Structure and chemical composition
The influenza virus has a spherical shape, with a diameter of 80-120 nm. Filamentous forms are less common. The helical nucleocapsid is a double helix ribonucleoprotein (RNP) strand 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 protein M, which connects the RNP to the double lipid layer of the outer shell and the 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 virus genome 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 each, 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 that stabilizes the structure of the virion. These antigens are detected in CSCs. The narrower specificity of type A virus is determined by two other surface antigens - hemagglutinin H and neuraminidase N, denoted 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 RTGA. The variability of hemagglutinin (H-antigen) determines the antigenic drift and shift of the influenza virus. Antigenic drift is understood as minor changes in the H-antigen caused by point mutations in the gene that controls its formation. Such changes can accumulate in the 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 that cause major epidemics and pandemics. Hemagglutinin is also a receptor by which the virus is adsorbed on sensitive cells, including erythrocytes, causing them to stick together , and is involved in the hemolysis of erythrocytes. 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 for 36-48 hours. The most sensitive to the influenza virus are the primary cultures of kidney cells of the human embryo and some animals. The reproduction of the virus in these cultures is accompanied by mild CPP, resembling spontaneous cell degeneration. Influenza viruses are adsorbed on glycoprotein receptors of epithelial cells, into which they penetrate by receptor endocytosis. Transcription and replication of the viral genome takes place in the cell nucleus. In this case, the read individual RNA fragments in the form of mRNA are translated to ribosomes, where virus-specific proteins are synthesized. After replication of the viral genome, a pool of viral RNA is formed, which is used in the assembly of new nucleocapsids.Pathogenesis
The 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 bloodstream, 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. Influenza causes intoxication of the body of varying severity.Immunity
The mechanism of anti-influenza immunity is associated with natural factors of antiviral non-specific protection, mainly with the production of interferon and natural killer cells. Specific immunity is provided by cellular and humoral response factors. The former are represented by macrophages and T-killers. The latter 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 and after 1.5 months. disappear from the blood of convalescents. Antibodies are found 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 an influenza infection, contrary to previous ideas, is measured in several decades. This conclusion was reached on the basis of 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 in 1977 only persons not older than 20 years. Thus, after suffering an influenza infection caused by type A influenza virus, 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 class antibodies to the corresponding virus subtype A. Immunity persists for 6-8 months.Epidemiology
The source of infection are sick people and virus carriers. The transmission of the pathogen occurs by airborne droplets. Influenza refers to epidemic infections that often occur in the winter and winter-spring months. Approximately every ten years, influenza epidemics take the form of pandemics, covering the population of different continents. This is due to the change in H- and N-antigens of 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 appeared, the influenza A (H3N2) virus, called the "Hong Kong virus", which continues to circulate to this day. In 1977, a type A (H1N1) virus joined it. This was unexpected, since an identical virus was already circulating in 1947-1957, and then was completely replaced by the "Asian" subtype. In this regard, a hypothesis arose that the shift variants of the virus are not historically new. They are serosubtypes circulating in the past years. The cessation of circulation of the influenza virus that caused another epidemic is explained by the collective immunity of the population that has developed to this antigenic variant of the pathogen. Against this background, selection of new antigenic variants occurs, the collective immunity to which has not yet been formed. It is not yet clear where the shift antigenic variants (serosubtypes) of influenza type A virus that have left active circulation in a particular historical period are stored for a long time. It is possible that the reservoir of such viruses are wild and domestic animals, especially birds, which are infected with human variants of influenza A viruses and keep them circulating 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 have a higher antigenic stability. Type B influenza viruses cause less intense epidemics and localized outbreaks. Influenza type C virus is the cause of sporadic diseases. The influenza virus is rapidly destroyed by temperatures above 56 ° C, UV radiation, disinfectants, detergents. It retains its viability 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 prophylaxis
