Influenza virus replication occurs in. Influenza viruses and influenza. Cultivation and reproduction
![Influenza virus replication occurs in. Influenza viruses and influenza. Cultivation and reproduction](https://i1.wp.com/bio-faq.ru/zzz/zzz010_clip_image004.jpg)
Adsorption
The famous “H5N1” stands for “hemagglutinin type five, neuraminidase type one” - these two proteins stick out on the surface of the influenza virus (in Fig. 1, hemagglutinin is green and neuraminidase is gray).
With the help of hemagglutinin, the influenza virus attaches to receptors on the surface of cells. The initial target of the virus is the cells of the ciliated epithelium of the respiratory tract, but this is not why we love it: hemagglutinin can attach to the receptors of many other cells, including red blood cells. If one virus attaches to two (adjacent) red blood cells at the same time, the red blood cells will stick together! Hence the name of the protein - “blood-gluing”.
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Implementation
The stupid cell absorbs the virus that has attached itself to it by phagocytosis - like, eats it. Why, why do children always put all sorts of nasty things in their mouths?! However, the virus is still inside the cell as food, inside a phagocytotic vesicle (in Fig. 2 - “endosome”). The endosome merges with the lysosome, a digestive vacuole is formed, protons are pumped into it from the cytoplasm to create an acidic environment (this process is shown in Fig. 2) - a little more, and we will digest the virus (with the words “protein food, what’s the difference”).
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Strip
But the virus is ready for this turn of events:
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- Hemagglutinin is modified under the influence of an acidic environment - its surface becomes hydrophilic, and it (previously attached to the receptor on the inner surface of the endosome membrane, now) is embedded inside this membrane.
- Protons pumped into the endosome pass through special channel proteins (M2 proteins, indicated in Fig. 1 and Fig. 3) through the lipid shell of the virus and reach the protein shell of the virus (in Fig. 1 - a circle of white balls - M1 proteins) . Because of this, the protein shell is destroyed (in Fig. 3, the M1 proteins of the destroyed protein shell are indicated as red stars).
- The lipid envelope of the virus (due to the penetrating action of hemagglutinin) fuses with the (lipid) membrane of the phagosome; The RNA of the virus ends up in the cytoplasm of the cell.
Virus replication
The virus RNA released into the cytoplasm is itself completely safe.
- Proteins cannot be made on it, because it is minus RNA (proteins are not encoded by it, but by the complementary plus strand, which does not yet exist).
- It is also impossible to make RNA on it - our cells generally do not have an enzyme capable of doubling RNA.
“Oh-ho-ho, you don’t have anything,” the flu virus grumbles, shaking his mustachioed head, “but it’s okay, I brought everything with me.” The virus brought with it the proteins PB1, PB2 and PA, which together form the viral RNA-dependent RNA polymerase - it can duplicate RNA. But bad luck! Any polymerase needs a primer to start working, but the forgetful flu didn’t take it with it! Everything is over?!
Calm down, don't panic! - With these words, the whole company (8 viral RNAs and 3 viral enzymes) is sent to the cell nucleus. There the flu gets full service:
- primers for viral RNA replication (to obtain plus RNA) are sections cleaved from cellular RNA;
- processing: sections that served as primers for RNA synthesis - these were caps, so the modification of the 5" end was carried out at the very beginning; at the end of the synthesis, polyadelation of the 3" end occurs;
- splicing: some viral RNA containing information for two proteins is cut into two parts.
In this way, plus RNAs are synthesized, which can serve as templates for the synthesis of viral proteins and viral minus RNAs.
Then everything is simple: the stupid cell, using its own ribosomes from its own amino acids, synthesizes the proteins of the virus, including RNA-dependent RNA polymerase. Influenza minus RNA is also vigorously produced inside the nucleus. The assembly of viral particles occurs in the cytoplasm, on the inner surface of the cell membrane. The finished virus leaves the cell by exocytosis (budding), neuraminidase bites the last thread that connects the cell and the newborn virus... A new (evil) little life comes out into the world!
