Hepatitis b virus general virology and laboratory diagnosis

This slide is about overview of Hepatitis B virus with special reference to serological markers used for hepatitis b viral infection diagnosis.

1. Hepatitis B virusHepatitis B virus
Shyam Kumar Mishra
Assistant Professor, Dept. of Microbiology
Institute of Medicine, TU
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2. Viruses causing hepatitis???Viruses causing hepatitis???
Hepatitis Viruses- A, B, C, D, E, and G (G.
Baker V)
Coxsackie virus
Yellow fever virus
Adenovirus
Paramyxovirus
Rubella virus
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3. Hepatitis virusesHepatitis viruses
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4. Family- HepadnaviridaeFamily- Hepadnaviridae
◦Avihepadnavirus
Bird HBV
Shanghai duck HBV
Ross goose HBV
China duck HBV
Heron HBV
◦Orthohepadnavirus
Mammalian HBV
Human HBV
Ground squirrel HBV
Woodchuck HBV
Hepatitis B virusHepatitis B virus
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5. HepadnaviridaeHepadnaviridae
Morphology
◦3 morphological forms by EM
Spherical form
Tubular or filamentous form
Complete form or Dane particles
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6. 6

7. ◦Spherical form
Most numerous
Small forms measuring 20-22 nm in diameter
Exclusively made up of HBsAg
◦Tubular or filamentous form
22 nm in diameter
200 nm long (variable)
Exclusively made up of HBsAg
Represent excess HBsAg (overproduction)
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8. Are
these
infectious
?????
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9. Complete form or Dane particles
◦Represent the intact Hepatitis B virus
◦Less frequently observed
Double-shelled
Spherical lipid-containing structure with an outer
diameter of 42-47 nm
This outer surface envelope represents surface antigen
(HBsAg)
It has three forms of the viral envelope proteins- L, M, S
Icosahedral nucleocapsid with a diameter of 27-
28 nm represents the inner shell of the virus.
It consists of
core antigen (HBcAg),
pre-core antigen (HBeAg) and
partially double-stranded DNA.
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10. 10

11. 11

12. Viral genome
◦The viral genome consists of two linear
strands of DNA held in a circular
configuration (3200bp in length)
◦One of the strands (+ strand) is incomplete
while the other is complete
◦This gives the appearance of partially double
stranded and partially single stranded DNA.
◦Associated with the plus strand is a viral
DNA polymerase
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13. Viral genomeViral genome
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The Hepatitis B virus genome is compact
and consists of four overlapping genes:
◦S gene
◦C gene
◦X gene
◦P gene
These genes code for different antigens.

14. Genes coding for antigens in HBVGenes coding for antigens in HBV
Gene Regions Antigen
S
(Having three regions S,
Pre-S1 and Pre-S2)
S
S + Pre-S2
S + Pre-S1 and S2
Major protein (S)
Middle Protein (M)
Large Protein (L)
C
(Having two regions C
and Pre-C)
C
C + Pre-C
HBcAg
HBeAg
P (Largest gene) Enzymatic activities-
DNA polymerase, Reverse
transcriptase activity, RNase H
activity
X HBxAg (Non-particulate antigen,
which can activate the transcription
of cellular and viral genes; contribute
to carcinogenesis by binding to p53;
HBxAg and its antibody are elevated
in patients with severe chronic
hepatitis and HCC. )
HBsAg
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15. ?Type I ?Type J
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HBV Genotypes

