Hepatitis A virus

Viral hepatitis A is an infectious disease of humans, characterized primarily by liver damage and clinically manifested by intoxication and jaundice. The hepatitis A virus was discovered in 1973 by S. Feinstone (et al.) using the method of immune electron microscopy and by infecting monkeys - chimpanzees and marmosets. The essence of the method of immune electron microscopy is that specific antibodies (convalescent serum) are added to the filtrate of the feces of a patient with hepatitis A and the sediment is subjected to electron microscopy. Due to the interaction of viruses with specific antibodies, they undergo specific aggregation. In this case, they are easier to detect, and aggregation under the influence of antibodies confirms the specificity of the pathogen. S. Feinstone's discovery was confirmed in experiments on volunteers.

The hepatitis A virus is spherical, the virion diameter is 27 nm. The genome is represented by single-stranded positive RNA with m. m. 2.6 MD. There is no supercapsid. The symmetry type is cubic - icosahedron. The capsid has 32 capsomeres, it is formed by four polypeptides (VP1-VP4). According to its properties, the hepatitis A virus belongs to the genus Heparnovirus, the family Picornaviridae. In terms of antigens, the hepatitis A virus (HAV - hepatitis A virus) is homogeneous. HAV reproduces well in the body of chimpanzees, baboons, hamadryas baboons and marmoset monkeys. For a long time, the virus could not be cultivated. Only in the 1980s was it possible to obtain cell cultures in which HAV reproduces. Initially, continuous cell lines of rhesus macaque embryo kidney (culture FRhK-4) were used for these purposes, and now a continuous cell line of green monkey kidney cells (culture 4647) is used.

According to the recommendations of WHO experts, the following nomenclature of hepatitis A virus markers has been adopted: hepatitis A virus - HAV antibodies to hepatitis A virus: anti-HAV IgM and anti-HAV IgG.

HAV is a small particle with a diameter of 27-30 nm, having icosahedral symmetry and possessing homogeneity. The electronogram obtained using the immune aggregation method reveals electron-dense particles with superficially located symmetrically arranged capsomers. With negative contrasting, both full and empty particles are revealed in the preparations. The nucleocapsid of HAV, unlike that of influenza, does not have surface protrusions and a membrane. It is also important that the HAV virion does not have a heart-shaped structure.

Based on its physicochemical properties, the hepatitis A virus is classified as belonging to the picornavirus family, the enterovirus genus with serial number 72. However, this taxonomy turned out to be too unusual, and the WHO considered it possible to retain the terminology “hepatitis A virus”.

Like all viruses of the Picornaviridae family, the hepatitis A virus contains ribonucleic acid. Some laboratories have demonstrated the possibility of cloning the hepatitis A virus genome, which opens up the prospect of obtaining vaccines.

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Hepatitis A virus resistance

The virus is relatively resistant to high temperatures, acids, fat solvents (no lipids), disinfectants, and tolerates low temperatures well. All this contributes to its long-term preservation in the external environment. At room temperature, it survives for several weeks, at 60 °C it partially loses its infectivity after 4-12 hours, and completely - after several minutes at 85 °C. It is highly resistant to chlorine, due to which it is able to penetrate into tap water through the barriers of water treatment facilities.

Summarizing all the data, we can characterize the hepatitis A virus as follows:

• the natural host is man;
• experimental animals - marmosets, chimpanzees;
• the source of infection is feces;
• the disease is epidemic and endemic;
• transmission route: feco-oral;
• incubation period - 14-40 days;
• transition to chronic hepatitis - not observed.

The immunological properties of HAV are as follows:

• Prototypic strains - Ms-1, CR-326, GВG. All are immunologically similar or identical;
• Antibodies - IgM and IgG, are produced in response to the introduction of structural proteins of the virus and are protective;
• I. The protective effect of human serum gamma globulin - prevents or attenuates the disease if administered before infection or during the incubation period.