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Structure and chemical composition
The influenza virus has a spherical shape, with a diameter of 80-120 nm. Thread-like forms are less common. The nucleocapsid of helical symmetry is a ribonucleoprotein (RNP) strand arranged in a double helix that forms the core of the virion. RNA polymerase and endonucleases (P1 and P3) are associated with it. The core is surrounded by a membrane consisting of the M protein, which connects the RNP with a lipid bilayer of the outer shell and styloid processes consisting of hemagglutinin and neuraminidase. Virions contain about 1% RNA, 70% protein, 24% lipids and 5% carbohydrates. Lipids and carbohydrates are part of the lipoproteins and glycoproteins of the outer shell and are of cellular origin. The genome of the virus is represented by a minus-strand fragmented RNA molecule. Influenza viruses types A and B have 8 RNA fragments. Of these, 5 encode one protein, and the last 3 encode two proteins each.Antigens
Influenza viruses A, B and C differ from each other in the type-specific antigen associated with RNP (NP protein) and the M-matrix protein, which stabilizes the structure of the virion. These antigens are detected in RSC. The narrower specificity of the type A virus is determined by two other surface antigens - hemagglutinin H and neuraminidase N, designated by serial numbers. Hemagglutinin is a complex glycoprotein with protective properties. It induces in the body the formation of virus-neutralizing antibodies - antihemagglutinins, detected in the RTGA. The variability of hemagglutinin (H-antigen) determines the antigenic drift and shift of the influenza virus. Antigenic drift refers to minor changes in the H-antigen caused by point mutations in the gene that controls its formation. Such changes can accumulate in offspring under the influence of selective factors such as antibodies. This ultimately leads to a quantitative shift, expressed in a change in the antigenic properties of hemagglutinin. With antigenic shift, a complete replacement of the gene occurs, which may be based on recombination between two viruses. This leads to a change in the subtype of hemagglutinin or neuraminidase, and sometimes both antigens, and the emergence of fundamentally new antigenic variants of the virus, causing major epidemics and pandemics. Hemagglutinin is also a receptor through which the virus is adsorbed on sensitive cells, including erythrocytes, causing them to stick together , and is involved in the hemolysis of red blood cells. Viral neuraminidase is an enzyme that catalyzes the cleavage of sialic acid from the substrate. It has antigenic properties and at the same time participates in the release of virions from the host cell. Neuraminidase, like hemagglutinin, changes as a result of antigenic drift and shift.Cultivation and reproduction
Influenza viruses are cultivated in chicken embryos and in cell cultures. The optimal environment is chicken embryos, in the amniotic and allantoic cavities of which the virus reproduces within 36-48 hours. The most sensitive to the influenza virus are primary cultures of human embryonic kidney cells and some animals. Reproduction of the virus in these cultures is accompanied by a mild CPE, reminiscent of spontaneous cell degeneration. Influenza viruses are adsorbed on glycoprotein receptors of epithelial cells, into which they penetrate through receptor endocytosis. Transcription and replication of the viral genome occurs in the cell nucleus. In this case, the read individual RNA fragments in the form of m-RNA are translated into ribosomes, where the synthesis of virus-specific proteins occurs. After replication of the viral genome, a pool of viral RNAs is formed, which is used in the assembly of new nucleocapsids.Pathogenesis
Primary reproduction of the virus occurs in the epithelial cells of the respiratory tract. Through the eroded surface of the mucous membrane, the virus enters the blood, causing viremia. The circulation of the virus in the blood is accompanied by damage to the endothelial cells of the blood capillaries, resulting in an increase in their permeability. In severe cases, hemorrhages are observed in the lungs, heart muscle and other internal organs. Influenza viruses, entering the lymph nodes, damage lymphocytes, resulting in acquired immunodeficiency, which contributes to the occurrence of secondary bacterial infections. With influenza, intoxication of the body of varying severity occurs.Immunity