16. Determining the genotype could be
helpful for predicting the outcome of
disease, and antiviral therapy in patients
with chronic hepatitis B.
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17. Clinical significance of GenotypingClinical significance of Genotyping
Genotype B appears to be associated
with less rapidly progressive liver disease
and cirrhosis and a lower likelihood, or
delayed appearance, of HCC than
genotype C or D.
Patients infected by genotype A are more
likely to clear circulating viremia and to
achieve HBeAg and HBsAg
seroconversion, both spontaneously and
in response to antiviral therapy.
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18. Impact of HBV genotypes on Impact of HBV genotypes on
response to Antiviral therapyresponse to Antiviral therapy
HBV genotypes D and C are associated with a lower
rate of favorable response to interferon alpha therapy
than genotypes A and B, respectively.
Rate of resistance to lamivudine is higher in patients
with HBV genotype A infection than in patients with
genotype D infection.
No difference in the risk of lamivudine resistance -
Genotype B and genotype C.
In patients with genotype C infection; however,
virological response is worse during lamivudine therapy,
and is also less durable after the discontinuation of
therapy than in patients with genotype B infection.
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19. HBsAg (Australia antigen)HBsAg (Australia antigen)
Blumberg et al (1965) – in Australian
aborigine
HBsAg contains a group-specific antigen,
a, with two pairs of mutually exclusive
subdeterminants, d/y and w/r
Four phenotypes of HBsAg
◦Adw, ayw, adr, ayr
◦Useful in epidemiologic investigations
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20. HBsAg sub-types and HBV HBsAg sub-types and HBV
DNA genotypes in NepalDNA genotypes in Nepal
Subtypes
Ayw - 47%
Adw - 34.3%
Adr – 4%
Genotypes
 D (69%)
A (22%).
(Shrestha SM, Shrestha S, Shrestha A, Tsuda F, Endo K, Takahashi M, et al. High prevalence
of hepatitis B virus infection and inferior vena cava obstruction among patients with
liver cirrhosis or hepatocellular carcinoma in Nepal. J Gastroenterol
Hepatol. 2007;22:1921–8.)
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21. Hepatitis B virus mutantsHepatitis B virus mutants
Pre-core mutants
◦They have defect in precore region of C gene
which leads to their inability to synthesize
HBeAg.
◦Patients may be diagnosed late and they tend
to have severe chronic hepatitis that
progresses more rapidly to cirrhosis.
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22. Escape mutants
◦Mutation in the S gene leading to alteration of
HBsAg (mutation in the “a” antigen).
◦Pose problem in the diagnosis of the disease.
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23. YMDD mutation
◦Patients on lamivudine therapy may develop
resistance to the drug due to mutation in the
YMDD (tyrosine-methionine-aspartate-
aspartate) locus present in the HBV reverse
transcriptase region of polymerase gene.
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24. PropertiesProperties
HBV and HBsAg – stable at (-20
0
C)
- stable to repeated freezing and thawing
HBV (but not HBsAg) is sensitive to
higher temp (100
0
C -1 min)
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25. Hepatitis B carriersHepatitis B carriers
Simple carrier –
◦HBeAg -
◦HBsAg + (low titer)
◦large volume of blood is required for
transmission
Super carrier –
◦HBeAg +
◦Highly infectious (0.00001 ml of plasma can
transmit infection)
in
blood
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26. Mode of transmissionMode of transmission
Entry of the virus into the blood through
skin, mucous membrane or body fluids
Main routes of transmission
Blood transfusion (blood/ blood products
containing HBV)
Use of HBV contaminated needles, blades
Sexual intercourse
Perinatal
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27. Chance of transmission following a
contaminated needle prick injury
◦HBV- 30%
◦HCV- 3%
◦HIV – 0.3%
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28. ReceptorsReceptors
Transferrin receptor
Asialoglycoprotein receptor molecule
Human liver endonexin
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29. PathologyPathology
Microscopically, spotted parenchymal cell degeneration,
with necrosis of hepatocytes, a diffuse lobular
inflammatory reaction, and disruption of liver cell cords
Kupffer cell hyperplasia, panlobular infiltration with
mononuclear cells
Ballooning or acidophilic bodies (Councilman-like bodies)
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30. Clinical FeaturesClinical Features
Onset is slow, usually insidious but more
severe.
Incubation period - 6 weeks to 6 months
Fever is less common and of low grade,
jaundice is rarely seen in children but is
persent more often in adults.
The course of acute HBV can be divided
into three phases-
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31. Preicteric phase
Icteric phase
Convalescent phase
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32. Onset of jaundice is often preceded by
GI symptoms
Outcome ranges from complete recovery
to progression to chronic hepatitis and
rarely, death due to fulminant disease
In adults,
◦65-80% of infections are inapparent
◦90- 95% of all patients recover completely
80-95% of infants and young children–chronic
carriers
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33. Hepatic complications
◦Very few cases may proceed to complications
such as fulminant hepatitis or cirrhosis or
HCC.
◦Chronic carriers are at high risk of developing
hepatocellular carcinoma
Fulminant HBV disease is associated with
superinfection by other agents (HDV)
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34. Extrahepatic complications
◦During the prodromal phase, a serum
sickness-like syndrome characterized by
arthritis, rash, angioedema, and rarely,
hematuria and proteinuria may develop in 5-
10% of patients.
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35. The average mortality~ 0.5-2%
Post-transfusion hepatitis (10-15%)
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37. HBV PrevalenceHBV Prevalence
Type 1 pattern (low endemicity) – carrier
rate <2%
Type 2 pattern (Intermediate endemicity)
– 2-8%
Type 3 pattern (high endemicity) - >8%
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38. Exposure
Infection
Death (1%)
(Fulminant
hepatitis)
Recovery (90-
95%)
(Immune)
Persistent infection
(HBsAg in serum ~
> 6 mths)
Immune
(Clearance)
Chronic active hepatitis
Cirrhosis
Hepatocellular carcinoma38