The physicochemical characteristics of NAU are as follows:

• Morphology: shell-less spherical particle with cubic symmetry, capsid consists of 32 capsomeres;
• Diameter - 27-30 nm;
• Density in CsCl (g/cm3) - 1.38-1.46 (open particles), 1.33-1.34 (mature virion), 1.29-1.31 (immature virions, empty particles);
• Sedimentation coefficient - 156-160 mature virions;
• Nucleic acid is a single-stranded, linear RNA;
• Relative molecular weight - 2.25 106-2.8 106KD;
• The number of nucleotides is 6,500-8,100.

The stability of HAV under physical and chemical influences is as follows:

• Chloroform, ether - stable;
• Chlorine, 0.5-1.5 mg/l, 5 °C, 15 min - partial inactivation;
• Chloramine, 1 g/l, 20 °C, 15 min - complete inactivation;
• Formalin, 1:4000, 35-37 °C, 72 h - complete inactivation, 1:350, 20 °C, 60 min - partial inactivation.

Temperature:

• 20-70 °C - stable;
• 56 °C, 30 min - stable;
• 60 °C, 12 h - partial inactivation;
• 85 °C, 1 min - complete inactivation;
• Autoclaving, 120 °C. 20 min - complete inactivation;
• Dry heat, 180 °C, 1 hour - complete inactivation;
• UFO, 1.1 W, 1 min - complete inactivation.

The presented data show that in its physicochemical properties, the hepatitis A virus is closest to enteroviruses. Like other enteroviruses, HAV is resistant to many disinfectant solutions and is completely inactivated within a few minutes at 85 °C and autoclaving.

It has been proven that the hepatitis A virus can reproduce in primary and continuous monolayer lines of human and monkey cell cultures. Particularly active reproduction of the hepatitis A virus in in vitro cultures is observed when using liver extracts from sick monkeys as the starting material. It should be noted, however, that in all experiments on the reproduction of the hepatitis A virus on in vitro cultures, attention is drawn to the long incubation period during primary passages (up to 4-10 weeks), subsequently the accumulation of viral genetic material increases, but the absolute values remain very insignificant, which gives grounds for many researchers to speak of incomplete replication of the hepatitis A virus in tissue cultures.

Summarizing the literature data on the reproduction of the hepatitis A virus in extra-tissue cultures, it can be said that the fact of long-term survival of HAV in vitro is beyond doubt. Optimal conditions for a stable high level of virus replication have not been finally identified, and this hinders the study of its biological properties, obtaining a source of reagents for the production of diagnostics and vaccine design.

At the same time, more optimistic judgments about this problem can be found in the literature. The solution of all issues related to the cultivation of the hepatitis A virus is a matter of the near future. When studying the optimal conditions for the reproduction of HAV in the culture of embryonic kidney cells of the rhesus macaque, two phases were identified: the phase of production of the infectious virus (up to 6-8 days at the level of the 5th passage) and the phase of intensive accumulation of the viral antigen. It was also shown that the most significant accumulation of the viral antigen occurs under the conditions of the so-called roller cultivation (rotating flasks). This method opens up wide opportunities for obtaining cultural antigen in large quantities, and, consequently, the source material for the preparation of diagnostic systems and the manufacture of vaccines will appear.

Epidemiology of hepatitis A

The hepatitis A virus is highly pathogenic for humans. According to the WHO (1987), infection with just one virion is enough to cause the disease. However, the practical infectious dose is probably much higher. The only source of infection is an infected person. The virus is excreted in large quantities with feces 12-14 days before the onset of jaundice and during the 3 weeks of the icteric period. No significant differences in the excretion of the pathogen in patients with icteric, anicteric and asymptomatic forms of hepatitis A have been found. The route of infection is fecal-oral, mainly waterborne, as well as household and foodborne. The route of infection is fecal-oral, mainly waterborne, as well as household and foodborne. The main (primary) route of transmission of the virus is waterborne. Infection by airborne droplets is also possible. The susceptibility of the population is universal. Mostly children under 14 years of age are affected. The disease has a pronounced autumn-winter seasonality.

