DEPARTMENT OF BIOCHEMISTRY

HEPATITIS B

By : MHB
Introduction

Hepatitis B virus (HBV) infects over 300 million individuals worldwide, resulting in over
250,000 deaths annually. HBV causes acute and chronic hepatitis. This depends on the
immune response of the body to HBV.

Hepatitis B virus:

HBV is the major member of the hepadnaviridae family . Other members of this family
include woodchuck, ground squirrel, and duck hepatitis viruses. Virus has enveloped virion
containing partially double- stranded, circular DNA genome. Replication is through RNA
intermediate. Virus encodes and carries a reverse transcriptase.HBV infects the liver and to
lesser extent, the kidney and pancreas of only humans and chimpanzees. The receptors for
this virus are found on hepatocytes.

HBV STRUCTURE

HBV is a small, circular, partially double stranded DNA of only 3200 bases. Although a DNA
virus, it encodes a reverse transcriptase and replicates through an RNA intermediate.

The virion , also called Dane particle, is 42 nm in diameter. HBV virion includes a polymerase
with reverse transcriptase, ribonuclease H activity and a P protein attached to the genome ,
which is surrounded by the hepatitis B core antigen( HBcAg) and an envelope containing the
glycoprotein hepatitis B surface antigen ( HBsAg). A hepatitis B e antigen ( HBeAg) protein is
a minor component of the virion. This is because that HBeAg is primarily secreted into
serum. The HBeAg and HBcAg proteins share most of their protein sequence.

Figure (1 ) : HBV- HBsAg particles .

HBsAg- containing particles:

These are released into the serum of infected people and outnumber the actual virions.
HBsAg particles lacking DNA and can be spherical or filamentous . They are immunogenic .
The HBsAg , originally termed Australia antigen, includes three glycoproteins (L, M, S )
encoded by the same gene and read in the same frame but translated into protein from
different AUG start codons. All share the same C-terminal amino acid sequences. All three
forms of HBsAg are found in the virion. The S glycoprotein is the major component of HBsAg
particles. It self-associated into 22-nm spherical particles which also contain small amounts
of M glycoproteins. The filamentous particles of HBsAg contain mostly S but also small
amounts of the M and L glycoproteins and other proteins and lipids. The L glycoprotein is an
essential component for virion assembly and promotes filament formation and the retention
of these structures in the cell. The HBsAg glycoproteins contain the group-specific (termed
a) and type –specific determinants of HBV (termed d, or y and w or r ) . combination of thee
antigens (e.g., ady, adw) result in eight subtypes of HBV that are useful epidemiological
markers.

Figure (2) : HBV- HBsAg particles .

Anti-HBc

Anti-HBc is found lifelong in person who have been infected with HBV and is a marker of
past and current viral infection. In acute infection, high levels of IgM anti-HBc appear during
the incubation period after the appearance of HBsAg, persists for 3-4 months and are then
replaced by IgG anti-HBc.

HBeAg and anti-HBe:

HBeAg is a marker of infectivity associated with the Dane particles, found during the
incubation period and the acute phase of clinical hepatitis B. Anti-HBe develops during
recovery.

HBV REPLICATION

HBV is replicating through these steps:

1- HBV attaches to the hepatocyte's membrane by HBsAg glycoproteins.

2- The core of the virus enters the cytoplasm.
3- The partially double stranded DNA is completed.
4- The genome is derived into the nucleus.
5- DNA transcription is controlled by cellular transcription elements found in the
hepatocytes.
6- DNA transcribed into 3 major bases (2100, 2400, and 3500) and 2 minor bases (900
bases ) of mRNAs.
7- mRNAs enter the cytoplasm.
8- The 3500-base mRNA encodes the HBc and HBe antigens, the polymerase, and the
protein primer for DNA replication and also acts as the template for replication of
the genome.
- 2100-base mRNA encodes for S and M glycoprotein from different in phase start
codon.
- 2400-base mRNA encodes the L glycoprotein , and overlaps the 2100-base
mRNA .
- 900-base mRNA encodes X protein which may promotes viral replication. The
actual function is not known yet.
9- Core assemble around 3500- base mRNA.
 10- RNA- dependent DNA polymerase has reverse transcriptase and ribonuclease H
activity but lacks the integrase activity of the retrovirus enzyme. This help to
synthesize negative-strand DNA on a protein primer.
11- The RNA is degraded by the ribonuclease H activity as positive-strand DNA is
synthesized from the negative-sense DNA template.
12- Core associate with HBsAg in cell membrane.
13- The virion is then released from the hepatocyte by exocytosis and not by cell lysis.

