Hepatitis B Virus: Structure, Pathogenesis, and Diagnosis

A neonate develops yellowish eyes and skin after a month of life. When screened for hepatitis, the neonate is positive for hepatitis B infection, possibly by transmission from the mother. Hepatitis from the hepatitis B virus (HBV) is responsible for acute and chronic conditions in children and adults. The virus belongs to the family hepadnaviridae; hepa is related to liver cells and DNA because its genomic material is DNA.  

Structure of HBV

• Small (approx. 3.2 kb) in size.
• Enveloped.
• The genome is partially double-stranded DNA as its positive strand is incomplete. The negative-strand consists of 4 genes; S, C, P, and X. S gene codes HBsAg and HBeAg, P codes for DNA polymerase with reverse transcriptase activity, and X codes for X-protein that regulates transcription. Similarly, the C gene codes for capsid or core proteins.
• Under an electron microscope, three particles of HBV appear in the serum of patients; spherical (22 nm diameter), filamentous or tubular, and spherical, double-walled with 42 nm diameter, commonly known as Dane particle.
• Nucleocapsids surround the DNA. Proteins like HBV core protein (HBcAg) attached to the genome are also enclosed by nucleocapsid, whereas the virus’s envelope consists of HBsAg and HBeAg.
• HBsAg: This antigen consists of three glycoproteins, namely S, L, and M. The same gene encodes these glycoproteins. The S glycoprotein is the major component of HBsAg, which self-associates into 22-nm spherical particles released from the cells. The filamentous particles of HBsAg in the serum contain a large amount of S and only a tiny amount of L and M glycoprotein and other proteins and lipids. The glycoprotein L is essential for virion assembly and formation of filamentous particles, and retention of the structures in the cell.

Genomic arrangements

• The genome of HBV is circular DNA, approximately 3,200 base pairs in length. 
• It is capable of independent replication.
• The genetic arrangement of HBV has two distinct features; proteins encoded from overlapping frames and all regulatory sequences residing in protein-encoding sequences.
• The genome has four open reading frames (ORFs). These codes represent the genes that code nucleocapsid protein (core), an envelope protein (s), replicase protein (polymerase), and a protein involved in virus gene expression (X). 
• The ORFs, as mentioned above, overlap; it is a distinct feature of the Hepatitis B virus that helps in encoding 50% (in mass) more unique protein than other DNA viruses.
• Unlike other viruses, the cis-acting regulatory elements are found inside the coding genes themselves instead of in the non-coding section.

 

Pathogenesis of Hepatitis B

Virulence factors

• Resistant to extreme temperatures. It can be stored at 37℃ for 60 minutes, for seven days at 44℃, and at room temperature for six months. But are sensitive to high temperatures and are killed when heated to 60℃  for 10 hours and 100℃ for 1 minute.
• HBsAg is a very stable antigen. The same gene encodes the 3 glycoproteins (S, L, and M) of this antigen. S protein being the highest. These are responsible for attachment to host serum albumin, helping in the infection of hepatocytes.
• HBsAg helps in antigenic differentiation of the HBV as HBsAg contains group-specific antigen d or y and w or r. Combining these specific antigens gives rise to 4 subtypes of HBV; ADW, ADR, AYW, and AYR.
• And there is a total of eight genotypic variations (A, B, C, D, E, F, G, and H).
• Although the virus is DNA, formation of RNA with the help of reverse transcriptase mediates the replication. And replication occurs in the nucleus of the infected host cell.

Transmission of Hepatitis B

The transmission is generally in the following ways;

• Sexual intercourse with an infected person.
• Blood transfusion of an infected individual to healthy individuals.
• Organ transfusion without proper screening.
• From the infected mother to the fetus.
• Needle sharing and use of contaminated blades.

Incubation period

The incubation period of hepatitis B infection varies from a few weeks to 6 months.

