HEPATITIS VIRUS

A. T. Aman, MD., MSc., SpMK, PhD.

DEPT. oF MICROBIOLOGY,
Fac. oF MEDICINE
GADJAH MADA UNIVERSITY,
YOGYAKARTA

Lecture for FM, Univ. of Tadulako, June 2015

 Introduction
1. Viral Hepatitis is an ancient diseases (5th century
BC)
2. MacCallum & Bauer (1947):
- “hepatitis A” for “infectious hepatitis”
- “hepatitis B” for “serum hepatitis”

3. This terminology: adopted by WHO (1973)

distinguish 2 infectious agents of hepatitis.
4.. The extensive study (late 20th century): many
discoveries:
*. Specific & sensitive serologic test for
Hepatitis B virus (HBV).
*. Pathogenesis and epidemiology of HBV.
*. Development of save and effective vaccine.
*. Additional agent (s): HCV etc.
 Viral Hepatitis: classification

• 1. Hepatitis A virus.
• 2. Hepatitis B virus.
• 3. Hepatitis C (1, 2 & 3 to be discussed)
• 4. Hepatitis D virus.
• 5. Hepatitis E virus.
• 6. Others are Suspected, but not confirm as
human pathogen: GB virus C/hepatitis G, TT virus
(TTV), and SEN virus.
• EPIDEMIOLOGY ■ About 44% of cases of
cases of viral hepatitis are caused by
HAV, 49% by hepatitis B (HBV), and 7%
by hepatitis C (HCV).
 II. Hepatitis B virus (1)
• A. Clinical Appearance:
– a viral infection of liver with very variable
– In most patients: subclinical, No symptoms/ jaundice.
– Other patients: symptoms, No jaundice (anicteric
hepatitis)
– 25 to 35 %: symptoms with jaundice (icteric hepatitis).
• The symptoms Vary considerably:
– From: mild & transient to severe & prolong
– Incubation: from 6 wks - 6 months
• Acute Hepatitis B:
– Most infant: No Clinical manifestation
– 1-5 yrs: 5-10% with Clinical signs
– > 5 Yrs /adult: 33- 50% with Clin. Signs
– Clinical Signs/ Symptoms: not specific
 2. Hepatitis B virus (2)
• Clinical Signs/ Symptoms: not specific
*. Symptoms: fatigue, myalgia, anorextia,
-nausea, vomiting.
*. Physical signs:
- jaundice, dark urine, clay color stool,
- hepatomegali.
- Low grade fever (< 39.50C)
*. Lasted: 1 to 2 weeks
*. Icteric: 1 month.
 2. Hepatitis B virus (3)
• Chronic Hepatitis B:
– Patients who have HBsAg post. For > 6
moths.
– Likely (majority) to remain post. For life.
– About 29%: become neg. after 12 yrs.
– Chronic active: can develop cirrhosis

• The Chronic symptoms:

– Vague and non-specific.
– HBV virion, DNA pol. Activity, HBV DNA
may be detected.
– Host factor that determine chronicity /
resolution: not known.
 Epidemiology HBV infection
*. Hepatitis B infection: one of the most
common infectious diseases in the
world.
*. WHO estimates: >2 billion people
infected with HBV at some time in their
live.
*. Survey (2000) by WHO: 350 million
people worldwide are carrier of HBV,
- the majority: in Asia and Africa.
*
 Transmission of HBV

*. The main mode of HBV transmission are: ->

vertical (mother to child).
- If the mother is HBeAg post, the risk of
transmission to infant: 90%.
- If the mother is HBsAg post., HBeAg neg.
the rate of transmission: 10%.
*. via sexual activity (homo- and hetero-sex)
• Injection drug use
• Physical contact with infected bodily fluids.
General Features of Hepatitis B virus

*. Belongs to family of Hepadnaviriday

• Partially dsDNA virus: 3,200 bp,
circular, overlapping gene.
• Has enveloped
• Complete virion: an outer envelope,
composed of surface antigen (HBsAg)
that surrounds a nucleocapsid or core.
 Virologi (1)

Classification of HBV virus:

