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Contents

Overview

  • Hepatitis C virus
  • Hepatitis A virus
  • Hepatitis E virus
  • Hepatitis B virus
  • Hepatitis Delta virus

Hepatitis presentation

  • Clinical presentation: acute hepatitis with marked onset of fever, headache, weakness, myalgia and loss of appetite
  • Above symptoms appear before the onset of dark urine, which is followed by jaundice and clay-coloured faeces
  • On physical examination, patient's liver may be enlarged or tender.

Definitions

  • Hepatitis (liver inflammation): elevated serum aminotransferases (transaminases)
  • Acute hepatitis: short term and/or severe. Typical presentation - malaise, anorexia, vomiting, right upper quadrant pain
  • Chronic hepatitis: lingering or lasting >6 months, can be severe. Defined as liver function test abnormalities without cirrhosis
  • Fulminant hepatitis: develops quickly, high mortality rate.
  • Cirrhosis: scarring from infection, toxins, etc
  • Jaundice: Yellowing of the skin/eyes due to raised levels of bilirubin in the blood due to liver damage.

Viral hepatitis

  • A number of agents can cause hepatitis including : drugs, autoimmune disease, a secondary manifestation of systemic illness, connective tissue disease, and viruses.
  • As well as the hepatitis viruses, many viruses are capable of causing liver inflammation (e.g. EBV, CMV, HSV, HHV6 (herpes), yellow fever virus).

Hepatitis viruses

Hepatitis virus Genomic material Transmission Incubation period Mean (days) Chronic infection? Virus classification
A RNA Enteric 2-6 weeks 30 No Picornavirus
B DNA Parenteral 1-6 months 112 Yes Hepadnavirus
C RNA Parenteral 1-5 months 49 Yes Flaviviridae
D RNA Parenteral 2-6 weeks 21 (if HBV positive) Yes Unclassified (satellite virus)
E RNA Enteric 3-6 weeks 38 No Calcivirus-like

Notes

  • Enteral/enteric/faecal-oral = eat/drink the virus. Often there is contamination of food or water source with viral particles that exited people in their stools.
  • Parentral = blood-to-blood (includes mother-to-baby: vertical) and sex
  • Parenteral = B, C, D
  • Enteric = A, E
  • Enteric = acute infection only (no chronic)
  • Parenteral = both acute and chronic disease.
  • Hep D is unclassified - a unique virus
  • Incubation period = how long it takes from getting it to being sick.
    • Shorter ones = enteric transmission (weeks)
    • Longer ones = parentreral transmission (months)
    • Exception: Hep D -- can only get it if you already have Hep B or you co-transmit Hep B and D.
  • RNA virus = genomic material made up of RNA, made into protein.
  • DNA virus = transcribed to RNA, which is then made into protein

Further virus information including serum diagnosis

HAV HBV HCV HDV HEV
Genome ssRNA dsRNA ssRNA ssRNA ssRNA
Family Picorna Hepadna Flavi Delta Calici
Transmission Fecal/Oral Blood/Sex Blood Blood Fecal/Oral
Serum Dx IgM(+) core IgM Ab(+) IgM(+) IgM(+)
Peak ALT 800-1000 1000-1500 300-800 1000-1500 800-1000
Chronicity No 3-10% ~75% Yes No
HCC No Yes Yes Yes No

Note

  • This forms the basis of a very important tutorial on hepatitis virus diagnosis.
  • Antibody testing (serological) is used for diagnosis. Generally within 1-2 weeks of exposure to pathogen, you make IgM that stay for weeks-months, disappear in a year.
  • IgG is slower to make, retained for life.
  • Can use either of these (acute phase=IgM or seroconversion=IgG).
  • Viruses in general are hard to grow in the lab, so we use antibody tests the most.
  • We have IgM tests for all but C (determine recent infection)
  • Acute hepatitis: ALT = 50x-100x normal
  • Chronic is lower ALT

Estimates of Acute and Chronic Disease Burden for Viral Hepatitis, United States

HAV HBV HCV HDV
Acute infections (x1000)/year 125-200 140-320 35-180 6-13
Fulminant deaths/year 100 150 ? 35
Chronic infections 0 1-1.25 million 3.5 million 70,000
Chronic liver disease deaths/year 0 5,000 8-10,000 1,000

Notes

  • Few HAV infections every year (at least that are recognised; but can occur in clusters/outbreaks)
  • HBV/HCV are also causes of acute infections. Most HCV infections are asymptomatic.
  • Fulminant = HAV, HBV, HDV (not really HCV).
  • The chronic infections are a major cause of morbidity for HBV, HCV, HDV

Hepatitis C virus

History

  • Following the discovery of HAV and HBV, a third type of infectious hepatitis was recognised that was responsible for most cases of post-transfusion hepatitis
  • Virus was finally identified in 1989 by Choo et al.
  • The discovery is an elegant demonstration of molecular virology
  • Main cause of non-AB hepatitis.
  • Molecular cloning of virus -- made guesses about the genes and then checked that they got it right (didn't have a way to sequence it at the time). Novel method at the time.

