Poxviruses: Human smallpox virus

The Poxviridae family includes two subfamilies: Chordopoxvirinae, which includes vertebrate pox viruses, and Entomopoxvirinae, which unites insect pox viruses. The subfamily of vertebrate pox viruses, in turn, includes 6 independent genera and several unclassified viruses. Representatives of each genus have common antigens and are capable of genetic recombination. Genera differ from each other in the percentage content and properties of DNA, the location and shape of threadlike structures on the outer shell of the virion, resistance to ether, hemagglutination properties and other characteristics.

[1], [2], [3]

Structure Poxviruses: Human smallpox virus

Representatives of the genus Orthopoxvirus - viruses of smallpox, smallpox monkeys and vaccinia. The virus of smallpox causes a particularly dangerous infection of a person, which by the efforts of the world community was eliminated in the mid-1970s. XX century. The monkeypox virus is pathogenic not only for primates: cases have been described in humans that are reminiscent of smallpox in the course of the current. Given this circumstance, it is useful to have general ideas about microbiology of smallpox.

The most studied representative of the genus Orthopoxvirus is the vaccinia virus, which occurred either from the cowpox virus or from the smallpox virus. It is adapted to the human body and for a long time was used as the first live virus vaccine.

The variola virus and other representatives of this genus are the largest of all known animal viruses. This is one of the most highly organized animal viruses, approaching the structure of some structures to bacteria. The virion has the form of a brick with somewhat rounded corners and a size of 250-450 nm. It consists of a well-distinguishable core (nucleoid, or nucleus) containing a genomic double-stranded linear DNA molecule with a molecular weight of 130-200 MD associated with proteins. On both sides of the nucleoid are oval structures called protein bodies. The core and lateral bodies are surrounded by a distinctly distinct surface shell with a characteristic furrowed structure. The core wall consists of an inner smooth membrane 5 nm thick and an outer layer of regularly arranged cylindrical subunits. The virus has a chemical composition reminiscent of bacterial: it contains not only protein and DNA, but also neutral fats, phospholipids, carbohydrates.

Poxviruses are the only DNA-containing viruses that reproduce in the cytoplasm of the host cell. The cycle of reproduction of the virus consists of the following main stages. After adsorption on the surface of a sensitive cell, the virus penetrates the cytoplasm through receptor-mediated endocytosis, and then a two-stage "stripping" of the virion takes place: first, the outer membrane breaks down under the action of cell proteases, partial transcription and synthesis of early-day mRNAs that code for the protein synthesis responsible for further stripping. Parallel to this, vDNA replication takes place. Child DNA copies are transcribed, late mRNA is synthesized. Then a translation is conducted, and about 80 virus-specific proteins with a molecular weight of 8 to 240 kD are synthesized. Some of them (about 30) are structural proteins, the rest are enzymes and soluble antigens. A peculiarity of the reproduction of poxviruses can be considered the modification of their cellular structures, which turn into specialized "factories", where the gradual maturation of new virus particles occurs. The ripe virus progeny leaves the cell either with its lysis or by budding. The cycle of reproduction of smallpox viruses takes about 6-7 hours.

The smallpox virus has haemagglutinating properties; Hemagglutinin consists of three glycoproteins. The most important antigens are: NP-nucleoprotein, common for the whole family; thermolabile (L) and thermostable (C), as well as soluble antigens.

Poxviruses withstand drying for many months at room temperature, are resistant to ether, in 50% ethanol at room temperature are inactivated for 1 hour, and in 50% glycerol solution at 4 ° C are stored for several years. Resistant to most disinfectants: 1% phenol or 2% formaldehyde at room temperature inactivates them only for 24 hours, 5% chloramine for 2 hours.

A human being is susceptible to the smallpox virus, as well as monkeys. When experimental infection in the brain of newborn mice develops a generalized infection that ends lethal; for adult mice, the virus is non-pathogen. It reproduces well in chick embryos when infected with the chorionellantoic membrane, in the amnion, in the yolk sac and the allantoic cavity. On the chorionallantoic membrane of 10-12-day-old chick embryos, the smallpox virus produces small white plaques; the vaccinia virus causes large lesions, with a black cavity in the center caused by necrosis. An important differential sign of variola virus is the maximum temperature of virus multiplication in a chicken embryo at 38.5 ° C.

Primary and transplantable cell cultures derived from humans, monkeys and other animals are sensitive to variola virus. On the culture of cells of tumor origin (HeLa, Vero), the smallpox virus forms small proliferative types of plaque, while when infected with the smallpox virus of the monkeys of Vero cells, circular, lytic-centered plaques are identified. In the kidney cells of the pig embryo, the smallpox virus can cause a clear cytopathic effect, which is not the case when the cells are infected with the monkeypox virus. In HeLa cells, the smallpox virus causes round-cell degeneration, while monkeypox and camel pox viruses cause degeneration with the formation of multinucleated cells.

[4], [5]

Pathogenesis

People who recover after smallpox are immune to life. Long-term resistant immunity is formed after vaccination. 2 basically humoral, virus neutralizing antibodies appear only a few days after the onset of the disease, but they do not interfere with the progressive spread of skin manifestations: the patient can die at the pustular stage, having a high level of antibodies in the blood. Antibodies that appear on the 8th-9th day after immunization and reach maximum titers after 2-3 weeks also respond to the artificial immunity created by vaccination.