*. HBV:
-. Family: Hepadnaviriday
-…> Hepatotropic DNA Virus.
*.Characteristic of Genome:
- DNA, ca. 3 Kb (3.200 bp).
- Relaxed circular.
- Partially duplex.
 Virologi (2)

*. HBV Virion:
- Envelop (40-42 nm): outer lipoprotein
composed of multiple glycoprotein, mainly
surface antigen (HBsAg)
- Nucleocapsid or core: 180 C-protein,
Icosahedral symetric capsids.
• Inside, core:
– one partially dsDNA (genome)
– core protein (HBcAg)
– Hepatitis e antigen (HBeAg)
– DNA polymerase
Virion- genome (3)

Virion

Core

Genome
 Virologi (4)
Subtypes:
*. Sequent polimorfisme on gene encoding
surface glicoproteins (S).
-. 4 subtypes: adw, ayw, adr dan ayr.
-. NO important differences in their biology
or pathogenesis.
-. Immunization against any subtype:
*. Cross-protective against all serotypes
*..> because commonality of neutralizing
and immunodominant a determinant.
*
 Virologi (5)
Genome:
*. A relaxed circular partially dsDNA:
- One DNA strand (Neg. strand): 3.2 Kb, the 5’end
covalently linked to virus encoded protein.
- The post.strand: 5’end is fixed, attached to an
oligoribonucleotide but the 3’end anywhere along
the genome
- - The DNA structure: important for DNA
synthesis.
- DR1 and DR2: important for DNA synthesis.

*. Contains 4 long ORFs: S/preS, C, P and X.

 Virologi (7)
ORF and Antigen (cont’d):
*. Core (C) protein:
- Two in-frame AUG at ORF coding for C protein
- 1: 21 kDA polypetide. > HBcAg
- 180-240 copies protein
- Resistant to exogenous nuclease
- 2: 5’ upstream, encodes preC protein (24kDa) with
signal for secretory pathway, after processing (via
Golgi, protease)..> 16 kDa protein (HBeAg): function
unknown, no relation with replication.
- Present in peripheral blood: viremia, high infectivity.
 Virologi (6)
ORF and Antigen:
*. Gene for S protein:
- Embedded in larger ORF
- Two upstream (& in phase) AUG initiation
codon
- They called preS region: preS1 & preS2
subregion.
- Initiation from preS, produce protein that
fuse with S protein.
- preS1 (L) protein: 39 kDa, involved in
receptor binding
- preS2 (M) protein: 31 kDa, unknown
function.
 Virologi (9)
Subviral particle:
*. Host invected cells produce: virions & subviral
particles
*. Subviral particle contains:
- Host derived lipoprotein.
- Envelope glycoprotein: mainly S protein,
with some M & L protein.
- No C/P protein
- Ratio Subviral: virion = 1000-10,000: 1.
….> S protein abundant ..> HBsAg.
- Function: unknown, May: protect virion
from anti- HBs antibodies.
 Virologi (10)
REPLICATION:
1. Incoming virion, interact with receptor, fuse
with membrane. Its host receptor (s) is
unknown.
2. Nucleocapsid present in cytosol,
transported to nucleus.
3. The genome ..> ccc DNA
4. cccDNA as template for sintesis by RNA
pol. II, produce genomic and subgenomic
transcript.
5. No integration of DNA into genome.
 Virologi (10a)
REPLICATION: DNA integration.
5a. Integrated DNA play no role in the
replication.
5b. Integrated DNA found in: hepatoma,
some chronic hepatitis, its role ?.
5c. No viral protein involve in the
integration.
5d. Might be: host-catalyzed illegitimate
recombination.
5e. Integrants: multiple rearrangement.
 Virologi (11)
REPLICATION:
6. RNA transcript transported into cytosol.
7. Translation to produce viral protein.
8. RNA packaging
9. Synthesis of viral DNA (Neg. strand).
10. Synthesis of post. Strand of DNA.
11. Transported to ER to produce virions
or to nucleus.
 Pathogenesis HBV (1)

• Severity of diseases correlates with

cellular host response to infected cells.

