Hepatitis B Virus Christian A. García Sepúlveda MD PhD Laboratorio de Biología Molecular Facultad de Medicina Universidad Autónoma de San Luis Potosí

Hepatitis B VirusChristian A. García Sepúlveda MD PhDLaboratorio de Biología MolecularFacultad de Medicina Universidad Autónoma de San Luis Potosí

2

Hepatitis B Virus

Introduction

• 250 million people infected worldwide.

• In areas of Africa and East Asia, 50% of the population may be seropositive, 5-15% may be chronically infected (carriers).

• Carriers are 200x more likely than non-carriers to develop primary hepatocellular carcinoma.

• 300,000 cases per year in the US; 4,000 fatalities.

• 70-90% of maternal-neonatal infections result in chronic infection.

8% - High: Early childhood infection, lifetime risk of infection 60%

2-7% - Intermediate: Infection at all ages, lifetime risk of infection 20%-60%

<2% - Low: Infection as adult, lifetime risk of infection <20%

3

Hepatitis B Virus

Taxonomy

Hepadnavirus family has primate (HBV) as well as Rodent (WHV) and avian (DHBV) representatives.

HBV and WHV have 80% homology (nt)

HBV and DHBV have 40% homology (nt)

Hepadnavirus must have existed before the speciation of birds and mammals.

DHBV possesses the smallest genome of known animal viruses (3021 bp).

4

Hepatitis B Virus

Taxonomy

Baltimore Classification: Group VII for dsDNA-RT integrating viruses.

Enveloped virion containing partial double-stranded circular DNA genome

Replication occurs through an RNA intermediate

Virus encodes and carries a reverse transcriptase

Virus encodes several antigenically and clinically predictive important proteins

5

Hepatitis B Virus

Morphology

Virion also referred to as Dane particle.

42 nm enveloped virus.

Expresses surface antigens (HBsAg).

Core antigen (HbcAg) located in the center (nucleocapsid).

Minor core component e antigen (HBeAg) antigenically distinct from HBcAg.

22 nm spheres and filaments are not infectious, outnumber the actual virions (subviral).

HBsAg = 4 phenotypes : adw, adr, ayw and ayr

HBcAg = inner core protein (a single serotype)

HBeAg = secreted protein; function unknown

25 nm

22 nm

100 – 700 nm

6

Hepatitis B Virus

Morphology

Outer plasma membrane derived from host cell (envelope)

Surface antigens embedded in envelope are glycoproteins.

Three sizes (L, M & S) glycoproteins.

Icosahedral core assures stability (HBcAg)

7

Hepatitis B Virus

Morphology

Three Surface Glycoproteins:

The small protein (SHBs) is encoded by the S region.

- 80-90% of Surface GP.

Large protein (LHBs) encoded bypre-S1, pre-S2, and S region.


Middle protein (MHBs) encoded bythe pre-S2 and S regions.


The SHBs is the most common form of these proteins.

8

Hepatitis B Virus

Genome

“Dane particles” enclose a single circular, incomplete double stranded DNA genome.

Regulatory and structural sequences for viral transcription included.

Extensive overlap of genes.

HBV genome adopts two forms: rc-DNA and cccDNA

In the virion, the genome exists as rcDNA (transcriptionally useless but great at saving information).

In the hepatocyte, the genome exists as cccDNAwhich is transcriptionally active and serves as a reference to replicate more rcDNA.

rcDNA

cccDNA

9

Hepatitis B Virus

Genome

rcDNA

cccDNA

rcDNA

Relaxed circular DNA

Partially double-stranded

L strand or (-)DNA strand is complete and has a short 5’ terminal redundancy covalently coupled to a protein (polymerase).

S strand or (+)DNA strand is incomplete, only 2/3 of the L strand, 5’ end has RNA primer and the length of the 3’ end is variable.

3’ end of L strand overlaps 5’ end of S strand.

10

Hepatitis B Virus

Genome

rcDNA

cccDNA

cccDNA

Covalently Closed Circular DNA

After cell entry (+)DNA strand is completed.

5’ RNA oligo is removed from the (+)DNA.

Pol and 3’ short terminal redundant sequences are removed from (-)DNA.

Two strands are covalently ligated by host repair enzymes.

Strands are stabilized with host cell nucleosomes and might be supercoiled.

Remains as a plasmid within hepatocytes.

Used as a replication/transcription template.