Cellular immunity plays no less important role than circulating antibodies. It has been established that antibodies do not occur in persons with hypogammaglobulinemia of antibodies, but they become immune to smallpox virus. This cellular immunity is based on the activity of T-cytotoxic lymphocytes.

[6], [7], [8]

Epidemiology

The source of the infection is a sick person. The vast majority of those not vaccinated against smallpox or unskilled people are susceptible to this infection. Smallpox is usually transmitted by airborne droplets, but infection can not be excluded by contact (through clothing, towels, bedding, household items). The patient is contagious to others during the entire period of development of the rash, up to the disappearance of the last crusts, but is most dangerous in the first 8-10 days, when there are lesions on the mucous membranes.

[9], [10], [11]

Symptoms

The entrance gate of the infection is the mucosa of the upper respiratory tract. Primary multiplication of the virus occurs in the lymphoid tissue of the pharyngeal ring, then for a short time the virus penetrates into the blood and infects the cells of the reticuloendothelial tissue (CMF). The virus multiplies there, and again there is viremia, but more intense and prolonged. The dermatotropic action of the virus is associated with its ability to penetrate from the bloodstream into the epidermis, cause early proliferation of subulate cells and the characteristic degeneration of Malpighian cells.

The incubation period is 8-18 days. Smallpox begins acutely: headaches, muscle aches, prostration, fever. After 2-4 days on the mucous membrane of the mouth and skin there is a characteristic rash - all elements almost simultaneously, are localized more on the face and extremities. The rash passes through the stages of the macula, papules, vesicles and pustules, then a crust (scab) is formed, after which the scar remains a scar. With the appearance of a rash, the temperature decreases and rises again at the stage of pustules. From the appearance of the rash to the loss of crusts is about 3 weeks. With such a classic severe course (variola major), the lethality during epidemics can reach 40%: with a milder form of the disease - alastrim (variola minor) - the lethality does not exceed 1-2%.

Diagnostics

Diagnosis of natural smallpox can be carried out by virososcopic, virologic and serological methods. The most effective and fastest method is direct electron microscopy of the material taken from the elements of the rash to the stage of pustules, since the amount of the virus at this stage is sharply reduced. When light microscopy in preparations from the contents of vesicles, large cells with Gvarnieri bodies are found, which are cytoplasmic inclusions of the oval form near the nucleus of the cell, usually homogeneous and acidophilic, less granular and with irregular contours. Tauri Guarnieri are "factories" where the smallpox virus multiplies. In smears prepared from the contents of small vesicles and stained by the method of M. Morozov, virions of smallpox, the Paschen body, are found.

To isolate and identify the virus, 12 to 14 day old chick embryos are infested into the chorion allantoic membrane, where the virus forms small whitish plaques, and infects cell cultures to detect a cytopathic effect, the establishment of a haemadsorption reaction, or immunofluorescence. Material for infection - blood, nasopharyngeal discharge, scrapings of skin elements of the rash, crusts, as well as sectional material.

The specific antigen of the smallpox virus can be detected in smears-prints from rash elements and nasopharyngeal discharge by indirect immunofluorescence. In the material of the elements of the rash, the antigen can be determined by immunodiffusion, RSK or IFM.

After the first week of the disease, it is possible to detect virus neutralizing, complement-binding antibodies and hemagglutinins. The presence of complement-binding antibodies is considered the most reliable sign of smallpox, since they rarely persist for longer than 12 months in vaccinated patients.

[12], [13], [14], [15], [16], [17], [18]

Treatment

For the purpose of specific treatment and prevention, metisazone (marborane) is used, a drug that suppresses intracellular reproduction of the smallpox virus. It is especially effective in the early stages of the disease and in the incubation period.

The history of human civilization remembers many epidemics and pandemics of smallpox. Only in Europe until the end of the XVIII century. From smallpox killed at least 150 million people. After receiving E. Jenner (1796) vaccine against smallpox began an active fight against this disease, which resulted in its complete elimination. Smallpox was eradicated in the Soviet Union in 1936, but at the expense of imported cases it was registered before 1960. In 1958, at the initiative of the USSR delegation, a resolution was adopted at the WHO assembly to eradicate smallpox all over the world, and in 1967 WHO accepted an intensified program to eradicate smallpox. The USSR, the USA, and Sweden provided extensive financial assistance to this program. The USSR not only provided assistance to specialists working in many endemic countries, but also donated about 1.5 billion doses of smallpox vaccine. A vaccine was used, represented by a live vaccinia virus, grown on a calf, then purified and dried. Good results were also given by the culture and embryonic (ovovaccine) live vaccines. For the prevention and treatment of complications that sometimes occur during vaccination, an antipoietic donor immunoglobulin (10% solution in a physiological solution of the gamma-globulin fraction of blood of donors specially revaccinated against smallpox) and human immunoglobulin titrated for the content of anti-inflammatory antibodies were used.

[19], [20]