Hepatitis viruses

Dr. Tarek Mahbub Khan
MBBS, M.Phil (virology)
Assistant Professor

Learning objectives
• Definition of hepatotropic virus and examples
• Outline of classification and characteristics of
hepatitis viruses
• Structure, epidemiology, pathogenesis, clinical
outcome and laboratory diagnosis of:outcome and laboratory diagnosis of:
– Hepatitis A virus
– Hepatitis B virus
– Hepatitis C virus
– Hepatitis D virus
– Hepatitis E virus
– Hepatitis G
14/1/2018 2Dr.Tarek/tarekviro@yahoo.com/2015

• Viral hepatitis is a systemic
disease caused by hepatitis
viruses that produce acute
inflammation of the liver
clinically characterized by
VIRAL HEPATITIS
clinically characterized by
fever, gastrointestinal
symptoms like nausea,
vomiting and jaundice.
14/1/2018 Dr.Tarek/tarekviro@yahoo.com/2015 3

HEPATOTROPIC VIRUSES
• Viruses which infects hepatocytes are hepatotropic
• Two broad classification:
• Primary hepatotropic : Infects liver cell primarily:• Primary hepatotropic : Infects liver cell primarily:
– e.g., Hepatitis A, B, C, D, E and G viruses
• Secondary hepatotropic : Hepatitis occurs as a
consequences to infection of other organs:
– e.g., CMV, Yellow fever virus, HSV, Dengue viruses

AN OVERVIEW OF HEPATITIS
VIRUSES
Viruses Genome On-set of
infection
Transmission Chronicity
HAV RNA Children Feco-oral No
HBV DNA Adult, Parenteral, YesHBV DNA Adult,
children
Parenteral,
Sexual, Vertical
Yes
HCV RNA Adult Parenteral, sexual Yes
HDV RNA Adult Parenteral, sexual Yes
HEV RNA Adult Feco-oral No

HEPATITIS A VIRUS
(cause infectious hepatitis)
• Hepatitis A is non-envelop RNA virus
• ssRNA genome
• Possess seven genotypes (only one serotype)
• Virus family: Picornaviridae
• Genus: Hepatovirus
• Can be killed by autoclaving, boiling for 5 minutes,
heating food (1850 F for 1 minute), disinfection by
bleach (1:100 concentration)

• Incubation periods: 10-50 days
• Children are asymptomatic (80-95%) in relation to adult
(10-25%)
• Viremia : Transient• Viremia : Transient
• Virus in stool: 2 weeks before and 2 week after jaundice
• Onset of infection is abrupt
• ALT level remains high up to 1-3 weeks
14/1/2018

Epidemiology of HAV infection
• Transmission is mainly fecal-oral route. Sexual and
intravenous route is not common
• Common in families, institutions, summer camp, day• Common in families, institutions, summer camp, day
care, over crowded and poor sanitary conditions
• Sources of infections: Raw oysters, undercooked foods,
frozen strawberries and non-human primates

• Virus attaches with a glycoprotein receptor (HAV cell
receptor 1 glycoprotein-HAVCR1) on hepatocyte
• HAV itself is not cytopathic
• Cellular immunity particularly CD8+T cell , plays a key• Cellular immunity particularly CD8+T cell , plays a key
role in cell injury
• IgM appears in blood at the onset of symptoms
• IgG confers lifelong immunity against all strains
14/1/2018

Immunological and biological events in HAV infection

Laboratory diagnosis
• SAMPLES: Blood, Stool, bile , liver
• COMON METHODS: ELISA, PCR, Hybridization
• SEROLOGICAL MARKERS (Anti-HAV):• SEROLOGICAL MARKERS (Anti-HAV):
• IgM: Acute infection, decline to non-detectable range by 3-6 months
• IgG: Indicates past infection, detected even after decades

