Hepatitis A virus

Viral hepatitis A is a human infectious disease characterized by a predominant liver damage and manifested clinically intoxication and jaundice. Hepatitis A virus was detected in 1973 by S. Feinstone (and others) 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 (serum of convalescent) are added to the filtrate of the feces of the patient with hepatitis A and the precipitate 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. The discovery of S. Feinstone was confirmed in experiments on volunteers.

Hepatitis A virus has a spherical shape, the diameter of the virion is 27 nm. The genome is represented by a single-stranded positive RNA with a mass of 2.6 MD. Supercapsid is absent. The symmetry type is cubic - icosahedron. The capsid has 32 capsomers, it is formed by four polypeptides (VP1-VP4). By its properties, the hepatitis A virus belongs to the genus Heparnovirus, the family Picornaviridae. Antigenically, hepatitis A virus (HAV - hepatitis A virus) is homogeneous. HAV multiplies well in the body of chimpanzees, baboons, hamadryls and gambling monkeys (marmosets). For a long time, the virus could not cultivate. Only in the 1980s. It was possible to obtain cell cultures in which HAV propagates. Initially, for these purposes, transplanted kidney lines of the rhesus monkey kidney (culture FRhK-4) were used, and now - the transplanted line of kidney cells of green monkeys (culture 4647).

According to the recommendations of WHO experts, the following nomenclature of markers of the hepatitis A virus was adopted: hepatitis A virus-NAU antibodies to hepatitis A virus: anti-NAV IgM and anti-NAV IgG.

HAV is small particles with a diameter of 27-30 nm, having icosahedral symmetry and possessing homogeneity. On the electron diffraction pattern obtained by the method of immune aggregation, electron-dense particles with superficially located symmetrically arranged capsomers are detected. With negative contrasting, both complete and empty particles are detected in the preparations. Nucleocapsid NAV, unlike that of influenza, does not have surface protrusions and membranes. It is also important that the virion HAV does not have a heart-shaped structure.

According to its physicochemical properties, the hepatitis A virus is attributed to the family of picornaviruses, the genus of enteroviruses with serial number 72. However, such a taxonomy turned out to be too unusual, and WHO considered it possible to leave the terminology "hepatitis A virus".

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

[1], [2], [3], [4], [5], [6], [7],

Resistance of the hepatitis A virus

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

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

• the natural master is man;
• experimental animals - marmozet, chimpanzee;
• source of infection - feces;
• disease - epidemic and endemic;
• the transmission route is fecal-oral;
• incubation period - 14-40 days;
• transition to chronic hepatitis - not noted.

Immunological properties of NAV are as follows:

• Prototype strains - Ms-l, CR-326, GVG. 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 trophic action of human serum y-globulin - protects or attenuates the disease under the condition of administration prior to infection or during the incubation period.

Physico-chemical characteristics of NAU are as follows:

• Morphology is a non-shell spherical particle with cubic symmetry, the capsid consists of 32 capsomers;
• The diameter is 27-30 nm;
• The density in CsCl (g / cm3) is 1.38-1.46 (open particles), 1.33-1.34 (mature virion), 1.29-1.31 (immature virions, empty particles);
• The sedimentation coefficient is 156-160 mature virions;
• Nucleic acid - 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 NAV in physicochemical effects is as follows:

• Chloroform, ether - is 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 ° С, 72 hours - complete inactivation, 1: 350, 20 ° С, 60 min - partial inactivation.

Temperature:

• 20-70 ° C - stable;
• 56 ° С, 30 minutes - it is stable;
• 60 ° С, 12 hours - partial inactivation;
• 85 ° С, 1 min - full inactivation;
• Autoclaving, 120 ° C. 20 minutes - complete inactivation;
• Dry heat, 180 ° C, 1h - complete inactivation;
• UV, 1.1 W, 1 min - complete inactivation.

The presented data show that the physicochemical properties of the hepatitis A virus are the closest to enteroviruses. Like other enteroviruses, HAV is resistant to many disinfectant solutions, completely inactivated for several minutes at 85 ° C and autoclaved.