Introduction

  • Member of Flaviviridae (including GBV-C/HGV, yellow fever virus, Dengue fever virus)
  • Family:Flaviviridae
  • Genera:
    • flavivirus, e.g. yellow fever virus,
    • pestivirus, e.g. bovine viral diarrhoea virus,
    • hepacivirus, HCV (only member)
    • GBV-C (HGV) unclassified
    • HCV is a unique member of this genus based on evolutionary studies.
  • Approximately 200 million chronic carriers world-wide
  • In Australia, India and US approximately 1-2% of the population are infected with HCV
  • Australia --250,000 250,000
  • United States 4,000,000
  • India-15,000,000
  • Most common reason for liver transplant (~50% in Australia and US)
  • There are 6 genotypes of the virus which demonstrate some geographical distribution.

Epidemiology

  • Most people don't know they have it. About 1% of Australia have it.

10,000 new cases every year in Australia.

  • Hep C has become the leading cause of liver transplantation. Large and growing burden of disease
  • Approximately 200 million chronic carriers world wide and in Australia there are 260,000 people infected with HCV
  • There are 66 genotypes of the virus which demonstrate some geographical distribution.
  • In Australia and US HCV infection is now the most common reason for liver transplantation

HCV genotypes - world view

4HMBPath1.png

  • Very variable genotypes worldwide - its strains change.
  • 4 in egypt due to past vaccination. Everywhere else - global spread.
  • South Africa genotype--5, Vietnam 5, Vietnam--6 and Egypt 4
  • Genotype 1 is by far the most common genotype
  • Genotype 3 is the second most common genotype accounting for around 20% of infections

HCV epidemiology in Australia

HCV epidemiology in Australia
  • Distribution of HCV genotypes in Australian patients -- subtype subtype 3a is more prevalent than other developed countries
  • Pie chart showing genotypic distribution of Australian HCV infected patients. PCR products were amplified from 296 HCV infected patients and sequenced.

Transmission

  • Can't be transmitted by intermediate vector (digested by mosquitoes etc) - must be from blood-to-blood. This is the link to the major risk group - injecting drug use. In the past, transmitted this via blood products and organ transplants, but this has nearly completely stopped since screening.

Can be transmitted vertically (maternal blood and broken skin on the baby). Sexual transmission: rare, almost exclusively homosexual men and cotransmission with HIV.

  • Not really associated with sexual transmission except in rare circumstances.
  • Route of infection --parenteral is the most prevalent, unlike

other flaviviruses HCV is not transmitted by arthropod vectors

  • High rates in IVDU, haemophiliacs, recipients of unscreened blood transfusions
  • Screening of blood products (from 1990) has greatly reduced transmission of HCV.
  • Sexual and mother--to--baby transmission does occur, but is not common –– eg Low prevalence (1-5%) among long-term partners

Sources of infection for persons with hepatitis C

4HMBPath3.png

Perinatal transmission of HCV

  • Transmission only from women HCV-RNA positive at delivery
    • Average rate of infection 6%
    • Higher (17%) if woman co-infected with HIV
  • No association with
    • Delivery method
    • Breastfeeding
  • Infected infants do well
    • Severe hepatitis is rare

EM of HCV

  • Morphology: Spherical, lipid envelope virus, diameter ~50nm

Genome and protease cleavage

HCV genome and protease cleavage. Some of these proteins are targets of our antiviral therapies
  • Virus genome: positive-sense RNA molecule 9.5kb
  • Translated as a large 3000 residue polypeptide which is then cleaved by both cellular and viral proteases
  • Some of these proteins are targets of our antiviral therapies
  • Nucleotide sequence highly variable, the most divergent isolates share 60% nucleotide sequence
  • Up to 5% sequence difference between HCV RNA molecules is present in the blood of a single individual at any one point -- termed quasispecies
  • Cope with changes (adaptation) through diversity
  • Due to this:
    • 1) By making mistakes, it helps them evade the immune system (selected for hypervariability). Allows chronic infection to occur
    • 2) Very hard to vaccinate (even harder than HIV)