Management
• TREATMENT: Supportive
• PREVENTION:
• Improvement of personal hygiene and sanitation
• Treatment with 0.5% sodium hypochloride solution• Treatment with 0.5% sodium hypochloride solution
• Vaccination: Formalin inactivated vaccine
• HAV Ig can be given to person within 2 weeks of infection

Because
– Most infected people are
contagious 10-14 days before
symptoms occurs
Why hepatitis A spread
readily in a community?
symptoms occurs
– 90% of the infected children
and 25%-50% of infected
adults have inapparent but
productive infections

HEPATITIS B VIRUS
(serum hepatitis)
• DNA envelope virus
• Icosahedral capsid
• Family: HepadnaviridaeFamily: Hepadnaviridae
• dsDNA genome with incomplete positive strand
• Virus posses reverse transcriptase that acts in later part of
replication
• DNA integrates with host cell DNA by enzyme integrase

HOSTS OF HEPATITIS B VIRUS
Woodchuck
Ground squirrel
Duck
Human

Basic structure of hepatitis B virus
•Antibodies are formed against all types of viral antigens
•HBsAg is a part of the viral envelope

HBVParticleHBVParticle

Electron microscopic picture of HBV

STRUCTURE OF HEPATITIS B VIRUS
• DANE PARTICLE: A 42 nm complete virion of HBV
• VIRAL CORE:
– Contains a partially double stranded DNA of only 3.2 kbp
– Enzymes: Protein kinase and DNA polymerase having both
reverse transcriptase and ribonuclease H activityreverse transcriptase and ribonuclease H activity
– A P protein attached to its genome
– An icosahedral capsid formed by hepatitis B core antigen (HBcAg)
– HBeAg shares most of its protein sequence with HBcAg
• VIRAL SURFACE:
– An envelope containing three forms of glycoprotein hepatitis B surface
antigen (HBsAg)

HEPATITIS B VIRUS GENOME
‘S’ gene: HBsAg
Four open reading frame (ORF)
‘P’’ gene: Polymerase Enzyme
‘X’ gene:
Transactivation of transcription
‘C’ genes: HBcAg, HBeAg

HEPATITIS B SURFACE ANTIGEN
• HBsAg containing particles are released into the serum and
outnumber the actual virion.
• Originally termed as Australian Antigen
• This antigen is immunogenic
• HBsAg includes three glycoproteins (L, M and S)• HBsAg includes three glycoproteins (L, M and S)
• S glycoprotein is the major component of HBsAg particle
• The glycoproteins of HBsAg have group specific and type
specific determinants:
– Group specific determinants: ‘a’
– Type specific determinants: ‘d’ or ‘y’ and ‘w’ or ‘r’
• Combination of these antigens forms eight subtypes of HBV

HBV REPLICATION
• HBV replication is unique because:
– Defined tropism for liver
– Small genome
– Replicates through an RNA intermediate– Replicates through an RNA intermediate
– Produce and releases antigenic decoy particles
(HBsAg)

ATTACHMENT AND ENTRY
• ATTACHMENT to the hepatocytes is mediated by HBsAg
glycoprotein and several liver cell receptors:
– Transferrin receptor
– Asialoglycoprotein receptorAsialoglycoprotein receptor
– Human liver annexin V
• ENTRY: HBsAg binds to polymerized human serum albumin
and other serum proteins that may facilitate virus uptake by
the liver

TRANSFER OF CORE TO THE NUCLEUS
• HBV core is transfer to the nucleus probably through microtubules.
• Capsid deliver the genome alongside with its own RNA polymerase
inside the nucleus.
• DNA REPAIR: incomplete positive strand is made complete with host
DNA polymerase and a cccDNA of HBV is formed.DNA polymerase and a cccDNA of HBV is formed.
• EARLY TRANSCRIPTION: This cccDNA is transcribed to several different
length mRNA (pre-genomic RNA) by virion RNA polymerase:
– 3500- base mRNA: encodes HBcAg, HBeAg, DNA polymerase and primer
– 2400-base mRNA : encodes large (L) HBsAg glycoprotein
– 2100-base mRNA : encodes medium (M) and small (S) glycoproteins
– 900-base mRNA : encodes the ‘X’ protein

