Poxviruses: Human smallpox virus

The Poxviridae family (English pox - smallpox + viruses) includes two subfamilies: Chordopoxvirinae, which includes vertebrate pox viruses, and Entomopoxvirinae, which unites insect pox viruses. The vertebrate pox virus subfamily, 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 membrane of the virion, resistance to ether, hemagglutinating properties and other features.

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Structure Poxviruses: Human smallpox virus

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

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

Smallpox virus and other representatives of this genus are the largest of all known animal viruses. It is one of the most highly organized animal viruses, approaching bacteria in the structure of some structures. The virion is brick-shaped with slightly rounded corners and measures 250-450 nm. It consists of a clearly distinguishable core (nucleoid, or core) containing a genomic double-stranded linear DNA molecule with a molecular weight of 130-200 MDa, associated with proteins. On both sides of the nucleoid are oval structures called protein bodies. The core and lateral bodies are surrounded by a clearly distinguishable surface membrane with a characteristic grooved structure. The wall of the core consists of an internal smooth membrane 5 nm thick and an outer layer of regularly arranged cylindrical subunits. The virus has a chemical composition similar to that of bacteria: it contains not only protein and DNA, but also neutral fats, phospholipids, and carbohydrates.

Poxviruses are the only DNA-containing viruses that replicate in the cytoplasm of the host cell. The virus reproduction cycle consists of the following main stages. After adsorption on the surface of a sensitive cell, the virus penetrates the cytoplasm by receptor-mediated endocytosis, and then a two-stage "undressing" of the virion occurs: first, the outer membrane is destroyed by cellular proteases, partial transcription and synthesis of early mRNAs encoding the synthesis of the protein responsible for further undressing occur. In parallel with this, replication of vDNA occurs. Daughter copies of DNA are transcribed, late mRNAs are synthesized. Then translation occurs, and about 80 virus-specific proteins with a molecular weight of 8 to 240 kDa are synthesized. Some of them (about 30) are structural proteins, the rest are enzymes and soluble antigens. A feature of poxvirus reproduction is their modification of cellular structures, which are transformed into specialized "factories" where new viral particles gradually mature. Mature viral offspring leave the cell either during its lysis or by budding. The reproduction cycle of smallpox viruses takes about 6-7 hours.

The smallpox virus has hemagglutinating properties; hemagglutinin consists of three glycoproteins. The most important antigens are: NP-nucleoprotein, common to the entire family; heat-labile (L) and heat-stable (C), as well as soluble antigens.

Poxviruses can withstand drying (especially in pathological material) for many months at room temperature, are resistant to ether, are inactivated in 50% ethanol at room temperature within 1 hour, and are preserved in 50% glycerol at 4 °C for several years. They are resistant to most disinfectants: 1% phenol or 2% formaldehyde at room temperature inactivate them only within 24 hours, 5% chloramine - within 2 hours.

Humans and monkeys are susceptible to the smallpox virus. When experimentally infected, the brain of newborn mice develops a generalized infection that ends fatally; the virus is not pathogegenic for adult mice. It reproduces well in chicken embryos when infecting the chorioallantoic membrane, amnion, yolk sac, and allantoic cavity. On the chorioallantoic membrane of 10-12-day-old chicken embryos, the smallpox virus produces small white plaques; the vaccinia virus causes larger lesions with a black depression in the center caused by necrosis. An important differential feature of the smallpox virus is the maximum temperature of virus reproduction in the chicken embryo of 38.5 °C.

Primary and continuous cell cultures obtained from humans, monkeys and other animals are sensitive to the smallpox virus. On the cell culture of tumor origin (HeLa, Vero), the smallpox virus forms small plaques of the proliferative type, while when Vero cells are infected with the monkeypox virus, round plaques with a lytic center are detected. In pig embryo kidney cells, the smallpox virus is capable of causing a clear cytopathic effect, which does not occur when these cells are infected with the monkeypox virus. In HeLa cells, the smallpox virus causes round-cell degeneration, while the monkeypox and camelpox viruses cause degeneration with the formation of multinucleated cells.

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Pathogenesis

People who have recovered from smallpox retain immunity for life. Long-term stable immunity is also formed after vaccination. 2 is mainly humoral, virus-neutralizing antibodies appear within a few days after the onset of the disease, but do not prevent the progressive spread of skin manifestations: the patient can die at the pustular stage, having a high level of antibodies in the blood. Antibodies are also responsible for the artificial immunity created by vaccination, appearing on the 8-9th day after immunization and reaching maximum titers after 2-3 weeks.