• The virus enters through blood, and as it has an affinity for hepatocytes, it binds and replicates in those cells. The replication leads to the production of viral antigens that activate the cytotoxic T cells like CD8 lymphocytes, which causes various immunological reactions.
• The stage (acute or chronic) of infection depends upon the cytotoxic T-cell response of the individual. If the reaction is strong, the person can be free from the disease, and if the response is not strong, these individuals could be chronic carriers. Chronic case occurs more in children (neonates and infants) than in adults. Hepatocellular carcinoma is the result of persistent infection of the hepatocytes.  

NOTE:  When a patient tests positive for hepatitis for more than six months, the condition is chronic.

Replication

The replication of the hepatitis b virus has the following steps:

• Firstly, the infection begins with low-affinity attachment to heparan sulfate proteoglycans (HSP)– glycoprotein present in the basement membrane and secretary vesicles.
• After that, high-affinity attachment occurs to NTCP (sodium taurocholate co-transporting polypeptide) a carrier protein (glycoprotein) present in the basolateral membrane of the hepatocyte.
• Likewise, the internalization occurs by endocytosis, and the uncoating of the viral envelope facilitates the translocation of capsid with relaxed circular DNA (rcDNA) into the nucleus of the host. 
• Similarly, rcDNA repairs into covalently closed circular DNA (cccDNA). 
• The cccDNA acts as a template for the transcription of mRNA.
• The mRNA translates the viral protein. 
• The viral protein (core, surface, and polymerase protein) and capsid enclose pre-genomic RNA assembles in the cytoplasm.
• The pg-RNA converts into DNA by reverse transcriptase in the viral capsid and may return back into the nucleus to repeat the replication process.
• Then the complete viral particles release from the infected host cell.

Clinical signs and symptoms

The clinical signs and symptoms of HBV depend on various factors like; age, immune system, and level of HBV in the body.

Acute HBV infection: Symptoms of HBV are as follows:

During the pre-icteric stage       

• Anorexia
• Malaise
• Fatigue

During the icteric phase

• Nausea,
• Vomiting
• Jaundice with liver tenderness
• Dark-colored urine
• Although the symptoms may be similar to HAV infections, the severity is high in HBV infections, leading to fatal.

NOTE: Icteric- of, relating to, or affected with jaundice.

Chronic Hepatitis B infections: Most chronic hepatitis cases are asymptomatic, but if symptoms develop, it is similar to acute hepatitis and can be a sign of liver damage. It may lead to

• liver cirrhosis.
• and liver cancer.

Diagnosis of Hepatitis B

Sample collection and Transport

Blood serum is the desired sample to diagnose hepatitis B infection. For obtaining serum, Collect the blood in vials without anticoagulant. Allow the blood to clot and centrifuge to get the serum. Transport the sample as soon as possible.

Direct Microscopy 

Electron microscopy can detect the antigen-specific to HBV

Antigen detection

Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) are the acute Hepatitis B infection markers.

1. HBsAg: It is the surface antigen of hepatitis B and is also called Australia antigen or hepatitis-associated antigen. HBsAg generally appears before the onset of symptoms and peaks during overt disease. In patients who successfully clear the HBV infection and do not progress to the chronic carrier stage, HBsAg typically is undetectable 4 to 6 months after infection. The presence of HBsAg even after six months indicates chronic infection.
2. HBeAg: HBeAg is a soluble protein in the core of the hepatitis B virus (HBV). It is generally considered to be a marker of HBV replication and infectivity. HBeAg arises during incubation and is present during the prodrome, early acute disease, and certain chronic carriers. Its presence in chronic carriers indicates a high likelihood of transmissibility; conversely, the absence of HBeAg indicates a low likelihood of transmission.

1. HBcAg: The test for HBcAg is not readily available.

Detection of Antibodies

Hepatitis B panel tests are necessary for a definitive diagnosis of the infection.