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40. Individuals at RisksIndividuals at Risks
Parental drug abusers
Sexual contact with an acute or
chronically infected person
Infants born to HBV infected mothers
Occupational contact with blood
Organ transplant patients
Institutionalized populations
Worldwide 300 million HBV carriers
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41. Which of the following serologic profiles
would be most consistent with acute
HBV infection?
◦HBsAg (-), anti-HBs (+), Total anti-HBc (+), HBeAg (-), anti-HBe (+)
◦HBsAg (+), anti-HBs (-), Total anti-HBc (-), HBeAg (-), anti-HBe (+).
◦HBsAg (+), anti-HBs (-), Total anti-HBc (+), HBeAg (+), anti-HBe (-).
◦HBsAg (-), anti-HBs (+), Total anti-HBc (-), HBeAg (-), anti-HBe (-).
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42. HBV antigensHBV antigens
HBsAg:
◦Reaches a peak during pre-icteric phase
Detectable in blood about a month after exposure
HBcAg:
◦Sequestered within an HBsAg coat (Not secreted and does not
circulate in blood)
◦Not detectable in serum of patients with HBV infection
◦Can be detected in hepatocytes by immunofluorescence
HBeAg:
◦Non-particulate soluble antigen possessing a signal protein
which enables it to be secreted.
◦Present in circulation
◦Indicates viral replication
Viral DNA polymerase:
◦Viremic stage of Hepatitis B
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43. Antibodies to HBVAntibodies to HBV
Anti-HBs:
◦Antibody to HBsAg
◦Indicates past infection with and immunity to
HBV, presence of passive immunity from
HBIG, or immune reponse from HBV vaccine
Anti-HBe:
◦Antibody to HBeAg
◦Presence in serum of HBsAg carrier suggests
lower titer of HBV
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44. Anti-HBc
◦Antibody to HBcAg
◦Infection with HBV at some undefined time in
the past
◦Not protective
IgM Anti-HBc
◦IgM class antibody to HBcAg
◦Indicates recent infection with HBV, positive
for 4-6 months after infection
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51. Laboratory diagnosisLaboratory diagnosis
Microbiological/ Serological tests
Biochemical tests
Histology and immunohistology
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53. Propagation and assay of HBV in Propagation and assay of HBV in
Cell CultureCell Culture
HBV has been successfully grown in
primary cultures of normal adult or fetal
human hepatocytes; however,
susceptibility wanes as the cells
differentiate.
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54. ProphylaxisProphylaxis
Active immunization
◦Recombinant subunit vaccine
◦HBsAg is used as vaccine candidate which is
prepared in Baker’s yeast by recombinant
technology by cloning the S gene into the
yeast chromosome.
◦IM over deltoid region
◦10-20mcg/dose
◦0, 1, 6 months
◦Target (Antibody titer) > 10 IU/mL
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55. Passive immunization (HBIG)
◦For immediate protection
◦0.05-0.07 mL/kg body weight
◦Two doses; 30 days apart
◦Short term protection lasting for 100 days
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56. Combined immunization
◦HBIG and vaccine
Guideline for post-exposure prophylaxis
◦AntiHBs >10 IU/mL, no further treatment
◦Vaccinated but antibody level <10,
Start HBIG immediately
Vaccine: Single dose given within 7 days of
exposure.
◦Not Vaccinated,
HBIG and full course of vaccine are needed.
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57. Hepatitis DHepatitis D
1977, Rizzetto et al.
Delta virus
Defective RNA virus
Can replicate only when HBV infection
persists in the host
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58. Spherical
36 nm particle with an outer coat
composed of the HBsAg surrounding the
circular ssRNA genome.
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59. InfectionInfection
Coinfection
Superinfection
No association between HDV and HCC
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60. DetectionDetection
Delta antigen in liver cell nuclei
Occasionally in serum
Anti-delta antibodies in serum
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61. PreventionPrevention
Immunization with HBV vaccine
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62. Virus Hepatitis A Hepatitis B Hepatitis C Hepatitis DHepatitis
E
Family PicornaviridaeHepadnaviridaeFlaviviridaeUnclassifiedUnclassified
 
Genus Hepatovirus  Orthohepadnavirus Hepacivirus  Deltavirus  Hepevirus 
Virion 27 nm, icosahedral42 nm, spherical60 nm, spherical35 nm, spherical30–32 nm,
icosahedral
Envelope No Yes (HBsAg) Yes Yes (HBsAg)No
Genome ssRNA dsDNA ssRNA ssRNA ssRNA
Genome size 7.5 kb 3.2 kb 9.4 kb 1.7 kb 7.6 kb
Stability Heat- and
acid-stable
Acid-sensitiveEther-sensitive,
acid-sensitive
Acid-sensitiveHeat-stable
TransmissionFecal-oral Parenteral Parenteral Parenteral Fecal-oral
Prevalence High High Moderate Low, regionalRegional
Fulminant diseaseRare Rare Rare Frequent In
pregnancy
Chronic diseaseNever Often Often Often Never
Oncogenic No Yes Yes ? No
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