Primary infection

  • Viremia - highest at onset of hepatitis
  • Antibodies appear 6-12 weeks after the onset of hepatitis
  • Spontaneous resolution of chronic liver disease rare (<2%)
  • Liver damage is immunologically mediated and is thought to be initiated by activation of virus specific CTL

Disease course (untreated)

4HMBPath5.png

Serologic pattern of acute HCV infection with recovery

4HMBPath6.png

  • Serologic pattern of acute HCV infection with recovery.
  • Good news for this person! No virus present in the blood, liver function test normal, cleared infection. This happens 30% of time.
  • Mostly symptomless. Viraemia precedes the development of the acute illness -- can identify acute infection before the antibodies have reached max.

Serologic pattern of acute HCV infection with progression to chronic infection

4HMBPath7.png

  • 70% of people develop chronic infection

Diagnosis

  • Serology (not useful in acute phase
    • EIA measures IgG directed against recombinant HCV proteins
    • Measures infection not immunity
  • Answer: PCR (qualitative or quantitative) - especially in acute infection

Viral load

HCV load - frequency distribution
  • HCV load - a predictor of response to therapy
    • Patients with low titre more likely to respond
  • Long-term response can be correlated to viral dynamics on therapy

Management for chronic HCV infection

  • Assess for biochemical evidence of CLD
  • Assess for severity of disease and possible treatment
    • Vaccinate against hepatitis A and B
  • Counsel to reduce further harm to liver
    • Limit or abstain from alcohol

Treatment with interferon (IFN)

  • Response outcomes
    • peg-interferon + ribavirin
    • sustained response 50-80%
  • Interferon can reduce the chance of progression to HCC
  • Viral factors associated with the likelihood of IFN response
    • Genotype other than 1 (1b)
    • Small number of quasispecies
    • Low viral load
  • Virological non-response can be predicted at week 12 based on viral load (<2 log loss)
  • In future - positive predictive value of response may be determined as early as week 4

Antiviral therapy

  • Antiviral therapy
  • Marketed: Ribavarin, PEG-IFNa
  • Phase 3: protease inhibitor, polymerase inhibitor, other IFNa
  • Will be a curable infection within a decade or so
  • There is no vaccine for HCV.

NEW SLIDE HERE: diagram

Hepatitis A virus

History

  • Infectious/epidemic hepatitis known for centuries, recorded in China several thousand years ago
    • Called infectious for historical reasons. Common in places where clusters of people could be infected.
  • Associated with wars e g “jaunisse des camps” during the Napoleonic Wars
  • The virus was first isolated by Purcell in 1973
  • Not long after that, antibody tests for diagnosis were developed

Epidemiology

  • HAV -common cause of hepatitis in developing countries
  • HAV is very contagious, spread by faecal-oral route. Outbreaks frequently associated with consumption of shellfish
  • Large epidemic in 1988, Shanghai, China -310,746 cases
  • Clinically, very variable -Asymptomatic; 25-50% adult infections; but >90% childhood infections
    • High subclinical/clinical ratio : a minor proportion will be symptomatic (this is age-dependent: in 0-5 you don't notice it). Therefore in the developing world, it is almost always transmitted asymptomatically; so they're nearly all immune protected -- but if you're not seroconverted and you get it at age 20, there are symptoms.

Geographic distribution of HAV infection

4HMBPath8.png

  • Varies by race etc because aboriginals have higher transmission rate

Classification

HAV classification
  • In vitro, grows in a variety of cell lines, but rather poorly
  • HAV is a HAV is a Picornavirus, formerly classified in the genus Enterovirus
    • Small RNA virus. Can grow it; but it's difficult to do this
  • Sequence homology shows it does not belong in this genus and it is classified in a genus of its own: Hepatovirus
    • Initially thought it was an enterovirus, but sequencing revealed they are in different viral genus within the family of Picornaviruses (pico RNA virus: small virus)

Clinical importance of HAV

  • Incubation period from 10 -50 days
  • Self-limited with no chronic viremia
  • 99% cases recover, death in ~0.1% cases - rare fulminant cases
  • Liver damage occurs after viral replication has stopped
  • Some cases experience permanent liver damage
  • Virus establishes disease in hepatocyte, replicates, causes some degree of cytolysis, but most injury occurs because of the immune response: T cells etc have friendly fire.