EXIT OF THE mRNA TO THE
CYTOPLASM
• mRNA exit to the cytoplasm through nuclear pore
• mRNA is read on host cell ribosome to translate different
proteins including DNA polymerase
• FUNCTIONS OF HBV DNA POLYMERASE:• FUNCTIONS OF HBV DNA POLYMERASE:
1. Priming: start synthesizing a negative DNA strand from the RNA
strand
2. Ribonuclease H activities: Cleaves the RNA strand that has been used
to synthesize DNA strand except a little RNA at the 3’ region of mRNA
3. Completion of genome synthesis

FINAL STEPS IN REPLICATION
• ENCAPSIDATION:
– 3.5 kb-base RNA (pre-genomic RNA) is encapsidized by structural
protein (HBcAg)
• SYNTHESIS OF NEGATIVE STRAND:
– Use 3.5 kb RNA as template
– Catalyst by DNA polymerase with reverse transcriptase activity.– Catalyst by DNA polymerase with reverse transcriptase activity.
– RNAase H will cleave the initial RNA template except few nucleotides
• SYNTHESIS OF POSITIVE STRAND:
– The small RNA primer at the 5’ end of the newly synthesized negative
DNA strand will start synthesizing positive strand
• RELEASE:
– Provirus traveled through ER and Golgi body and acquire its envelope
and released by budding

REPLICATION OF HBV

HEPATITISB PREVALENCE

SPREAD OF HBV IN THE BODY

PATHOGENESIS OF
HEPATITIS B INFECTION
• Transmission: Parenteral, Sexual ,Vertical
• Liver damage:
1. Virus reaches the hepatocytes and attached by receptor and
displayed on cell surfacedisplayed on cell surface
2. Cytotoxic T lymphocyte mediates an immune attack against
viral infected cells
3. Results in inflammation and cell necrosis –Hepatitis
4. Antigen-antibody complex can be deposited in different organs
and produce inflammation (eg. Arthritis,vasculitis)

CLINICAL OUT COME OF

OUTCOMES OF AGE OF
INFECTION

CLINICAL FEATURES
• Two clinical types
1. Acute hepatitis
2. Chronic hepatitis
• Incubation period: 10-12 weeks• Incubation period: 10-12 weeks
• Many cases are asymptomatic
• Fever, anorexia, nausea, vomiting and jaundice
• Dark urine, pale feces, arthralgia, arthritis, rash
• Most chronic carriers are asymptomatic

CLINICAL AND SEROLOGICAL EVENTS OF
HBV

SIGNIFICANT OF HBV
SERO-MARKERS
Viral markers Significance
HBsAg Acute infection, if persists for more than six
months: chronic infection
Anti-HBs Protection: by natural infection or vaccination
Anti-HBcAg IgM: Acute infection
IgG: Past or chronic infection
HBeAg Active viral replication and high chance of
transmissibility
Anti-HBeAg Disease recovering
HBV-DNA Active viral replication and high chance of
transmissibility

LABORATORY DIAGNOSIS
1. Sample: Blood(serum plasma)
2. Serology:
– Methods: Agglutination test, ICT, ELISA
– Acute infection: HBsAg, anti-HBcAg (IgM), Anti-HBs
– Chronic infection or carrier: HBsAg (>6 months), Anti-HBcAg,
HBeAg(active viral replication)HBeAg(active viral replication)
– Window period: Anti-HBcAg (IgM)
3. PCR:
– HBV-DNA in acute and chronic infection. Used for prognostic
parameter. Viral load >105 copies/ml: Active infection
4. SGPT, Serum bilirubin: Increased

