnosisDiagnosis

• Clinical

• Contact tracing

• Anti-HAV IgM (acute hepatits A)

• Only HAV IgG (natural immunity/vaccination

• Virus RNA in serum/plasma and feces

• Polyclonal IgM

HAV epidemiologyHAV 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

Endemic - epidemicEndemic - 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

Is hepatitis A virus also Is hepatitis A virus also transmitted via blood ?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?

HAV vaccineHAV 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

HAV controlHAV 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

HAV genotyping and heterogeneityHAV 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

Viral heterogeneity (cont´d)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

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

Local data over 20 years in Malmö, SwedenLocal 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

Local data over 20 years in Malmö, SwedenLocal data over 20 years in Malmö, SwedenMethodsMethods

• 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.

Figure 1. Hepatitis A PCR, primers in VP3/VP1 Figure 1. Hepatitis A PCR, primers in VP3/VP1 and VP1/2A systemsand VP1/2A systems

HA12170-2192

HA73003-3024

HAV62839-2857

HAV73357-3380

VP3 2A

HA32618-2640

21672166-2191

HAV82890-2914

HAV93264-3286

1st PCR

2nd PCR31072208

VP1

Arauz-Ruiz et al, 2001 (JMV) Grinde et al, 1997 (JMV)

Results (1)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)

Dominant geno- and subtypesDominant 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

Results (2) 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)

Genotype IGenotype 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.

Less commonLess 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)

HAV summaryHAV 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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Hepatitis A