1.       IgM anti-HBc (IgM class antibody to HBcAg)
The first antibody to appear is IgM antibody to hepatitis B core antigen (IgM anti-HBc). The presence of the IgM anti-HBc antibody is diagnostic of acute HBV infection. IgM anti-HBc is first detectable in the serum shortly before the onset of symptoms at a time generally concurrent with the onset of elevated transaminase levels.

2.       IgG anti-HBc (IgG class antibody to HBcAg)
IgG anti-HBc indicates that the patient has a history of infection with HBV. Within several weeks, the IgM anti-HBc disappears, and IgG anti-HBc is detected. IgG anti-HBc may remain present for life.

3.       Anti-HBs
The presence of anti-HBs is likely to be protective against repeated HBV exposure, and the anti-HBs may persist for life.

You may also like to read: Interpretation of Hepatitis B Serologic Test Results

4.       Anti-HBe
When viral replication slows, and infectivity declines, the HBeAg disappears, and antibodies to hepatitis B e antigen (anti-HBe) may be detected. HBeAg to anti-HBe seroconversion occurs early in patients with acute infection, before HBsAg to anti-HBs seroconversion. Anti-HBe may persist for years.

Molecular Diagnosis

The genome and DNA polymerase activity are detectable during the incubation period of the infection. HBV PCR demonstration is helpful during the treatment of chronic stage and in diagnosing causes of liver failure.

Other Tests

Liver function tests that help in determining abnormalities in the liver are also carried out to diagnose hepatitis. High levels of ALT and AST are found during the infection. Similarly, levels of serum bilirubin also indicate the intensity of jaundice. 

Treatment of Hepatitis B

No specific antiviral therapy is available to treat acute hepatitis B infection. Treatment of symptoms is the only measure in treating acute disease. The antiviral therapy is recommended for chronic cases. The treatment with interferons and nucleoside analogs is the standard method used. These do not cure HBV but only prevent morbidity and complications related to HBV infections. 

Interferon: Interferon-alpha is the most common interferon used in treating hepatitis. The interferon prevents the progression of acute infection to chronic and neutralization of viremia.

Nucleoside analogs: Nucleoside analogs such as lamivudine, adefovir, and telbivudine are widely used in antiviral therapy. These analogs halt the replication of HBV. These are bio-available and highly effective antiviral therapy available.

Prevention and Control

The preferable method to prevent and control HBV infections is both vaccines and immunoglobulins .

• Immunoglobulin: The passive immunization just before or after exposure to the virus with HBIg. It is obtained from the patients who have recovered from the infection and contains a high titer of HBsAb. It is recommended to those in recent contact with infected people, who are to be in household contact with the infected people, infants born with HBsAg positive mothers, and those in sexual contact with acutely infected patients.
• Vaccines: Plasma-derived and recombinant DNA HBV vaccines are the two types of vaccines available with HBsAg that generates anti-HBs. Recommended vaccine schedule for adults is at 0,1 and 6 months, and infants are at birth, at 1-2 months, and 6-18 months. 

Why HBcAg is not detected in Serum?

References

• Abbas, Naaz & Arshad, Yousra & Shakoori, Abdul. (2006). Mutations in the hepatitis B virus core gene and its efficacy as a vaccine – A Review.. Proc. Pakistan Congr. Zool. 26. 103-129. 
• Krajden, M., McNabb, G., & Petric, M. (2005). The Laboratory Diagnosis of Hepatitis B Virus. Canadian Journal Of Infectious Diseases And Medical Microbiology, 16(2), 65-72. https://doi.org/10.1155/2005/450574.
• Maepa, M., Roelofse, I., Ely, A., & Arbuthnot, P. (2015). Progress and Prospects of Anti-HBV Gene Therapy Development. International Journal Of Molecular Sciences, 16(8), 17589-17610. https://doi.org/10.3390/ijms160817589.
• Parija, S. (2012). Textbook of Microbiology and Immunology (2nd ed.,) Page 550-555.Elsevier, a division of Reed Elsevier India Private Limited.