For the prevention of influenza, rimantadine is used, which suppresses the reproduction of influenza type A virus. For passive prophylaxis, human anti-influenza immunoglobulin obtained from the blood serum of donors immunized with the influenza vaccine is used. Human leukocyte interferon has a certain effect. Live and inactivated vaccines are used for vaccination. With the introduction of live vaccines, both general and local immunity are formed. In addition, interferon induction is noted. At present, inactivated vaccines of various types have been obtained: virion, subunit, split and mixed. Virion vaccines are obtained 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 low reactogenicity and high immunogenicity. Cleaved or disintegrated vaccines are obtained from a purified suspension of virions by treatment with detergents. However, there is as yet no consensus on the benefit of any one of these vaccines. Inactivated vaccines induce an immune response in the system of general and local humoral immunity, but to a lesser extent than live vaccines induce the synthesis of interferon. Many years of experience in the use of live and inactivated vaccines indicates that the antigenic mismatch of vaccine strains with epidemic strains is the main, but not the only reason low effectiveness of influenza vaccination. 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, severe general intoxication. Influenza is often accompanied by the occurrence of severe complications - secondary bacterial pneumonia, exacerbation of chronic lung diseases. Influenza pathogens belong to the Orthomyxoviridae family. It includes three genera of viruses - A, B, C. The influenza virus has a spherical shape, its dimensions are 80-120 nm. Sometimes filamentous virions are formed. The genome is formed by a single-stranded negative strand of 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, P3 involved in genome transcription and virus replication. The nucleocapsid has helical symmetry. Above the capsid membrane is a layer of matrix protein (M protein). On the outer, supercapsid membrane, 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. According to the internal nucleoprotein antigen, three types of influenza viruses are distinguished - A, B, C, which can be determined in the RSK. Type A viruses that infect humans have three types of hemagglutinin (HI, H2, H3) and two types of neuraminidase (N1, N2). Depending on their combinations, there are variants of influenza A viruses - H1N1, H2N2, H3N2. they are determined in the hemagglutination inhibition reaction with the corresponding sera. Influenza viruses are easily cultivated in chicken embryos and various cell cultures. The maximum accumulation of viruses occurs after 2-3 days. In the external environment, the virus quickly loses its infectivity through drying. At a low temperature in the refrigerator it is stored for a week, at -70 ° C - much longer. Heating leads to its inactivation after a few minutes. Under the influence of ether, phenol, formalin quickly collapses.Virological diagnostic method
Nasopharyngeal swabs, nasal discharge taken with dry or wet sterile cotton swabs in the first days of the disease, sputum were used as material for research. Viruses can be found in the blood, cerebrospinal fluid. In fatal cases, pieces of affected tissues of the upper and lower respiratory tract, brain, etc. are taken. Nasopharyngeal swabs are taken on an empty stomach. The patient should rinse the throat three times with sterile saline sodium chloride solution (10-15 ml), which is collected in a sterile wide-mouthed jar. After that, a piece of sterile cotton wool is wiped with the back wall of the pharynx, nasal passages, then it is dipped into a jar with flushing. You can take the material with a sterile swab moistened in sodium chloride solution, which is carefully wiped with the back wall of the throat. After taking the material, the swab is immersed in a test tube with physiological saline, to which 5% of inactivated animal serum is added. In the laboratory, swabs are rinsed in liquid, squeezed against the wall of the test tube and removed. The drain is kept in a refrigerator for settling, then the middle part of the liquid is taken into sterile test tubes. Antibiotics penicillin (200-1000 units/ml), streptomycin (200-500 μg/ml), nystatin (100-1000 units/ml) are added to the material to destroy the 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 infects 10-11-day-old chicken embryos. The material in a volume of 0.1-0.2 ml is injected into the amniotic or allantois cavity. Infect, as a rule, 3-5 embryos. Embryos are incubated at the optimum 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, use the methods of adsorption of viruses on chicken erythrocytes, treatment with a 0.2% trypsin solution 