Hepatitis B virus

Hepatitis B is an infectious disease of humans characterized by selective damage to the liver by a virus. This form of hepatitis is the most dangerous in its consequences among all known forms of viral hepatitis. Its causative agent is the hepatitis B virus (HBV).

The hepatitis B virus antigen was first discovered by B. Blumberg in 1964 in the blood serum of an Australian aborigine, and the pathogen itself was discovered in 1970 by D. Dane (et al.) and was called Dane particles, since there was no complete certainty that it was really a virus and not its components. Subsequently, all doubts disappeared, since genomic DNA and viral DNA-dependent DNA polymerase were discovered in the composition of Dane particles. The virion contains three main antigens, for which the following designations were introduced in 1974:

• HBsAg - superficial, or soluble, or Australian antigen.
• HBcAg - core antigen (cor-antigen).
• HBeAg is an e antigen localized in the core of the virion and, unlike HBcAg, is not only present in the virion but also circulates in the blood in free form or as a complex with the anti-HBeAg antibody. It is released into the blood from hepatocytes during active HBV replication.

The surface antigen, HBsAg, exists in three morphologically distinct variants: 1) is a supercapsid of the whole virion; 2) is found in large quantities in the form of spherical particles with a diameter of 20 nm; 3) in the form of 230 nm long threads. They are chemically identical. HBsAg contains one common antigen, a, and two pairs of mutually exclusive type-specific determinants: d/y and w/r, which is why there are four main subtypes of HBsAg (and, accordingly, HBV): adw, adr, ayw, and ayr. Antigen a ensures the formation of general cross-immunity to all subtypes of the virus.

The virion itself, the Dane particle, is spherical and 42 nm in diameter. The virion supercapsid consists of three proteins: the major (basic), large, and medium. The genome is enclosed in the capsid and is represented by double-stranded circular DNA with a molecular weight of 1.6 MD. The DNA consists of approximately 3200 nucleotides, but its "plus" strand is 20-50% shorter than the "minus" strand. The virus-specific protein is covalently linked to the 5' end of the long strand. The 5' ends of both strands are complementary and form "sticky" sequences 300 nucleotides long, due to which the strands close into a ring. The G + C content in the virion DNA is 48-49 mol %. In the core of the virion, in addition to genomic DNA, there is viral DNA-dependent DNA polymerase. The minus strand of HBV DNA contains only four genes (S, C, P, and X), but they are organized very compactly. The S, C, P, and X genes overlap significantly and control the synthesis of the following products. The S gene encodes the synthesis of the major envelope protein and contains all the information about the surface antigen HBsAg. In addition, it encodes the synthesis of the middle and large envelope proteins. The proteins contain a common COOH end, but their translation begins with three different initiator codons. The C gene encodes the synthesis of the capsid proteins (HBcAg and HBeAg); although these proteins are encoded by a single gene, their translation pathways are different. The P gene is the largest. It includes part of all three other genes and encodes enzymes necessary for viral replication. In particular, it encodes reverse transcriptase, the RNase H enzyme domain, and the 5'-terminal protein of the minus strand. Gene X encodes proteins that regulate the expression of all viral genes, in particular a 17 kD protein that is a transactivator of gene transcription.

The proteins that form the surface antigen exist in glycosylated (gp) and nonglycosylated forms. Glycosylated are gp27, gp33, gp36, and gp42 (the numbers indicate m.w. in kDa). The HBV supercapsid consists of the major, or core, S protein (92%); the middle, M protein (4%), and the large, or long, L protein (1%).

• The major protein, p24/gp27, or core protein (protein S), is the major component of the HBV envelope. In the absence of other envelope proteins, it polymerizes to form 20-nm-diameter spherical particles composed of 100 polypeptide molecules.
• The large protein, p39/gp42, or long protein (protein L), is present in all three forms of HBsAg. It plays an important role in virion morphogenesis and in their exit from the cell. The L protein contains the sequence of protein M, which is supplemented at the N-terminus by sequences of 108 (ayw) or 119 (adw, adr, ayr) amino acid residues encoded by the npe-Sl region of the S gene.
• The middle protein, gp33/gp36, or M protein, is also present in all three morphological forms of HBsAg. The M protein contains at its N-terminus a region of 55 amino acid residues encoded by the pre-52 region of the S gene. It is assumed that this region plays an important role in the recognition of liver cells of a limited range of hosts (humans, monkeys, and chimpanzees) by the hepatitis B virus. The sequences of proteins encoded by the npe-S regions of the S gene are highly immunogenic, and their determinants are located on the virion surface. Therefore, antibodies against these antigens play an important role in the formation of immunity against hepatitis B.