Morphology and genome

  • Single-stranded, positive linear RNA molecule, 7478 nucleotides
  • Genome structure similar to other Picornaviruses
  • 732 bp 5’ untranslated region precedes the AUG translation start codon
  • Single long open reading frame of 6681 nucleotides that encodes a polyprotein of 2227 aa
    • Single, long, open reading frame. Makes the whole thing then chops it up into little bits.
  • Hepatitis A virus
    • 28 nm in diameter
    • non enveloped
    • spherical particles

Genome and protease cleavage

4HMBPath10.png

  • Contains three functionally distinct segments
  • Encodes a series of protein which are important for its own machinery. Encodes a polymerase that allows it to copy itself; makes proteases to help chop up the polypeptide that it synthesises all in one go.

Diagnosis

  • HAV is not distinguishable from other forms of viral hepatitis
    • Acute is acute w.r.t. symptoms -- need to do an assay.
  • Liver function tests ALT/ASTALT/AST are sensitive measures of liver damage
    • ALT>AST in viral, but can't distinguish acute HAV from acute HBV, HDV etc
  • Enzyme immunoassays -detection of IgM antibody specific to HAV - indicates ongoing or recent infection
    • Look for IgM antibodies to virus, or do an acute and convalescent IgG assays to identify seroconversion.

Vaccines

  • Inactivated HAV vaccine (Havrix or Vaqta)
    • Vaccine has been around for 15 years. Administered in a 2-dose schedule, with a booster at 1-5 years for lifelong immunity
    • Protective efficacy rate 95--100% of recipients have protective levels of 100% of recipients have protective levels of antibody within 1 month of receiving first dose
    • Nearly 100% immunogenic in healthy subjects -- in published studies, 94%-100% of children protected against clinical hepatitis A after after equivalent of one dose
  • Protective antibodies seen by 15 days in 85%
    • Safe, well tolerated
    • Dose & Schedule --2 doses at 0 and 6-12 months
  • Prevention of HAV infection with normal human immune globulin (IG) 0.02 ml/kg IMI (90% effective for 2-6 months)
  • Can also give passive immunisation for the lifespan of the infection, if you don't seroconvert and you need the antibodies.

Hepatitis E virus

Introduction

  • Important cause of enterically transmitted hepatitis
  • In developed countries, cases usually have history of travel to HEV-endemic areas
  • Normal course of infection: acute illness similar to HAV, except during pregnancy (third trimester) - 15-30% mortality
  • HEV = big cause of epidemic. Unique association with pregnancy - pregnant women with HEV get a very severe illness with high mortality rate. Exact mechanism not known.
  • Not endemic in Australia.
  • We have good diagnostic immunoassays.

Diagnosis

  • HEVAb EIA can detect IgG and IgM

Geographic distribution

4HMBPath11.png

Epidemiology

  • HEV is epidemic in Africa India Asia Middle East South America but not in Australia
  • Minimal person to person transmission - the virus is less contagious than HAV
  • Large epidemics are associated with faecal-oral (waterborne) spread
  • Host range different to other hepatitis viruses, chimps, several monkey species and pigs are susceptible to HEV infection --(rats and other animals could be vectors)
  • Enterically transmitted
  • Only causes acute infection
  • Doesn't cause chronic disease
  • Epidemics occur because of contamination of water sources.
  • There are close relatives that infect pigs etc. The virus recombines between the pig and human to evolve over time, but the main cause of human HEV is from people

Hepatitis B virus

Geographic distribution of chronic HBV infection

4HMBPath12.png

  • Thailand: 10% of babies born to mothers with HCV, if not immunised, will get it

Electron microscopy

  • HBV: DNA virus, transcribed to RNA then to protein.
  • Get diagram from the newslides.
  • 'Serum hepatitis' was distinguished clinically from 'infectious

hepatitis' in the 1930's

  • Virus transmitted
    • Serum --blood transfusions, transplants, IVDU blood transfusions, transplants, IVDU
    • Sexually
    • Mother to child
  • Incubation period 45-120 days
  • Now it is sequences and we can find out who has it.
  • Blood-blood transmission.
  • 10% carriage rate in NZ. Horizontal transmission = person-to-person in a household (scuffles, sharing toothbrushes, etc). Vertical transmission also occurs.

Morphology

HBV morphology
  • HBV is the prototype member of the family Hwpadnaviridae
  • Spherical enveloped particles 42-47nm diameter
  • Have two uneven strands of DNA:
    • (-) sense strand, 3.0 - 3.3kb
    • (+) sense strand, 1.7-2.8kb

Genome

HBV genome.
Very complex replication process. Reads in overlapping reading frames - blocks our understanding
  • All have same polarity, same 3' ends but different 5' ends
  • 4 known genes:
    • C - core protein C
    • P - polymerase
    • S - 3 polypeptides of surface antigen (preS1, preS2 and S) from alternative translation start sites
    • X - transactivator of viral transcription
  • Very complex replication process. Reads in overlapping reading frames - blocks our understanding.