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 aspirated sterile pipettes or syringe allantoisnu or amniotic fluid. In this, with the help of RHA, the presence of an infectious virus is determined. To do this, mix equal volumes (0.2 ml) of virus material and 1% suspension of chicken erythrocytes. A positive reaction (the presence of a virus in the material) is evidenced by the sedimentation of erythrocytes in the form of an umbrella. If there is a virus in the material that has hemagglutination properties, it is titrated using an expanded RGA, determining the titer of hemagglutination activity. With the help of this reaction, the titer of the hemagglutination virus is determined - the highest dilution of the material, which still gives the hemagglutination reaction. This amount of virus is taken as one hemagglutination 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. Accounting for the reaction is carried out after the formation of a sediment of erythrocytes in the control wells. A positive reaction is evidenced by a delay in hemagglutination in the test wells. Influenza viruses can be isolated using various cell culture lines - human embryo, monkey kidney, continuous dog 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. 1:8). In addition to this reaction, RGGads can be used, however, it is less sensitive and requires an immune serum titer of at least 1:160, as well as RSK, RN, PEMA, etc.Serological study
A serological test is used to confirm the diagnosis of influenza. It is based on determining a fourfold increase in 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 consumed simultaneously, the first of them is stored in a refrigerator at a temperature of -20 ° C. RTGA, RSK, RNGA are most often used. These reactions are set with special sets of standard viral diagnostic kits (reference strains of the influenza virus of various serological types). Since the sera of patients may contain non-specific inhibitors of hemagglutination, 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 put in test tubes (macroshtod) or in special plates for immunological studies. The reaction is considered positive when a compact, dense erythrocyte sediment with smooth edges is formed.Express Diagnostics
The method is based on the detection of specific viral antigens in the test material using immunofluorescence in direct or indirect RIF. Mucus is obtained from the nasal passages or the posterior pharyngeal wall, centrifuged, and smears are prepared from the sediment of cells of the cylindrical epithelium of the mucous membrane on glass slides. they are treated with immunofluorescent sera conjugated to fluorochromes, such as FITC (fluorescein isothiocyanate). When examining preparations using a luminescent 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.The epidemic season of 2017-2018 is on its way. Vaccinators prepare syringes, therapists prepare phonendoscopes, pharmacists stock up on “anti-influenza drugs”, and the population reads media reports and hopes to survive another seasonal viral attack with minimal losses. Over the years of active development of the information space, citizens have already become accustomed to the mysterious names H1N1 or H5N1, and some already know that the first is swine flu, and the second is bird flu. But until now, few of the ordinary patients - former and future - understand how the influenza virus works and how exactly it works. MedAboutMe will fill this gap.
How is the flu virus organized?
Influenza viruses belong to a separate family of orthomyxoviruses. Their genome does not contain double-stranded DNA, as in humans, but single-stranded RNA. Moreover, this chain consists of 8 separate fragments encoding in general only 11 proteins. Fragments of RNA even replicate, that is, multiply independently of each other. This is an important point that explains why influenza viruses change and form new varieties so easily. If two different strains of the influenza virus penetrated into one cell, then they can exchange separate sections of the genome, thus giving rise to new, previously non-existing reassortant viruses.
The shape of the virus is a sphere. At the very heart of this sphere are fragments of an RNA strand, each of which is associated with a set of proteins responsible for the replication of this particular fragment of the genome, that is, they represent 8 nucleoproteins. All these nucleoproteins are packaged in a nucleocapsid, a delicately screwed protein shell. And on top - and this is a special feature of the so-called enveloped viruses - there is another coating, which is called the supercapsid.
The supercapsid is an extremely important formation for the influenza virus. In fact, this is a lipid bilayer membrane, which includes several types of glycoproteins - complexes of proteins and carbohydrates. It is by glycoproteins that scientists determine what kind of strain of the influenza virus got into their test tube. It is thanks to these compounds that the virus enters the cell and multiplies. And, finally, some effective drugs against influenza are aimed at contact with glycoproteins.