MANAGEMENT OF
A. Acute infection: Supportive treatment
B. Chronic hepatitis B infectionB. Chronic hepatitis B infection
– Alpha interferon, Lamivudin, Adefovir
TREATMENT

C. Prevention strategy:
– Safe blood transfusion, safe sex, elective Caesarean
section
– Vaccines: Recombinant Hepatitis B vaccine contains
HBsAg antigens
PREVENTION
HBsAg antigens
– Prophylaxis: Hepatitis B immunoglobulin after accidental
exposure( eg. Needle stick injury)
– Newborn in infected mother: Both vaccine and HBIG
– Elective cesarean section

COMPLICATION OF
• Cirrhosis of liver
• Hepatocellular carcinoma• Hepatocellular carcinoma
– FACTORS:
• Integration of HBV-DNA into host cell DNA
• Transactivation of transcription by X gene expression

HEPATITIS C
• RNA envelope virus
• Family: Flaviviridae
• Genus: Hepacivirus
• Related to postranfusional NANB hepatitis
• Genome encodes:
– One core protein, two envelope glycoprotein and several NS
proteins
– Six major genotypes(Clades) and more than 100 subtypes

GENOME OF HCV VIRUS

SIGNIFICANCE OF GENOMIC
DIVERSITY
• Different genotypes(1-6) are prevalent in different parts of the world
• Virus undergoes sequence variation in chronic infection
• Treatment response depends on genotypes
• QUASI-SPECIES
a complex viral population presents in a single infected individual

EPIDEMIOLOGY
• 3% of the world population has been infected (WHO, 1997)
• Mode of transmission: Parental, sexual, saliva , organ transplant
• HIGH RISK INDIVIDUALS:• HIGH RISK INDIVIDUALS:
– Intravenous drug users
– Hemophiliacs (Multiple blood transfusion)
– High risk sexual partners
– Health workers

HCV INFECTION
• Incubation period: 6-7 weeks
• Antibody appears 8-9 weeks after exposure
• Antibodies are developed against core, envelope, NS3 and NS4
proteinsproteins
• 70-90% of infected individual develop chronic hepatitis
• 10-20% of chronic HCV infection leads to:
– Chronic active hepatitis
– Cirrhosis (20-50%)
– Hepatocellular carcinoma (5-25%)

• Hepatitis C viral infection is more dangerous.
• REASONS:
– 70-90% of HCV infections progress to chronic hepatitis
HCV OR HBV ?
WHICH ONE IS MORE DANGEROUS?
– 70-90% of HCV infections progress to chronic hepatitis
– Inapparent infection is more in HCV than HBV
– Delay in development of antibody even in low titer
– No vaccine is available due to antigenic diversity of HCV

• SAPMLE: Blood (Serum, Plasma)
• METHODS OF TEST: ELISA, ICT, RIBA, RT-PCR
• SEROLOGY (Anti-HCV):
– In 50-70% cases antibody appears with symptoms
– In other cases antibody appears in 3-6 weeks time
– Can not be distinguish between acute, chronic or resolved infection
• RT-PCR:
– Detects viral RNA
– Useful in prognosis after antiviral drug
– Used to detect genotypes to guide anti-viral therapy

ALGORITHAMS OF HCV INFECTION
DIAGNOSIS
Anti-HCV (Screening)
POSITIVE NEGATIVE
NEGATIVE
HCV-RNA/anti-HCV core Ab
Repeat after 3 months
POSITIVE

RECOMBINANT IMMUNOBLOT
ASSAY (RIBA)
RIBA detects more specific HCV antibodies. Test is
interpreted as positive (2 or more antigens) indeterminate(1
antigen) , negative (0 antigen). More specific than ELISA.
HCV-RNA has now replace the importance of RIBA.