NEW SLIDE HERE: Serology of HBV infection

  • HBsAg - indicates HBV present in body - a protein of the virus itself
  • HBeAg - active viral replication
  • HBV DNA - same
  • Anti-HBs (HBsAb) - indication of immunity (surface antibody)
  • Anti-HBe - coupled with loss of HBeAg, indicates seroconversion
  • Anti-HBc (IgM) -develops after acute infection
  • Anti-HBc (IgG) - develops after acute infection and may persist indefinitely
  • Core things - no antibodies for it (on the core)
  • E antigens = large amounts of viral replication. Antibodies for E indicate that the immune system is controlling it.

Pathogenesis

Hepatitis B pathogenesis.
  • HBV infection has 3 possible outcomes:
  1. Acute course, recovery and immunity (>90%)
  2. Chronic infection - carrier state with virus persistence (~10%)
  3. Fulminant hepatitis (~1%) with liver failure and death (80% of fulminant cases)

4HMBPath16.png

NEW SLIDE HERE: natural history with arrows

  • Natural history - depends on age.
  • From mother: low chance of acute, high chance of chronic. Unless given antibodies or vaccination to protect them.
  • But if you get it as an adult, less likely to have chronicity (more likely to be asymptomatic).
  • In those who do get chronicity have a very strong correlation with cirrhosis and liver failure/hepatocellular carcinoma (30 fold with C, 180 fold with B)

NEW SLIDE HERE: natural history with graphs

  • If you acquire infection vertically, the immune system relatively ignores the virus (have antibodies, not much T cells, not much inflammation, heaps of virus present, lots of antigen present, extends up to 20 years old). Get to a point where the immune system becomes active -- immunoactive phase. Good part: want to get rid of virus Bad: don't want to destroy the liver.
  • End up with flares of hepatitis, and very rapid fibrosis. Once you've got cirrhosis, the virus burns out a little bit (quiescence), but still present in the blood.

NEW SLIDE HERE: antiviral therapy for HBV

  • can't cure it. Want to control it or suppress it. Can give people things that slow replication.
  • There are also life-long antiviral drugs. Resistance of the virus to these drugs is very rare.

Treatment of chronic HBV infection

  • Currently approved therapies:
    • Interferon alpha
      • 5MU daily or 10MU three times weekly for 16 weeks
    • Lamivudine
      • 100mg daily for 12 months
    • Adefovir dipivoxil
      • "Rescue" therapy for patients with LMV resistant HBV

Prevention

  • Purified HBsAg from the blood of chronic carriers was originally used as a vaccine
  • Recombinant HBsAg vaccine produced in yeast now used - safe and reasonably effective
  • Effective vaccination campaigns:
    • Save ~1 million lives per year
    • Could eradicate the virus
  • Very good vaccine so far.

Hepatitis Delta virus

  • In 1977, a novel nuclear antigen was discovered in patients with chronic HBV infection -- called delta antigen (after the 3 known HBV antigens)
  • HDV is a defective transmissible pathogen dependent on HBV for replication. ~10 million infections world-wide.
  • Delta antigen found wherever HBV infection occurs and may

potentiate the effects of HBV infection

  • Fulminant hepatitis (mortality ~80%) is 10X more common than with HBV infection alone
  • The RNA appears to be packaged into a particle composed of L-HDAg and small-form hepatitis B surface antigen (HBsAg) of HBV - similar but not identical in structure to "Dane particles" (36-38 nm)

Features

  • Taxonomically placed in its own group within the class V negative sense RNA viruses, Deltavirus
  • HDV encodes a protein (the delta antigen) whose function is not known, but may be associated with the RNA genome
  • Circular negative sense ssRNA genome 1.7kb
  • No vaccine. HDV can be controlled by controlling HDV infection
  • Infection is a result of co-infection or superinfection

Summary

  • Memorise the tables describing the viruses (above)
  • Take home: RNA virus, DNA virus, Enteric, parenteral, weeks-months.

Quick quiz:

  • Which are transmitted person-person?
    • All
  • Which are enterally transmitted?
  • Chronic liver disease?
  • Curable with treatment?
    • Not A or E
  • Preventable by immunisation?
    • A,B,D,E