What unique compounds can be found on the surface of the supercapsid of the influenza virus?
- Hemagglutinin.
This is a compound with which the virus, firstly, recognizes the receptors of the cells of the host organism, and secondly, attaches to them. Antibodies to hemagglutinin are formed when a person becomes ill with a certain strain of the influenza virus and provides future protection against it. There are 16 subtypes of hemagglutinin.
- Neuraminidase.
This is an enzyme that, firstly, destroys the components of the protective layer of mucus on the mucous membranes of the respiratory tract and thereby facilitates the passage of the virus to the target cell. Secondly, neuraminidase is involved in the fusion of the viral particle with the cell. Finally, it ensures the release of new viral particles from the infected cell. If there were no neuraminidase, then the reproduction cycle would be limited to just one cell, even without any symptoms of the disease. Antibodies to neuraminidase are formed in our body as a result of vaccination - they do not allow the influenza virus to spread throughout the body. There are 9 subtypes of neuraminidase in influenza A viruses and one each in influenza B and C viruses.
- M2 protein.
This is the so-called ion channel, that is, an adjustable “hole” in the virus membrane through which ions can move. Since we are talking about ions, it means that we are talking about the charges that they carry, that is, when the ion channel is working, the pH inside the viral particle will change. M2-protein is intended for the transfer of protons, that is, the nuclei of the hydrogen atom that have a positive charge (H +).
So, the influenza virus, with the help of neuraminidase, made its way through the layer of mucus in the respiratory tract and reached the surface of the epithelial cell, more precisely, to the ciliated epithelium lining them. Neuraminidase has a special "pocket" with which it binds to small carbohydrate residues protruding from the cell membrane (oligosaccharides). In this case, the supercapsid of the virus comes into contact with the cell membrane and their lipid layers merge. As a result, the nucleocapsid, containing, as we remember, 8 segments of RNA, enters the cell, into its cytoplasm.
While the process of penetration of the nucleocapsid of the virus into the cell is underway, the M2 protein is actively working. It pumps protons into the virus, which means that the environment inside it becomes more and more acidic. As a result of these manipulations, the contents of the nucleocapsid penetrate into the cell nucleus. At the same time, segments of viral RNA are released in the form of complexes with proteins, which receive all the necessary resources of the cell at their disposal and start the production of new viruses. This is also a very thoughtful process, during which “temporary” mRNAs are formed, leaving the nucleus for the cytoplasm to organize the synthesis of viral proteins there. Then these proteins are transported to the nucleus, where, finally, the assembly of viral particles takes place. Part of the new genomic RNA is used for additional replication of the virus genome.
One can only admire the accuracy of assembling 8 different viral RNA segments into one future viral particle. It is impossible for two identical segments to enter the same nucleocapsid, and the mechanism of this process is still unknown. At this moment, the formation of reassortant viruses, which we discussed above, can just occur. Finally, the finished nucleocapsids move into the cytoplasm. When passing through the cell membrane, the freshly assembled nucleocapsid receives a supercapsid shell with the entire set of glycoproteins.
The entire cycle from the penetration of the virus into the cell to the release of new viral particles from it takes from 6 to 8 hours. Numerous viruses come out and infect neighboring cells. Less commonly, virions enter the bloodstream and spread throughout the body. The spread of the virus through tissues and organs is called viremia. The peak of influenza virus replication is observed in the interval from 24 to 72 hours from the moment the viral particles enter the epithelium of the respiratory tract.
When new virions are released, the cells in which they reproduced die. The inflammatory process breaks out. Therefore, with influenza, the upper respiratory tract is primarily affected, gradually the inflammation covers the trachea and bronchi. If viruses enter the bloodstream and spread throughout the body, the infection becomes generalized, intoxication of the body develops.
The danger of the flu lies in the fact that it affects the blood vessels and the nervous system. Against the background of infection with the influenza virus, there is a massive formation of reactive oxygen species (ROS), that is, free radicals that tend to oxidize everything that gets in their way.