Figure (3): HBV replication.

Figure (4): HBV replication (cont,).

Figure (5) : HBV replication (cont,).

Transmission of hepatitis B virus

HBV is transmitted by:

1- Blood
2- Milk and amniotic fluid
3- Semen and vaginal secretions
4- Saliva

Figure (6): risk factors for acute hepatitis B in USA, 1992 – 1993.

Pathogenesis and immunity

HBV enters the body and target hepatocytes, since its receptors are found predominantly on
these cells. The virus starts to replicate within 3 days from the infection. The incubation
period of the virus is range between (45 – 180).HBsAg and DNA may be detected in this
period.

Cell-mediated immunity and inflammation are responsible for causing the symptoms and
effecting the resolution of the infection. Interferon most likely initiates the response by
enhancing major histocompatibilty complex (MHC) antigen expression and the display of
peptides from HBs, HBc, and HBe antigens to cytotoxic killer T cells. The cytotoxic T cells
attack hepatocytes and cause the lyses of these cells. This lead to the increase of liver
enzyme in the serum .
An insufficient T-cell response to the infection generally results in the occurrence of the
mild symptoms, an inability to resolve the infection and the development of chronic
hepatitis.

Depending on the immune response, viral dose and the route of the infection, HBV can case
acute or chronic, symptomatic or asymptomatic disease.

Acute hepatitis B

90% of acute hepatitis can be resolved.

1% of acute hepatitis B lead to fulminant hepatitis. This may be fatal. It has severe symptoms
and indications of severe liver damage, such as ascites and bleeding.

9% of acute hepatitis may develop chronic hepatitis B.

Figure (7): Acute HBV infection with recovery( typical serologic course).
Chronic hepatitis B

The chances of becoming chronically infected depends upon age. About 90% of infected
neonates and 50% of infected young children will become chronically infected. In contrast,
only about 9%of immunocompetent adults infected with HBV develop chronic hepatitis B.

Figure (8): Progression to chronic HBV infection ( typical serologic course).

50% of people have chronic hepatitis B can be resolved. The rest either become
asymptomatic chronic carriers or people with chronic active hepatitis B.

In the chronic active hepatitis B, HBV is replicating and can cause cirrhosis or hepatic cell
carcinoma. Both are fatal with the absence of liver transplantation.

Diagnosis of hepatitis B

1- Clinical symptoms
These symptoms include :
- Jaundice
- Fatigue
- Abdominal and join pains
- Loss of appetite ,nausea and vomiting
2- Liver enzymes
Liver enzymes are detected in the serum during symptomatic phase. The lyses of
hepatocytes increases the liver enzymes , specially ALT.
3- Hepatitis B profile
Further more information can be obtained from serology and the presence of HBV
antigens. An acute infection can be distinguished from a chronic infection by the
presence of ant-HBc IgM.

Hepatitis B Profile interpretation

Table (1): Hepatitis B results interpretation.

TEST RESULT INTERPRETATION

HBsAg Negative susceptible

Anti-HBc Negative
Anti-HBs negative

HBsAg Negative Immune due to hepatitis

Anti-HBc Negative B vaccination
Anti-HBs Positive

HBsAg Negative
Anti-HBc Positive Immune due to natural
Anti-HBs Positive infection

HBsAg Positive
Anti-HBc Positive Acute infection
IgM anti-HBc Positive
Anti-HBs Negative

HBsAg Positive
Anti-HBc Positive Chronic infection
IgM anti-HBc Negative
Anti-HBs Negative

HBsAg Negative 1- False positive

Anti-HBc Positive 2- "Window stage"
Anti-HBs Negative 3- Chronic carriers
but with
undetected
 HBsAg level.