11

Hepatitis B Virus

Genome

The partially double-stranded relaxed circular DNA (rcDNA) indicated with thick black line. P covalently linked to the 5´ end of the (-)DNA. RNA primer (zigzag line) at the 5´ end of (+)DNA. The dashed line symbolizes the heterogeneous lengths of the (+)-strands.

DR1 and DR2 are the direct repeats. The outer circle symbolizes the terminally redundant pgRNA with ε close to the 5´ end, and the poly-A tail at the 3´ end. The precore mRNAis nearly identical, except it starts slightly upstream. The relative positions of the open reading frames for core (C), P, preS/S, and X are shown inside.

12

Hepatitis B Virus

Genome



13

Hepatitis B Virus

Genome



14

Hepatitis B Virus

Genome



15

Hepatitis B Virus

Genome



16

Hepatitis B Virus

Genome



17

Hepatitis B Virus

Genome



18

Hepatitis B Virus

Genome



19

Hepatitis B Virus

Open Reading Frames (ORFs)

There are 4 open reading frames in the same strand (+)DNA

• S – the surface antigen

• Three different polypeptides producedfrom three different alternative translation ATGs.

• C - the core protein

• And its minor (preC) component

• P - the polymerase

• Has a Terminal Protein (TP) domain.

• X – the viral transcription transactivator.

• Conserved in all mammals (but not avian) hepadnaviruses.

• Though not essential in transfected cells, it is required for infection in vivo.

20

Hepatitis B Virus


HBV Genome has an inner (+)DNA strand that is almost circular and has varying lengths.

(+)DNA strand has a DR2 and DR1 cassette at the 5’ end.

21

Hepatitis B Virus


An outer (-)DNA strand is full length and close to 3.5 kb long.

It also has a DR1 at the 5’ end and a DR2 at the 3’ end.

(-)DNA DR1 and DR2 complement (+)DNA DR1 and DR2.

It is covalently associated to a Terminal Protein Domain of the Polymerase.

22

Hepatitis B Virus


Most of the HBV genome is dedicated to encoding the Polymerase (ORF P).

Surface antigen (S) is encoded as a single mRNA transcript which includes:

- pre-S1

- pre-S2 and

- ORF S

Alternate start codons lead to their translation.

Core protein and minor component ara encoded by first ORF (ORF C).

Last ORF codes for X protein (ORF X).

23

Hepatitis B Virus


Each ORF is transcribed into seperate mRNAs.

Each mRNA is translated to produce the different proteins.

24

Hepatitis B Virus

Life Cycle

25

Hepatitis B Virus

Replication

Replication of the hepadnaviral genome can broadly be divided into three phases:

(1) Infectious virions contain a partially double-stranded circular but not covalently closed DNA genome of about 3.2 kb in length (relaxed circular, or rcDNA) inside their inner icosahedric core.

26

Hepatitis B Virus

Replication

(2) Upon infection, the RC-DNA is converted, inside the host cell nucleus, into a plasmid-like covalently closed circular DNA (cccDNA).

27

Hepatitis B Virus

Replication

(3) Genomic and subgenomic RNAs are transcribed by cellular RNA polymerase II from cccDNA.

28

Hepatitis B Virus

Replication

- Pregenomic RNA (pgRNA) selectively packaged into progeny capsids and is

reverse transcribed by the P protein into new rcDNA genomes.

29

Hepatitis B Virus

Replication

- Matured rcDNA containing-but not immature RNA containing nucleocapsids can be used for intracellular cccDNA amplification, or be enveloped and released from the cell as progeny virions.

30

Hepatitis B Virus

Genotypes

First DNA sequence for HBV publshed in 1979 by Galibert et al.

Genotypes are defined as a group of nt sequences that exhibit ≥ 92% homology and diverge from other sequences by ≥ 8%.

Phylogenetic tree of HBV genotypes and subtypes.

Genotypes geographical distribution

A & D relatively ubiquitous.

D rare in N Europe & Americas.

G is least known, posibly ubiquitous.

B & C are predominanty Asian

E Subsaharan Africa

F South/Central America

H Central America and Southern US

31

Hepatitis B Virus

Genotypes

First DNA sequence for HBV publshed in 1979 by Galibert et al.

Genotypes are defined as a group of nt sequences that exhibit ≥ 92% homology and diverge from other sequences by ≥ 8%.

Phylogenetic tree of HBV genotypes and subtypes.