The synthesis of viral proteins is tightly controlled at the level of transcription and translation. During transcription of the viral genome, two types of mRNA are synthesized:

• the smaller one - 2100 nucleotides - encodes the main and middle proteins of the membrane;
• large - 3500 nucleotides, i.e. longer than the genomic DNA itself; it contains terminal repeats 100 nucleotides long.

This type of mRNA codes for the capsid protein and the products of the P gene. It is also a matrix for the replication of viral DNA. The genome contains enhancers (transcription amplifiers) - regulatory elements that activate the expression of all viral genes and act primarily in liver cells. In particular, the S gene is expressed at a very high level only in liver cells and under the influence of steroid hormones. This circumstance explains why chronic hepatitis B and liver cancer (hepatoma) are observed in men more often than in women, whose steroid hormone levels are lower.

Other regulatory elements of the hepatitis B virus modulate (control) the levels of synthesis of individual proteins. For example, the large protein is synthesized only in small quantities. Most of it is on the surface of infectious virions. But the main protein and, to a lesser extent, the middle protein are synthesized in huge quantities and leave the cells as part of surface antigen particles, which are many times more abundant in the blood serum than mature virions. The number of surface antigen particles can be 1011 -1013 per 1 ml of blood (several hundred μg).

The hepatitis B virus has been isolated into a new family of viruses - Hepadnaviridae, genus Orthohepadnavirus. Similar hepadnaviruses have been found in various animals (ground squirrels, marmots, chipmunks, Peking ducks).

Hepadnaviruses reproduce in a somewhat unusual manner. In particular, the replication of genomic DNA occurs through an intermediate link - RNA, i.e. with the mechanism of reverse transcription.

Life cycle of the hepatitis B virus.

• Adsorption on the cell.
• Penetration into the cell via the mechanism of receptor-mediated endocytosis (coated pit -> coated vesicle -> lysosome -> release of the nucleocapsid and penetration of the viral genome into the hepatocyte nucleus).
• Intracellular reproduction.

During penetration into the cell, the short ("plus") DNA chain is lengthened (completed). In the nucleus, the cellular DNA-dependent RNA polymerase synthesizes RNA of 3500 nucleotides (pregenome) and mRNA, which are smaller in size, for the synthesis of viral proteins. Then the pregenome and viral DNA polymerase are packaged into a newly synthesized capsid, which is transferred to the cytoplasm. Here, reverse transcription of the pregenome occurs. A new "minus" DNA strand is synthesized on it. After the synthesis of the "minus" DNA strand is complete, the pregenomic RNA is destroyed. Virion DNA polymerase synthesizes a "plus" strand on the "minus" strand. The viral DNA, now double-stranded, can exist in the cell for quite a long time and return to the nucleus for the next replication cycle. If the new viral particle does not undergo further replication, then the formed nucleocapsid, passing through the cell membrane, is covered by a supercapsid, buds off from the cell, and the elongation of the short "plus" DNA chain immediately stops. That is why the length of this thread varies. In a typical acute form of hepatitis B, the following serological markers appear in the blood sequentially: HBsAg, HBeAg and antibodies (IgM, IgG): anti-HBcAg. anti-HBeAg and anti-HBsAg.

The hepatitis B virus does not contain an oncogene, but it has been established that, when introduced into a cell chromosome (into different parts of it), viral DNA can induce various genetic rearrangements in them - deletions, translocations, amplifications, which can cause the development of liver cancer - one of the most severe consequences of viral hepatitis B.

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Hepatitis B virus resistance

The hepatitis B virus is highly resistant. It remains viable for 3 months at room temperature and for several years when frozen. The virus is completely inactivated by autoclaving (120 °C), boiling for 30 minutes, dry heat at 180 °C for 60 minutes, and at 60 °C for 10 hours. It is resistant in an acidic environment, but is destroyed in an alkaline environment. The virus dies when treated with H2O2, chloramine, formalin, phenol, and UV irradiation.

Pathogenesis and symptoms of hepatitis B

The virus is carried directly to the liver by the hematogenous route. Autoimmune humoral and cellular reactions play an important role in the pathogenesis of hepatitis. It is assumed that the damage to hepatocytes is associated not so much with the direct action of the virus itself, but with the immunological reactions of the host, arising in connection with the modification of the cell membrane by viral proteins, which induce the appearance of autoantibodies to liver cells. Therefore, developing chronic hepatitis and cirrhosis of the liver can be considered an autoimmune disease.

Cellular autoimmune reactions to viral proteins contained in the hepatocyte membrane are mediated by T-cytotoxic lymphocytes and other liver killer cells. Therefore, acute liver dystrophy can be considered as a rejection reaction of a kind of heterotransplant.