4- Liver biopsy
Hepatitis B is confirmed, and prognosis is assessed ,by liver biopsy. In asymptomatic
chronic carriers, you can often see "ground glass cell" on liver biopsy. These cells
contain large amounts of HBsAg being synthesized. Other individuals with chronic
hepatitis with various degree of lever inflammation on the biopsy. The amount of
inflammation , and the presence of fibrosis or cirrhosis correlate with the worse
prognosis.

Figure (9): Australia antigen carrier, ground glass hepatocytes .

HBV can also be detected in the laboratory by immunohistochmeistry.

Figure (10): Cirrhosis and hepatitis B antigen positive.

HBV transmitted by HBsAg- negative blood

HBV can be transmitted by HBsAg- negative blood , if the donors are:

1- Carriers with HBsAg below the detection level.

2- Carriers with mutated HBV : point mutation in the pre-core region may results in
inability to synthesize HBsAg. Fulminant hepatitis developed in recipients of HBsAg-
negative blood from donors infected with this mutated virus. In all of these donors,
high levels of anti-HBc were present .
3- Donors with "diagnostic window". This stage happens after the clearance of HBsAg
but before anti- HBsAg has become detected. In this phase, anti-HBc Ag and often
anti-HBeAg can be detected.

About 50% of cases of hepatitis B that could be transmitted by blood from HBsAg-negative
donors can be prevented by screening for anti-HBc. Testing all blood donations for anti-
HBcAg as well as anti-HBsAg has decreased the incidence of HBV infection by transfusion in
the USA.

During the early stage of the incubation period , neither anti-HBcAg nor HBsAg can be
detected . The donor's washed red cells were given to 32 healthy volunteers at a time when
the donor was HBsAg negative. Between 36 and 76 days later, the donor turned HBsAg
positive and subsequently developed hepatitis. Of 32 subjects who received the donor's
blood, 19 developed HBsAg and 14 of these contracted hepatitis. A further nine subjects
developed anti-HBs.

Evidence of Occult Hepatitis B Virus Infection among Omani Blood

Donors

Two hundred Omani HBsAg-negative donors were screened for anti-HBc. Those found to be
positive were investigated for HBV DNA by polymerase chain reaction (PCR). HBsAg was
retested on these sera following an immune complex dissociation technique. The results
showed that HBsAg was present in 2.8% of the donors. Forty-one out of 200 (20.5%) HBsAg-
negative donors were positive for anti-HBc. Of these, 37 (90.2%) had detectable anti-HBs,
including 22 with anti-HBs levels greater than 100 mIU/ml, 12 with levels between 10 and
100 mIU/ml, 3 with levels less than 10 mIU/ml, and 4 had no anti-HBs at all.
All 159 anti-HBc-negative donors were negative for HBsAg after dissociation (S/N < 2).
All anti-HBc-positive donors were originally negative for HBsAg, but following immune
complex dissociation, eleven sera subsequently gave positive results for HBsAg, whereas 8
gave readings just over the cutoff. HBV DNA was not detected in this group. This findings
indicate that testing donors for HBsAg alone is not sufficient to eliminate HBV from the
blood supply in Oman. Immune complex dissociation of sera, either prior to testing or as an
integral part of a commercial assay, will increase the detection rate of HBsAg-positive
donations. This is strongly recommended where HBV prevalence is high at least in first-time
donors. The use of nucleic acid testing on pooled sera is not recommended for HBV due to
the possibility of low viral loads that may be involved. Individual testing would be required
which is time-consuming and expensive.

TREATMENT

In some cases, especially when the patient show no signs and symptoms or liver damage,
the doctor may suggest monitoring rather than treatment. Liver transplantation is the only
option in the end-stage of the disease that shows sever damage of the liver.