New World Genotypes F & H are on same phylogenetic branch as the WM Non-human Primate WM sequences…

Indictaes a zoonotic transmission of these genotypes to humans of Central and South America.

32

Hepatitis B Virus

Genotypes

Characteristics of HBV genotypes and subtypes

Main serotypes in bold bp aminoacids

33

Hepatitis B Virus

Genotypes and Clinical Outcome

A genotypes may lead to more Chronic Hepatitis than D genotypes in some countries.

A genotypes also easier to clear.

C genotypes progress more rapidly to cirrhosis and hepatocelular carcinoma.

F genotypes (Amazonian basin) linked to fulminant hepatitis in HDV coinfected Hosa.

G genotypes associated with increased liver fibrosis in HIV patients.

Genotypes A & B have higher chance of becoming HBeAg negative than Genotypes C & D.

In the Japanese population, Genotype B and C respond better to interferon treatment in contrast to Genotype A patients.

34

Hepatitis B Virus

Emergence of Viral Mutants

Mutations can arise in any part of the

35

Hepatitis B Virus

Serologic Course (Acute Infection w/recovery)

HBsAg

Can be detected in high levels in serum during acute or chronic HBV infection.

The presence of HBsAg indicates that the person is infectious.

The body normally produces antibodies to HBsAg as part of the normal immune response to infection.

HBsAg is the antigen used to make hepatitis B vaccine.

36

Hepatitis B Virus


Anti-HBs

The presence of anti-HBs is generally interpreted as indicating recovery and immunity from HBV infection.

Anti-HBs also develops in a person who has been successfully vaccinated against hepatitis B.

HBsAg is the antigen used to make hepatitis B vaccine.

37

Hepatitis B Virus


Total anti-HBc

Total hepatitis B core antibody.

Appears at the onset of symptoms in acute hepatitis B and persists for life.

The presence of anti-HBc indicates previous or ongoing infection with HBV in an undefined time frame.

38

Hepatitis B Virus


IgM anti-HBc

IgM antibody to hepatitis B core antigen.

Positivity indicates recent infection with HBV (≤6 months). Its presence indicates acute infection.

39

Hepatitis B Virus


40

Hepatitis B Virus


41

Hepatitis B Virus


42

Hepatitis B Virus


43

Hepatitis B Virus


44

Hepatitis B Virus


45

Hepatitis B Virus

HBV Testing

Testing recommended for:

- pregnant women - infants born to HBsAg-positive mothers - household contacts and sex partners of HBV-infected persons - healthcare workers, laboratory personel - persons infected with HIV

Serologic testing for hepatitis B surface antigen (HBsAg) is the primary way to identify persons with chronic infection with HBV infection (CDC, Sep 2008).

46

Hepatitis B Virus

HBV Testing (Chronic HBV Infection)

47

Hepatitis B Virus


48

Hepatitis B Virus


49

Hepatitis B Virus

HBV Testing (ELISA)

• Advantages 96 tests format Objective results Automatable Appreciable

sensitivity & Specificity

Narrower detection window

• Disadvantages Demands skill sets Decade old method Detection capability

surpassed by newer methods• Sero-conversion

detection panels

50

Hepatitis B Virus

HBV Testing (Rapid Tests)

Rapid Assay Observed Sensitivity (%), 95% CI *

Thermo stability (C)

Overall Ease of Use

Run Time (Minutes)

OraSure(OraQuick) 100, 94.2-100. Up to 30 5 20

AT FIRST (FIRST VUE) 100, 94.2-100 4-30 4 10

ALFA (Instant View) 100, 94.2-100 15-30 4 10

Axiom (axiom HCV) 98, 89.4-99.9 2-30 4 10

Core (Core HCV) 98, 89.4-99.9 4- 30 3 15

MedMira (MiraWell) 100**, 93.3-100 2-30 2 3

Biokit (Biorapid) 84, 70.9-92.8 2-25C/RT N/A 15

51

Hepatitis B Virus


Kit Insert Data

Biorapid HCV Biorapid HBsAg

Sensitivity .994 (.969-.999)

(N=176)

“able to detect HBsAg concentrations of 5IU/ml or higher”

Specificity .987 (.973-.995)

(N=472)

“>98% in studies with blood donors’ samples”

Storage Temp. 2-25C 2-8C

Testing Temp. “Room Temp.” “Room Temp.”