• There is no direct correlation between

viral load and severity of liver diseases.
 Pathogenesis HBV (1)

• Recovery of Acute HBV patients

dependent on the vigorous production of
antibody to S antigen, and vigorous CTL
response which is polyclonal and class I
restricted, reactive with HBV envelope,
nucleocapsid and polymerase region.
 Pathogenesis HBV (2)
HBV-related liver damage in acute
hepatitis, attribute to mostly to a virus
specific CTL response directed
against hepatocytes infected with
HBV.
• No clear evidence that HBV DNA is
oncogenic.
• Multiple factor may contribute to the
cancer development.
 DIAGNOSIS HBV infection

• Serologic test (RIA/ EIA) :

– Detect HBV antigens / Antibody
– Very sensitive and specific.
• Molecular Detection:
– Dot blot Hybridization technique:
detect HBV DNA
– RNA probe
– Amplification DNA using PCR.
Interpretation of test result
• The present of HBsAg: active
infection (acute/chronic)
– HBsAg detected: 3 to 5 wks before
symptoms/jaundice.
– HBV DNA: 3 to 5 wks before HBsAg
serum
• High titer (> 1: 1,000) IgM anti-HBc:
acute infection.
• Chronic Hepatitis: HBeAg, HBV DNA.
• HBeAg post.: more chance to transmit
sexually and perinatally.
 HBV TREATMENT
• No specific treatment for acute
HBV: Symptomatic and
supportive.
• Primary goal of treatment:
suppress viral replication, inhibit
progression to liver damage
before become cirrhosis.
• Antiviral: Alpha interferon; DNA
analogue.
 I. Hepatitis A virus (1)
• A. Feature:
– a viral infection of liver with very variable
– In most patients (90% <5 yrs): unicteric hepatitis.
– In adult: 40 to 70% : shows clinical manifestation.

• The Clinical Appearance:

– From: mild & transient to severe & prolong
– Incubation: from 15 to 50 days, average: 28 days.
– Incubation period is inversely correlate with the viral
inoculum size.
– Clinical Signs/ Symptoms: not specific
 I. Hepatitis A virus (2)
The symptoms (cont’d):
- Clinical Signs/ Symptoms: not specific
- Short prodromal/pre icteric phase:
several days to > a week.
- Mainly: anorexia, malayse, fatigue,
fever, abdominal pain.
- Less common symptoms: myalgia,
anthralgia, diarrhea, pruritus.
 Epidemiology HAV infection

*. Hepatitis A outbreak: first reported

about a century ago.
*. Majority affected: 5 to 30 yrs.
*. Worldwide: annual incidence: > 1.5
million cases..
*. Estimate cost: 1.5 to 3 billion US $..
* HAV is endemic, majority are infected in
developing countries.
 Transmission of HAV

*. The mode of HAV transmission:

fecal to oral.
*.Poor sanitation and poor
personal hygiene: promote viral
transmission.
General Features of Hepatitis A virus
*. Belongs to family of Picornaviriday.
• Viral shape: icosahedral.
• No envelope virion, with diameter: 27 to 28
nm.
• Genome: 7,478 nt. With single long ORF.
– Positive strand RNA.
• Resistant to environmental stress and stable
for a month. Infective after heating 10-12 hrs
at 600C.
• Resistant to ethyl ether and chloroform.
• Inactivation:
– In saline, 4 mnts, 700C
– In saline, immediately at 850C
DIAGNOSIS HAV infection (1)
• Samples:
– Blood for serologic test.
– Feces: HAV antigen, virus.
• First technique used: Immune
Electron Microscopy (IEM): difficult,
time consuming and expensive.
• Serologic test:
– Radioimmunoassay: detect HAV antigen,
early method. Now has been replaced by
ELISA.
– ELISA.
 DIAGNOSIS HAV infection (2)
• Molecular Detection:
– Molecular Hybridization technique:
Hybridization of HAV with cDNA of HAV.
• 10 fold more sensitive than Immunoassay.
RNA probe
– Dot blot hybridization: HAV-spesific
ssRNA probe.
– RT- PCR.
– For acute HAV infection: assay of anti
HAV IgM: method, solid phase antibody-
capture immuno assay.
Interpretation of (HAV) test result
• Anti HAV IgM: present in serum in early
infection to several months. Therefore,
IgM : indicate recent infection.
• Total Ig: measure IgM and IgG, IgA etc.
• IgG positive indefinitely.
• IgG positive, IgM negative ?, has
protection.
• Exogenous IgG ?.
– Transfussion ?.
Hepatitis C virus
 3. Hepatitis C virus
• Epidemiology:
– HCV distributed worldwide, ~ 170 million infected.
– Before screening: HCV was the major cause of
transfusion-associated hepatitis.
– Seen in USA and France: Infection rate decrease
with blood screening.
– Seroprevalence: ~ 3% (worldwide)
– Age distribution: most > 15 yrs
• Reinfection: No protective immunity after HCV
infection, ..> in hemophiliac: mixed infection was
detected.
General Features and Genome of HCV