The mechanism of anti-influenza immunity is associated with natural factors of antiviral nonspecific protection, mainly with the production of interferon and natural killer cells. Specific immunity is provided by factors of cellular and humoral response. The first are represented by macrophages and T-killers. The second are immunoglobulins, primarily antihemagglutinins and antineurominidase antibodies, which have virus-neutralizing properties. The latter, unlike antihemagglutinins, only partially neutralize the influenza virus, preventing its spread. Complement-fixing antibodies to the viral nucleoprotein do not have protective properties even after 1.5 months. disappear from the blood of convalescents. Antibodies are detected in the blood serum 3-4 days after the onset of the disease and reach maximum titers after 2-3 weeks. The duration of specific immunity acquired after influenza infection, contrary to previous beliefs, is measured in several decades. This conclusion was reached based on a study of the age structure of the incidence of influenza caused by the A (H1N1) virus in 1977. It was found that this virus, which had been absent since 1957, affected only people under 20 years of age in 1977. Thus, after suffering an influenza infection caused by the influenza virus type A, intense immunity is formed, strictly specific to the subtype of the virus (by H- and N-antigens) that caused its formation. In addition, newborns have passive immunity due to IgG antibodies to the corresponding virus subtype A. Immunity lasts for 6-8 months.Epidemiology
The source of infection is sick people and virus carriers. Transmission of the pathogen occurs by airborne droplets. Influenza is an epidemic infection that occurs more often in the winter and winter-spring months. Approximately every ten years, influenza epidemics become pandemics, affecting the population of different continents. This is explained by the change in the H- and N-antigens of the type A virus associated with antigenic drift and shift. For example, the influenza A virus with hemagglutinin NSW1 caused the Spanish flu pandemic in 1918, which claimed 20 million human lives. In 1957, the “Asian” influenza virus (H2N2) caused a pandemic that affected more than 2 billion people. In 1968, a new pandemic variant emerged, the influenza A (H3N2) virus, called the Hong Kong virus, which continues to circulate to the present day. In 1977, it was joined by the type A virus (H1N1). This was unexpected, since an identical virus had already circulated in 1947-1957, and was then completely replaced by the “Asian” subtype. In this regard, a hypothesis arose that shift variants of the virus are not historically new. They represent serosubtypes circulating in past years. The cessation of circulation of the influenza virus, which caused the next epidemic, is explained by the collective immunity of the population that has developed to this antigenic variant of the pathogen. Against this background, there is a selection of new antigenic variants, collective immunity to which has not yet been formed. It is not yet clear where the shift antigenic variants (serosubtypes) of the influenza A virus that came out of active circulation in one or another historical period are preserved for a long time. It is possible that the reservoir for the persistence of such viruses are wild and domestic animals, especially birds, which are infected with human variants of type A influenza viruses and maintain their circulation for a long time. At the same time, genetic recombinations between avian and human viruses occur in the body of birds, which lead to the formation of new antigenic variants. According to another hypothesis, influenza viruses of all known subtypes constantly circulate among the population, but become epidemically relevant only with a decrease in collective immunity. Influenza viruses of types B and C are characterized by higher antigenic stability. Influenza B viruses cause less intense epidemics and local outbreaks. Influenza virus type C is the cause of sporadic diseases. The influenza virus is quickly destroyed by temperatures above 56°C, UV radiation, disinfectants, and detergents. It remains viable for 1 day. at room temperature, on smooth metal and plastic surfaces - up to 2 days. Influenza viruses survive at low temperatures (-70°C).Specific prevention
For the prevention of influenza, rimantadine is used, which suppresses the reproduction of the influenza virus type A. For passive prevention, human anti-influenza immunoglobulin is used, obtained from the blood serum of donors immunized with influenza vaccine. Human leukocyte interferon has a certain effect. Live and inactivated vaccines are used for vaccine prevention. When live vaccines are administered, both general and local immunity are formed. In addition, interferon induction is noted. Currently, inactivated vaccines of various types have been obtained: virion, subunit, split and mixed. Virion vaccines are produced by high-quality purification of viruses grown in chicken embryos. Subunit vaccines are purified surface antigens of the influenza virus - hemagglutinins and neuraminidase. Such vaccine preparations are characterized by reduced reactogenicity and high immunogenicity. Cleaved or disintegrated vaccines are prepared from a purified virion suspension by treatment with detergents. However, there is still no consensus on the superiority of any one of these vaccines. Inactivated vaccines induce an immune response in the system of general and local humoral immunity, but induce interferon synthesis to a lesser extent compared to live vaccines. Many years of experience in the use of live and inactivated vaccines indicate that the antigenic mismatch of vaccine strains with epidemic ones is the main, but not the only reason low effectiveness of influenza vaccine prevention. In recent years, attempts have been made to create genetically engineered and synthetic influenza vaccines.Flu