LIMITATIONS OF THE
SEROLOGICAL TEST
• Limitations of the serological test:
– Long delay (even >6 months) in development of anti-HCV
antibodyantibody
– Reactive screening test does not distinguish between
current and past infection
– False reactive test result
– Cannot provide information about treatment response

MANAGEMENT
• TREATMENT
– Alpha interferon or pegilated interferon
– Lamivudine
– Relapse is common
• PREVENTION
– No vaccine developed
– Practicing safe sex, blood transfusion, prohibiting IV addiction,
safe organ donation may protect

HEPATITIS D VIRUS
• RNA envelop (HBsAg) virus
• Virus contains a single stranded negative sense RNA genome
• Is a defective virus: Needs HBsAg for effective infection• Is a defective virus: Needs HBsAg for effective infection
• One serotype
• RNA genome encode one core protein: Delta antigen

Co-infection and super
infection with HBV
• Simultaneous infection of HBV and HDV results in co-infection
• HDV infects in preexisting HBV infection results in super infection
• Fulminant hepatitis is associated with super-infection

• In co-infection
– HDAg, HDV-RNA and anti-HDV IgM
– All markers of HDV replication disappear in convalescence
stage, even anti-HDV antibody disappear within months
• In super-infection
– Persistence of high level of anti-HDV IgM and IgG, HDAg,
HDV-RNA

HEPATITIS E VIRUS
• Non enveloped positive sense ssRNA virus
• FAMILY: Hepeviridiae
• GENUS: Hepevirus
• Most common cause of water borne epidemic in Asia, Africa, Mexico
• 20% of pregnant women develops fulminant hepatitis
• Detection of anti-HEV antibody in blood helps diagnosis

HEPATITIS G VIRUS
• Belongs to Flaviviridae family
• RNA envelope virus
• HGV is also known as GB virus C
• Transmitted through parenteral and sexual route
• Have strong significance in HIV co-infection (35% cases)• Have strong significance in HIV co-infection (35% cases)
• HGV interfere HIV virus replication lower mortality rate
• Laboratory detection:
– GBV-C RNA detection
– Anti-GBV-C antibody

PATHOLOGIC CHANGES IN HEPATITISPATHOLOGIC CHANGES IN HEPATITIS
14/1/2018

Histology of Chronic hepatitis
14/1/2018
Right: Granular cytoplasm, ground-glass hepatocytes
Left: Immunoperoxidase test for HBsAg (HBsAg particles in
cytoplasm of hepatocytes

1. HAV is a picornavirus
2. HAV can be detected in the blood 2 weeks prior to jaundice
3. Anti-HBcAg IgM is an useful viral marker in acute HB
infection
4. Viral cytopathic effect is the main pathogenesis in liver4. Viral cytopathic effect is the main pathogenesis in liver
damage by HBV
5. Reverse transcription occurs in the initial stage of HBV
replication
6. 70-80% of HCV infection leads to chronic hepatitis
7. Quasi-species is observed in HEV infection

8. HDAg is detected in co-infection only
9. HDV genome is a ssRNA with positive polarity
10. HEV commonly infects adults
11. HEV is a flavivirus
12. Fulminant hepatic failure is frequently observed in pregnant12. Fulminant hepatic failure is frequently observed in pregnant
women with HEV infection
13. HGV is a DNA virus
14. HGV interfere with HIV replication
15. Anti-HBsAg is not detectable in chronic HBV infection

Reference
• Review of Medical Microbiology and Immunology. Warren Levinson,
12th May 2012. Mc Graw-Hill (Lange)
• Jawetz, Melnick and Adelberg’s Medical Microbiology
George F. Brooks, Karen C. Carroll, Janet S. Butel,
25th Mar 2010. Mc Graw-Hill (Lange)
• Bailey and Scott’s Diagnostic Microbiology.Betty A.Forbes,
Daniel F. Sahm, Alice S. Weissfeld,12th 2007. Mosby Elsevier
• Lippincott’s illustrated review Microbiology. Richard A Harvey,
Pamella C. Champe, Bruce D. Fisher, 2007. Lippincott William &
Wilkins

Hepatitis viruses

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