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Symptoms of Hepatitis A

The incubation period varies from 15 to 50 days, depending on the infective dose of the virus, but on average is 28-30 days. Once in the body, the hepatitis A virus multiplies in the regional lymph nodes, penetrates the blood, and then the liver cells and causes acute diffuse hepatitis, which is accompanied by damage to hepatocytes and reticuloendothelial elements of the liver and a decrease in its detoxification and barrier functions. Damage to hepatocytes occurs not due to the direct action of the virus, but as a result of immunopathological mechanisms. The most typical picture of hepatitis A is an acute icteric cyclic form: incubation period, prodromal (pre-icteric), icteric period and convalescence. However, in the foci of infection, a large number of patients with anicteric and asymptomatic forms of infection are detected, the number of which significantly predominates over icteric ones ("iceberg phenomenon").

Post-infection immunity is strong and long-lasting, caused by virus-neutralizing antibodies and immune memory cells.

Microbiological diagnostics of hepatitis A

Diagnostics of hepatitis A (except for infection of animals - chimpanzees, marmosets, baboons, which we do not have) is based on various immunological methods: RSC, immunofluorescence method, hemagglutination of immune adhesion (the complex of viral antigen + antibody in the presence of complement is adsorbed on erythrocytes and causes their gluing). However, the possibilities of using these methods are limited due to the lack of specific viral antigens, and the immunofluorescence reaction requires liver biopsy, which is undesirable. The method of immune electron microscopy is reliable and specific, but it is very labor-intensive. Therefore, so far the only acceptable immunological reaction is the method of immunosorbent analysis of the solid phase in the form of IFM or RIM, especially in the modification of "capture" of immunoglobulins of class M. In our country, a test system has been proposed for this purpose - "DIAGN-A-HEP". The principle of operation of this test system is as follows. First, antibodies to immunoglobulins of class M (antiimmunoglobulins M) are sorbed on the walls of polystyrene wells, then the patient's serum to be tested is added. If it contains IgM antibodies, they will bind to anti-antibodies of class M, then a specific viral antigen (hepatitis A virus) is added, which is obtained by growing in cell culture. The system is washed, and antiviral antibodies labeled with horseradish peroxidase are added to it. If all four components of the system interact, a four-layer "sandwich" is formed:

• antiimmunoglobulins M,
• immunoglobulins M (against the hepatitis A virus - in the patient's serum under study),
• viral antigen,
• enzyme-labeled antiviral antibodies.

To detect this complex, a substrate for the enzyme is added to the wells. Under the influence of the enzyme, it is destroyed, and a colored product is formed. The intensity of the color can be measured quantitatively using a spectrophotometer or photocolorimeter.

The advantage of the IgM "capture" method is that antibodies of this class of immunoglobulins appear during the primary immune response and indicate the active stage of infection, they disappear after the disease has been suffered. Antiviral antibodies belonging to the IgG class, on the contrary, persist for a long time after the disease has been suffered, providing acquired immunity. A DNA probe method has been proposed for detecting the hepatitis A virus: complementary vRNA DNA is used as a probe.

Treatment of hepatitis A

Due to the fact that interferon production is disrupted in viral hepatitis, treatment of hepatitis A is based on the use of interferon and the inducer of its endogenous synthesis, amixin.

Specific prevention of hepatitis A

Previously widely used seroprophylaxis of hepatitis A with gamma globulin did not justify itself, therefore the main emphasis was placed on conducting vaccination prophylaxis, vaccination against hepatitis A is carried out. For this purpose, various types of vaccines are being developed and are already being used. In Russia, an effective vaccine against hepatitis A was obtained back in 1995, and it is now being successfully used.