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

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Epidemiology

The source of infection is a sick person. The vast majority of people who have not been vaccinated against smallpox or have not had the disease are susceptible to this infection. Smallpox is most often transmitted by airborne droplets, but infection by contact is also possible (through clothing, towels, bedding, household items). The patient is contagious to others during the entire period of development of the rash, until the last crusts fall off, but is most dangerous in the first 8-10 days, when there are lesions on the mucous membranes.

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Symptoms

The entry point for infection is the mucous membrane of the upper respiratory tract. Primary virus reproduction occurs in the lymphoid tissue of the pharyngeal ring, then the virus briefly enters the blood and infects the cells of the reticuloendothelial tissue (RET). The virus reproduces there, and viremia occurs again, but more intense and prolonged. The dermatotropic effect of the virus is associated with its ability to penetrate from the bloodstream into the epidermis, causing early proliferation of spinous cells and characteristic degeneration of the cells of the Malpighian layer.

The incubation period is 8-18 days. Smallpox begins acutely: headaches, muscle pain, prostration, fever. After 2-4 days, a characteristic rash appears on the mucous membrane of the oral cavity and skin - all elements almost simultaneously, localized more on the face and limbs. The rash goes through the stages of macula, papule, vesicle and pustule, then a crust (scab) is formed, after which a scar remains. With the appearance of the rash, the temperature drops and rises again at the pustule stage. About 3 weeks pass from the appearance of the rash to the falling off of the crusts. With such a classic severe course (variola major), the mortality rate during epidemics can reach 40%: with a milder form of the disease - alastrim (variola minor) - the mortality does not exceed 1-2%.

Diagnostics

Smallpox can be diagnosed using virusoscopic, virological and serological methods. The most effective and rapid method is direct electron microscopy of material taken from the rash elements before the pustule stage, since the amount of virus at this stage decreases sharply. Light microscopy of preparations from the contents of the vesicles reveals large cells with Guarnieri bodies, which are oval-shaped cytoplasmic inclusions near the cell nucleus, usually homogeneous and acidophilic, less often granulated and with irregular outlines. Guarnieri bodies are the "factories" where the smallpox virus reproduces. In smears prepared from the contents of smallpox vesicles and stained using the M. Morozov method, smallpox virions - Paschen bodies - are found.

To isolate and identify the virus, 12-14-day chicken embryos are infected on the chorion-allantoic membrane, where the virus forms small whitish plaques, and cell cultures are also infected to detect the cytopathic effect, to set up a hemadsorption or immunofluorescence reaction. The material for infection is blood, nasopharyngeal discharge, scrapings of skin elements of the rash, crusts, as well as autopsy material.

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

Already after the first week of the disease, virus-neutralizing, complement-fixing antibodies and hemagglutinins can be detected. The presence of complement-fixing antibodies is considered the most reliable sign of smallpox, since they rarely persist in vaccinated individuals for longer than 12 months.

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Treatment

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

The history of human civilization remembers many epidemics and pandemics of smallpox. In Europe alone, at least 150 million people died from smallpox by the end of the 18th century. After E. Jenner (1796) received a vaccine against smallpox, an active fight against this disease began, which ended with its complete elimination. In the Soviet Union, smallpox was eliminated in 1936, but due to imported cases, it was registered until 1960. In 1958, on the initiative of the USSR delegation, a resolution was adopted at the WHO Assembly on the eradication of smallpox throughout the world, and in 1967, WHO adopted an intensified smallpox eradication program. The USSR, the USA, and Sweden provided extensive financial assistance to this program. The USSR not only provided assistance with specialists working in many endemic countries, but also donated about 1.5 billion doses of smallpox vaccine. The vaccine used was a live smallpox virus grown on a calf's sac, then purified and dried. Good results were also obtained with culture and embryonic (ovovaccine) live vaccines. For the prevention and treatment of complications that sometimes arise during vaccination, anti-smallpox donor immunoglobulin (10% solution in physiological solution of gamma-globulin fraction of blood of donors specially revaccinated against smallpox) and human blood immunoglobulin titrated for the content of anti-smallpox antibodies were used.

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