It is proved that the hepatitis A virus can be reproduced in primary and transplant monolayer culture lines of human and monkey cells. Especially active reproduction of hepatitis A virus in cultures in vitro is observed when using liver extracts of monkey patients as a starting material. It should be noted, however, that in all experiments on the reproduction of the hepatitis A virus on in vitro cultures, a long incubation period in primary passages (up to 4-10 weeks) attracts attention, further accumulation of viral genetic material is increasing, but absolute values remain very insignificant, which gives reason to many researchers to talk about incomplete replication of the hepatitis A virus in tissue cultures.

Summarizing the literature data on the reproduction of hepatitis A virus from non-root cultures, it can be said that the fact of a prolonged experience of HAV in vitro is unquestionable. Optimum conditions for a stable high level of viral replication have not been finally identified, and this constrains the study of its biological properties, the production of a source of reagents for the production of diagnostics and the design of vaccines.

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 for the near future. When studying the optimal conditions for HAV reproduction in the culture of kidney cells of the macaque-rhesus embryo, 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 is also shown that the most significant accumulation of the viral antigen occurs under the conditions of so-called roller cultivation (rotating bottles). This way opens up wide possibilities of obtaining a cultural antigen in large quantities, and consequently, a starting material for the preparation of diagnostic systems and the manufacture of vaccine preparations will appear.

Epidemiology of hepatitis A

Hepatitis A virus has a high pathogenicity for humans. According to the conclusion of the WHO (1987), for the onset of the disease, it is enough to infect only one virion. However, the practical infectious dose is probably much higher. The source of infection is only an infected person. The virus is released in large quantities with feces 12-14 days before the appearance of jaundice and within 3 weeks. Icteric period. Significant differences in the isolation of the pathogen in patients with icteric, jaundice and asymptomatic forms of hepatitis A have not been identified. The method of infection is fecal-oral, mainly aqueous, and also by household and food routes. The method of infection is fecal-oral, mainly aqueous, and also by household and food routes. The main (primary) route of transmission of the virus is water. It is also possible to airborne infection. The susceptibility of the population is universal. Mostly children under the age of 14 are ill. The disease has a pronounced autumn-winter seasonality.

[8], [9], [10], [11], [12], [13], [14], [15]

Symptoms of hepatitis A

The incubation period varies from 15 to 50 days, which depends on the amount of the infecting dose of the virus, but on average is 28-30 days. Once in the body, the hepatitis A virus multiplies in regional lymph nodes, penetrates into the bloodstream and then into 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 does not occur due to direct action of the virus, but as a result of immunopathological mechanisms. The most typical picture of hepatitis A is acute icteric cyclical form: incubation period, prodromal (pre-jaundiced), icteric period and convalescence. However, in the foci of infection, a large number of patients with jaundiced and asymptomatic forms of infection are identified, the number of which significantly prevails over icteric ("iceberg phenomenon").

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

Microbiological diagnosis of hepatitis A

Diagnosis of hepatitis A (except for the infection of animals - chimpanzees, marmoset, baboons, which we do not have) is based on various immunological methods: RSK, immunofluorescence method, hemagglutination of immune adhesion (the complex virus 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 absence of specific viral antigens, and the immunofluorescence reaction requires liver biopsy, which is undesirable. Reliable and specific is the method of immune electron microscopy, but it is very laborious. Therefore, while 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 the "capture" of immunoglobulins of class M. In our country, a test system - DIAGN-A-GEP is proposed for this purpose. The working principle of this test system is as follows. First, antibodies to immunoglobulins of class M (anti-immunoglobulin M) are sorbed on the walls of polystyrene lunches, then the test serum of the patient is added. If it has antibodies of IgM class, they will be bound by anti-T antibodies of M, then a specific viral antigen (hepatitis A virus) is added, which is obtained by cultivation in a 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" arises:

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

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

The advantage of the method of "capture" of IgM is that antibodies of this class of immunoglobulins appear at the primary immune response and indicate an active stage of infection, they disappear after the transferred disease. Antiviral antibodies belonging to the class of IgG, in contrast, persist for a long time after the disease, providing the acquired immunity. For the detection of hepatitis A virus, a DNA probe method is proposed: a complementary vRNA probe is used as a probe.

Treatment of hepatitis A

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

Specific prevention of hepatitis A

Previously widely applied seroprevention hepatitis A using gammaglobulin was not justified, however, the emphasis was placed on the holding immunization, carried vaccination against hepatitis A. To this end, various variants of vaccines are being developed and are already being used. In Russia, an effective vaccine against hepatitis A was obtained back in 1995, and now it has been successfully applied.