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 t e h 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 enzym This help to e. 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 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 . 180).HBsAg and DNA may be detected in this

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 Anti-HBc Anti-HBs

Negative Negative negative

susceptible

HBsAg Anti-HBc Anti-HBs HBsAg Anti-HBc Anti-HBs HBsAg Anti-HBc IgM anti-HBc Anti-HBs HBsAg Anti-HBc IgM anti-HBc Anti-HBs

Negative Negative Positive Negative Positive Positive Positive Positive Positive Negative Positive Positive Negative Negative

Immune due to hepatitis B vaccination

Immune due to natural infection

Acute infection

Chronic infection

HBsAg Anti-HBc Anti-HBs

Negative Positive Negative

1- False positive 2- "Window stage" 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 HBsAgnegative 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 antiHBcAg 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%) HBsAgnegative 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 longterm 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 fatigu worsening e, 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.

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