Sample Types S, P, WB S, P

Volume Needed

25µl 125µl

52

Hepatitis B Virus


Conclusions Biorapid Evaluation

Biorapid preformed poorly against panel specimens, but had no invalid results.

Also poor against clinical samples (84% sensitivity for HCV, 87.5% sensitivity for HBsAg) and had an 18% invalid rate with the HBsAg test.

Conclusion: Biorapid hepatitis rapid tests should be replaced by tests that perform better

53

Hepatitis B Virus

HBV Testing (Chemiluminescence)

• Limitations Limited suppliers Capital equipment

• Advantages Lasting

luminescence Increased sensitivity High precision when

automated Wider detection

limits

54

Hepatitis B Virus

HBV Testing (NAT)

• Advantages Direct detection of

viruses Higher sensitivity

than ELISA Closure of window

period of detection

• Limitations High skill sets High TAT Infra-structure Sample processing

step yet to be automated

Room for error Cost of single NAT:

10X ELISA

55

Hepatitis B Virus

HBV Testing (Blood Banks)

FDA recommendations for the management of donors and units based on an algorithm that permits the reentry of some donors when positive for NAT.

Nucleic Acid Testing (NAT) of Hepatitis B. Screening of donors of Whole Blood and blood components for transfusion (WB) and Source Plasma (SP) for the manufacture of injectable plasma derivatives.

Current mandatory scheme for blood banks based on HBsAg and HBcAg.

Recent introduction of FDA approved COBAS AmpliScreen HBV Test.

NAT still optional.

Centers will need to make decisions regarding donor/unit management based on test result combinations

How to intepret NAT results (optional) in the context of serological screening (mandatory).

56

Hepatitis B Virus


Consistent with current regulations and guidance documents:

Whole Blood/Components for transfusion is tested for

- Hepatitis B surface antigen (HBsAg) - Antibody to hepatitis B core antigen (anti-HBc)

Source Plasma for further manufacture is tested for

- HBsAg

FDA’s current position regarding NAT:

If a unit tests HBV NAT negative:

- Donor/unit management is c/w current FDA requirements and recommendations for HBsAg and anti-HBc.

- Units that test NAT and serology negative used.

If a unit tests HBV NAT positive:

- Units that test NAT and /or serology positive not used. - Donor indefinitely deferred.

57

Hepatitis B Virus


Category HBV NAT HBsAg

Anti-HBc Donor and Unit

1 PositiveRepeat Reactive/

neutralizedNon-Reactive

Unit Not Used, Donor Permanently

Deferred


neutralizedRepeat Reactive


Not neutralizedRepeat Reactive

4 Positive Non-Reactive Repeat Reactive

5 Positive Non-Reactive Non-Reactive Unit Not Used, Donor Indef

Deferred, May be reentered6 Positive

Repeat Reactive/

Not neutralizedNon-Reactive

Algorithm for Whole Blood and Blood Components for Transfusion

58

Hepatitis B Virus


Algorithm for Source Plasma

Category HBV NAT HBsAg Donor and Unit

1 Positive

Repeat Reactive/

Neutralized = Positive Unit not used, Donor

Permanently Deferred

2 Positive

Non Reactive

Unit not used, Donor Indefinitely Deferred,

Possible reentry

3 Positive

Repeat Reactive/

Not Neutralized

59

Hepatitis B Virus


During clinical trials of Roche’s COBAS AmpliScreen HBV NAT.

- donor follow-up studies showed that the maximum period of time that HBV DNA preceded HBsAg detection was 17 days, and anti-HBc detection was 48 days .

60

Hepatitis B Virus


Therefore, FDA recommends a minimum 6-month waiting period after a positive HBV NAT result with negative serology results prior to retesting.

A sample (not a donation) is collected at least 6 months after the index donation

For Whole Blood/Components for transfusion donors, sample tested for HBsAg, anti-HBc, HBV DNA by individual sample NAT

For Source Plasma donors, sample tested for HBsAg and HBV DNA by individual sample NAT.

If positive NAT obtained, donor should be permanently deferred, irrespective of serology results.

Only negative individual NAT and negative serologic test(s), collected at least 6 months after the index donation, qualifies the donor for reentry.

Documents

Hepatitis B Virus Christian A. García Sepúlveda MD PhD Laboratorio de Biología Molecular Facultad de Medicina Universidad Autónoma de San Luis Potosí