Influenza is an acute human respiratory disease that tends to spread epidemically. It is characterized by catarrhal inflammation of the upper respiratory tract, fever, and severe general intoxication. Influenza is often accompanied by severe complications - secondary bacterial pneumonia, exacerbation of chronic lung diseases. Influenza pathogens belong to the Orthomyxoviridae family. It includes three types of viruses - A, B, C. The influenza virus has a spherical shape, its size is 80-120 nm. Sometimes filamentous virions are formed. The genome is formed by a single-stranded minus-strand RNA, which consists of eight fragments, and is surrounded by a protein capsid. RNA associated with 4 internal proteins: nucleoproteins (NP) and high molecular weight proteins PI, P2, R3, involved in genome transcription and virus replication. The nucleocapsid has a helical type of symmetry. Above the capsid shell is a layer of matrix protein (M protein). On the outer, supercapsid shell, hemagglutinin (H) and neuraminidase (N) are located in the form of spines. Both glycoproteins (N and H) have pronounced antigenic properties. In influenza viruses, 13 different antigenic types of hemagglutinin (NI-13) and 10 variants of neuraminidase (N1-10) were found. Based on the internal nucleoprotein antigen, three types of influenza viruses are distinguished - A, B, C, which can be determined in RSC. Type A viruses that infect humans have three types of hemagglutinin (HI, H2, H3) and two neuraminidases (N1, N2). Depending on their combinations, variants of influenza A viruses are distinguished - H1N1, H2N2, H3N2. they are determined in the hemagglutination inhibition reaction with appropriate sera. Influenza viruses are easily cultivated in chicken embryos and various cell cultures. Maximum accumulation of viruses occurs after 2-3 days. In the external environment, the virus quickly loses its infectivity through drying out. At low temperatures in the refrigerator it is stored for a week, at -70 ° C - much longer. Heating causes it to inactivate after a few minutes. Under the influence of ether, phenol, formaldehyde, it is quickly destroyed.Virological diagnostic method
The material for research is swabs from the nasopharynx, nasal discharge, which is taken with dry or wet sterile cotton swabs in the first days of the disease, sputum. Viruses can be found in blood and cerebrospinal fluid. In case of fatal cases, pieces of the affected tissues of the upper and lower respiratory tract, brain, etc. are removed. Nasopharyngeal swabs are taken on an empty stomach. The patient should gargle three times with sterile saline sodium chloride solution (10-15 ml), which is collected in a sterile wide-necked jar. After this, wipe the back wall of the pharynx and nasal passages with a piece of sterile cotton wool, then dip it into a jar with rinsing. You can take the material with a sterile swab moistened in a sodium chloride solution, which is used to thoroughly wipe the back wall of the pharynx. After collecting the material, the swab is immersed in a test tube with physiological solution, to which 5% of inactivated animal serum is added. In the laboratory, swabs are rinsed in liquid, squeezed against the side of the tube, and removed. The drain is kept in the refrigerator to settle, then the middle part of the liquid is collected into sterile tubes. Antibiotics penicillin (200-1000 IU/ml), streptomycin (200-500 μg/ml), nystatin (100-1000 IU/ml) are added to the material to destroy accompanying microflora, kept for 30 minutes at room temperature and used to isolate viruses. having previously checked it for sterility. A sensitive method for isolating viruses that infect 10-11-day-old chicken embryos. Material in a volume of 0.1-0.2 ml is injected into the amniotic or allantois cavity. As a rule, 3-5 embryos are infected. Embryos are incubated at an optimal temperature of 33-34 ° C for 72 hours. In order to increase the number of virions in the test material, it is pre-concentrated. To do this, they use methods of adsorption of viruses on chicken red blood cells, treatment with a 0.2% trypsin solution in order to enhance the infectious properties of viruses, or precipitate them using special methods. After incubation, chicken embryos are cooled at a temperature of 4 ° C for 2-4 hours, then sucked off with sterile with pipettes or a syringe, allantoic or amniotic fluid. In this case, the presence of an infectious virus is determined using RGA. To do this, mix equal volumes (0.2 ml) of virus-resistant material and 1% suspension of chicken red blood cells. A positive reaction (the presence of a virus in the material) is indicated by the sedimentation of erythrocytes in the form of an umbrella. If there is a virus in the material that has hemagglutinous properties, it is titrated using an expanded RGA, determining the titer of hemagglutinous activity. Using this reaction, the titer of the hemagglutinating virus is determined - the highest dilution of the material that still gives the hemagglutination reaction. This amount of virus is taken as one hemagglutinous unit (HAU).Identification of influenza viruses using RTGA