The therapeutic goals in chronic hepatitis B are to prevent or decrease cirrhosis and
hepatocellular carcinoma in patient pre-cirrhotic or early cirrhotic disease and to stabilize
patient with end- stage cirrhosis. The treatment includes interferon or nucleoside
analogues( e.g. lamivudine, hepsera, telbivudine or baraclude).

1- Interferon:
This was the first drug approved in united states for the treatment of the chronic
hepatitis. Interferon is protein that mimics the cell's natural defenses against viral
infection. Interferon most likely initiates the response the enhancing the Major
Histocompatibility Complex antigen expression and the display of peptides from
HBs,HBc,and HBe antigens to cytotoxic killer Tcells. In a few cases IFN alpha
treatment eliminates the virus completely, although the infection can later return.
Two interferon medication are available, interferon alpha -2b (Intron A ) and
peginterferon alpha-2 a ( Pegasys). Intron A is given by injection several time a week.
Pegasys is given by injection once time a week.INF alpha treatment has several
disadvantages; it is expensive, it must be administrated by injection, there are side
effects, and INF alpha is poorly tolerated. The side effects include ; flue-like
symptoms, depression, rashes, other reaction and abnormal blood counts. High
doses could cause kidney, heart toxicity.
 2- Telbivudine (Tyzeka):
This is antiviral medication which prevent HBV from replication. It is taken in pill
form once a day. It has almost no side effects for up to a year. Studies show
telbivudine is more effective than are other treatments. However, the patients can
experience a sever worsening of symptoms when they stop taking the drug. Also,
telbivudine can cause drug resistant form of HBV, particularly when taken as a long-
term treatment.
3- Entecavir (Baraclude):
This antiviral medication is taken once a day as pill form. It is more effective than
lamivudine, but it may cause serious worsening of symptoms when the drug is
stopped.
4- Lamivudine(Epivir-HBV):
This is an older antiviral medication. It is similar to telbivudine, but it is slightly less
strong. It is taken usually in pill form once a day. It has less side effects during the
treatment. However, when the patents stop taken the drug, they may experience a
sever worsening of symptoms. In long-term treatment the lamivudine may cause
drug resistant form of HBV. The drug should not be taken by people with kidney
problems or a history of pancreatitis. If the patient shows fatigue, worsening
jaundice, bleeding while the treatment, the doctor should be informed.
5- Adefovir dipivoxil ( Hepsera):
This drug is taken in pill form once a day. It is effective in people who are resistant
to lamivudine. It has also less side effects, but the symptoms may worsen when the
treatment is stopped. This drug can cause kidney problems.

Anti-HBV immunoglobulin

If the person accidently exposed to HBV, he should be given HBV immunoglobulin within 24
hours. Further dose should be given one month later. Intravenous injection of HBV
immunoglobulin provides immediate protection against HBV infection, since the antibodies
are present in the circulation as soon as they have been given. It can also be given
neonatally to children of  HBsAg-positive mothers. The dose for adults is 600 IU, and for
child is 100 IU. The subject should be also vaccinated.
HBV vaccination:

Full immunization include three doses. The second dose is after one month from the first
dose, and the third is after 6 month from the first dose. Protective antibodies develop in 80%
- 97% of people receiving full immunization course. Most people appear to be protected for
15 years, however , in some people the protection may last no longer than 2-5 years.

REFRENCE

1- Mayne Philip D. clinical chemistry in diagnosis and treatment. Sixth ed. Arnold;
1994.
2- Whitby L.G, Smith A.F, Beckett G.J. Lecture notes on clinical chemistry. Fourth
ed. Blackwell; 1988.
3- Murray P.R, Rosenthal K.S, Kobayashi G.S, Pfaller M.A. Medical microbiology.
Third ed. Mosby; 1998.
4- http://en.wikipedia.org/wiki/Hepatitis_B.
5- http://www.gicare.com/diseases/hepatitis-b.aspx.
6- http://www.cdc.gov/hepatitis/b/bfaq.htm.