The incubation period lasts from 45 to 180 days, on average it is 60-90 days. The clinical course of hepatitis B is characterized by great diversity; the disease can proceed: in a latent form, detected only by laboratory methods, in a typical icteric form and in a malignant form, ending in death. The duration of the pre-icteric stage is from one day to several weeks. The icteric period, as a rule, is long and is characterized by well-defined symptoms (jaundice, hyperbilirubinemia, darkening of urine, yellowness of the sclera). The protracted form is observed in 15-20% of patients, and 90% of them develop chronic hepatitis B. Patients with a protracted form often have autoimmune processes, accompanied by an increased content of antihepatic antibodies, which are detected using immunosorbent assay (IFM). In children, hepatitis B proceeds in a milder form and often without the development of jaundice, in younger children - mostly asymptomatic.

Post-infectious immunity (humoral and cellular) is long-lasting, lifelong, and is caused by virus-neutralizing antibodies (anti-HBsAg) in the absence of surface antigen in the blood. Latent immunization is often observed due to repeated contact with HBV, which is the reason for the widespread immunity to the virus among the population. Usually, patients with acute hepatitis B recover completely as antibodies to it accumulate. However, in some cases, despite the high level of viral antigen in the blood (the circumstance that explains why parenteral infection occurs most often), antibodies to it are not produced. The virus remains in the liver, and a person becomes a chronic carrier for a long time, sometimes for life. This circumstance is obviously associated with a weak immune response. One of the most common outcomes of chronic hepatitis B is liver cirrhosis and liver cancer, which develops after a latent period of up to 30-50 years.

Epidemiology of hepatitis B

The source of infection with the hepatitis B virus is only a human. Contrary to previous ideas that infection with the hepatitis B virus occurs exclusively parenterally, it has now been proven that it is found in various secretions and excretions: in saliva, nasopharyngeal secretions, feces, tear fluid, in sperm, menstrual blood, etc. Thus, infection occurs not only parenterally, but also sexually and vertically (from mother to fetus), i.e., infection with the hepatitis B virus is practically possible in different ways.

Hepatitis B has killed as many people worldwide as during all the years of World War II. According to WHO, the number of HBV carriers ranges from 0.1 to 20% of the population of different countries or regions.

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Diagnosis of hepatitis B

Currently, the main method for diagnosing hepatitis B is the use of the reverse passive hemagglutination assay (RPHA) to detect the virus or its surface antigen, HBsAg. As already noted, the blood contains many times more surface antigen than the virus itself (100-1000 times). For the RPAHA reaction, erythrocytes sensitized with antibodies against the hepatitis B virus are used. If the antigen is present in the blood, the hemagglutination reaction occurs. RPAHA is simple, convenient, and very specific. Various immunological methods (RSK, RPHA, IFM, RIM, etc.) are used to detect antibodies to the viral antigen HBsAg. In addition, PCR variants are used to detect HBV and its antigens.

To detect antibodies to the viral antigen (HBsAg) in the patient's serum, various immunological methods can be used (CSC, RPGA, precipitation reaction, IFM, RIM, etc.).

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Specific prophylaxis of hepatitis B

Taking into account the high incidence of hepatitis B, as well as the fact that there are many HBV carriers in the world, WHO recommends that hepatitis B vaccinations are mandatory and should be administered in the first year of life. Two types of vaccines are proposed for vaccination. To prepare one of them, the plasma of virus carriers is used as raw material, since it contains the viral antigen in quantities sufficient for preparing the vaccine. The main condition for preparing this type of vaccine is its complete safety, i.e. complete inactivation of the virus, which is provided for by the vaccine preparation technology. To prepare another type of vaccine, genetic engineering methods are used, in particular, a recombinant clone of yeast producing the surface antigen of the hepatitis B virus is used to obtain the antigen material.

Both vaccines are highly effective (protect 95% of those vaccinated). The duration of post-vaccination immunity is at least 5-6 years. Vaccines have been created for adults, newborns and young children - the most important component of the fight against hepatitis B globally. The full vaccination course consists of three injections:

I dose - immediately after birth; II dose - after 1-2 months; III dose - until the end of the first year of life.

These vaccinations are included in the WHO expanded program of immunization and are combined with its implementation calendar (according to WHO recommendations, vaccinations against tuberculosis, poliomyelitis, hepatitis B, measles, tetanus, diphtheria, and whooping cough are administered in the first year of life).

Gammaglobulin containing antibodies against HBV is used for emergency passive immunoprophylaxis of individuals who have had contact with a patient with hepatitis B.

Interferon and amixin (to induce its endogenous synthesis) are used to treat hepatitis B (acute and chronic forms). The new drug lamivudine (synthetic nucleoside) is effective in treating chronic hepatitis B.