To do this, first prepare a working dilution of the viral material, which contains 4 GAO of the virus in a certain volume. The reaction is taken into account after the formation of a sediment of erythrocytes in the control wells. A positive reaction is indicated by a delay in hemagglutination in the test wells. Influenza viruses can be isolated using various cell culture lines - human embryo, monkey kidneys, continuous canine kidney cell line (MDCK) and others. In cell cultures, the cytopathic effect of viruses is manifested (the appearance of cells with scalloped edges, vacuoles, the formation of intranuclear and cytoplasmic inclusions), which ends with the degeneration of the cell monolayer. To identify the isolated viruses, RTGA is used (provided that the hemagglutinin titer in the culture fluid is at least 1:8). In addition to this reaction, you can use RGGads, however, it is less sensitive and requires an immune serum titer of at least 1:160 as well as RSK, RN, REMA, etc.Serological study
Serological testing is used to confirm the diagnosis of influenza. It is based on determining a fourfold increase in the antibody titer in the patient's serum. The first serum is obtained at the onset of the disease in the acute period (2-5-1 days of illness), the second - after the 10-14th day of the disease. Since the serums can be mixed at the same time, the first of them is stored in the refrigerator at a temperature of -20 ° C. Most often, RTGA, RSK, RNGA are used. These reactions are performed with special sets of standard viral diagnostics (reference strains of influenza virus of various serological types). Since patient sera may contain nonspecific hemagglutination inhibitors, they are first heated at a temperature of 56 ° C and also treated with a special enzyme (for example, neuraminidase) or solutions of potassium periodate, rivanol, manganese chloride, white tire suspension, etc. according to special schemes. ANDHemagglutination inhibition reaction
The hemagglutination inhibition reaction can be performed in test tubes (macromsh tod) or in special plates for immunological studies. The reaction is considered positive when a compact, dense sediment of red blood cells with smooth edges is formed.Express diagnostics
The method is based on identifying specific viral antigens in the test material using immunofluorescence in direct or indirect RIF. Mucus is obtained from the nasal passages or the back wall of the pharynx, centrifuged, and smears are prepared on glass slides from the sediment of columnar epithelial cells of the mucous membrane. they are treated with immunofluorescent sera conjugated to fluorochromes, for example, FITC (fluorescein isothiocyanate). When examining drugs using a fluorescent microscope, a characteristic green-yellow glow of influenza viruses is observed, which are localized at the onset of the disease in the nuclei of epithelial cells. Recently, it has been proposed to use ELISA, RZNGA, and PCR to indicate specific viral antigens.They are intracellular obligate parasites, meaning that they cannot replicate or pass on their genes without help. A single viral particle (virion) is itself inert. When a virus infects a cell, it uses enzymes and the bulk of the cell's structure to replicate.
Unlike what we see in cell division processes such as and, virus replication produces many progeny that destroy the host cell and then infect other cells in the body.
Viral genetic material
Viruses can contain single-stranded/double-stranded DNA or RNA. The type of genetic material found in a particular virus depends on its nature and function. The exact nature of what happens after a host is infected varies depending on the nature of the virus.
The replication process will be different for dsDNA, ssDNA, dsRNA, and single-stranded RNA viruses. For example, double-stranded DNA viruses usually must enter host cells before they can replicate. However, single-stranded RNA viruses replicate primarily into host cells.
Once a virus infects a host, components of the viral progeny are produced by cellular machinery, and assembly of the viral capsid is a non-enzymatic process. Viruses can usually only infect a limited number of hosts. The "lock and key" mechanism is the most common explanation for this phenomenon. Certain proteins on the virus particle must match certain receptor proteins on the cell surface of a particular host.
How do viruses infect cells?
The basic process of infection and replication of the virus occurs in 6 stages:
- Adsorption - the virus binds to the host cell.
- Entry - the virus introduces its genome into the host cell.
- Viral genome replication - The viral genome is replicated using the host's cellular structure.
- Assembly - viral components and enzymes are formed and begin to assemble.
- Maturation - viruses develop from the assembled components.
- Exit - new viruses break out of the host cell in search of new victims to infect.
Viruses can infect any type of cell, including