.Virion Morphology
• Filtration and EM: 50 nm, with spike-like
projection.
• Core: ~ 33 nm..
• Genome: plus-strand RNA, 9,500 nt .. >
encode polyprotein 3000 a.a.
• Cleavage by host and virus encoded
protease, produce: structural prot.
(core) and 2 glycoprotein E1 and E2)
and non-struct. Prot. NS2-NS5.
 General Features and Genome of HCV

• Genome: heterogenous (quasispecies),

cause by RNA pol.
• 6 Major genotype (12-6), the dominant world-
wide are 1,2,3.
• No serotypic classification (not available yet).
• Quasispecies: envade host immune
responses.
• Patient with more homogene, more
responsive to interferon.
 HCV: from infection to HCC

• Pathogenesis:
– Incubation: 6-8 wks.
– Exposure to anti HCV can be detected (by
ELISA): 8-9 wks.
– HCV RNA detected : 1-2 wks.
– Cirrhosis: 20 % of Infection (vary)
– HCC: 20-25 % of cirrhosis
– Infection to HCC: 10-30 years.
 HCV: from infection to HCC

• Mechanism of HCV infection to cause HCC

is still uknown.
• Reinfection:
– Upon infection, the host develop antibodies
against almost all virus antigen.
– The first: anti envelop (anti E2) and anti core, at
low concentr.
– In chronic (50% of cases) infected patient,
antibodies develop after months or years.
– Infection to develope HCC: 10-30 years.
 HCV: from infection to HCC

• Prospective study: post transfussion HCV

– 20-50% develop cirrhosis.
– 5-25 % develop HCC after 10-30 years
• Retrospective study:
– Cirrhosis in 15 after 10-15 yrs.
• Other study (in Japan) :
– Persistent HCV: cause 70% HCC
– Risk to HCC: HCV RNA post 100 X than RNA
neg.
 HCV: from infection to HCC

• Reinfection and chronicity:

– Study on cimpanzees: failure to mount early,
vigorous and sustein immune response..> high
rate of chronicity.
– During chronic: reinfection with heterologous HCV.
– HCV exists in complex population.

– HCV escape from host immune surveillace by:

• Emmergence of B- & T-cell epitope.
• Masking by association of HCV-lipid.
• Produce empty particle.
Hepatitis D Virus
 HDV

• It is a blood-borne viral hepatitis.

• a ssRNA viroid called the delta agent.
• A defective virus: cannot infect a
hepatocyte without the hepatocyte also
being infected with HBV.
• To infect, HDV must be coated with
HBsAg.
 HDV
• HBV and HDV may coinfect a person
simultaneously (coinfection)
• or HDV infect a patient after infection with
HBV /chronic HBV (superinfection) ..> HDV
is found only in concurrent HBV infections.

• Patients with HBV-HDV coinfection may

have more severe acute disease and a
higher risk of fulminant hepatitis (2–20%)
compared with patients infected with only
HBV (1%).
 HDV
• Chronic HBV infection occur less frequently
in persons with HBV-HDV coinfection.
• Chronic HBV carriers who acquire HDV
superinfection usually develop chronic HDV
infection.
• Chronic HBV carriers with HDV
superinfection, 70–80% developed chronic
liver diseases with cirrhosis compared with
15–30% of patients with chronic HBV
infection alone.
• Transmission: percutaneous exposures and
are usually seen in intravenous drug users.
 .

•THANK YOU
VERY MUCH