It should be understood that the influenza virus itself does not contain toxins. The toxic effect is exerted by compounds that our body produces in an attempt to protect itself from the virus. This reaction is so violent, and the place for the introduction of the virus is chosen so “successfully” that a person suffers from his own immune system. According to research, ROS trigger the processes of proteolysis - the destruction of proteins. This occurs in the airways at the border with air, resulting in a "respiratory" or "metabolic" burst.
Since the process of introduction and reproduction of the virus takes place in the respiratory tract, the walls of the capillaries located there (small blood vessels) suffer first of all. They become more brittle, permeable, which in severe cases leads to disruption of local circulation, the development of hemorrhagic syndrome and the threat of pulmonary edema. Against the background of damage to the vascular system, the blood supply to the brain may worsen and, as a result, a neurotoxic syndrome is formed.
The immune system at this time activates the production of a huge amount of cytokines - substances that trigger inflammatory reactions and have a cytotoxic effect. Normally, they should be engaged in the inactivation and elimination of infectious agents. But the scale of the process is so great that a systemic inflammatory response develops.
As a result, due to damage to the mucous membrane of the respiratory tract and blood vessels, the ability of the immune system to withstand external threats decreases, and the activity of protective blood cells of neutrophils decreases. In general, this leads to the activation of existing chronic diseases and increases the risk of bacterial infection. The most severe and common complication of influenza is pneumonia.
Different strains of influenza differ from each other, in particular, the ability to activate the massive production of ROS. Therefore, some types of flu are more severe, while others are easier. To a large extent, the state of the patient's body, his immune status, experience of acquaintance with other strains play a role. Some types of influenza are more dangerous for the elderly and children, while others more often affect the population in their prime.
To stop the process of virus replication in cells and its spread throughout the body, substances are needed that can interrupt its reproduction cycle, honed by evolution.
In 1961, scientists proposed to fight influenza viruses with amantadine. This compound was approved for use in 1966, and in 1993 rimantadine, its analogue, appeared. Amantadine (and rimantadine) are able to block the ion channels of the M2 protein. This stops the replication of the virus in the initial stages.
The drug was very effective against group A viruses, but had no effect on group B and C viruses. And in 2006, the US Centers for Disease Control and Prevention (CDC) published data on the extremely high resistance (resistance) of some virus strains to adamantanes, reaching up to 90%. The cause was point mutations in the genome of the virus that occurred during treatment with adamantanes. So today, rimantadine and its other analogues are considered ineffective drugs. Moreover, they were initially useless against viruses of groups B and C.
In 1983, neuraminidase inhibitors were developed - substances that block the enzyme's ability to trigger the release of new virions from an infected cell. This allows you to stop the replication and spread of the virus.
Neuraminidase inhibitors include oseltamivir (Tamiflu) and zanamivir (Relenza). Since 2009, another intravenous drug from this group, paramivir, has been approved for use in the United States. These drugs are, in fact, the only drugs designed specifically to fight the flu virus. But they should be taken within 24-48 hours from the moment of the first manifestations of the disease. Later they will be ineffective - numerous new viruses will already spread throughout the body.
All other so-called antiviral agents have no effect on the influenza virus itself or on individual stages of its penetration into the body, reproduction and spread.
- The influenza virus is a construct designed by nature to penetrate the body through the respiratory tract and equipped with all the necessary "master keys" for this.
- There are only a few types of drugs that act specifically on the influenza virus, taking into account the characteristics of its life cycle and structure. But one of these drugs is already ineffective, as the virus has adapted to it. And other types of drugs are effective only for a very short period after the onset of the first symptoms. The anti-influenza effect of other drugs has not been proven.
- Therefore, for the treatment of influenza, symptomatic therapy and monitoring of the patient's condition are used. In most cases, with the flu, it is enough to simply lie down at home, taking drugs to reduce the high temperature if it has grown to 39 ° C, and other means to alleviate the patient's condition. It is important to prevent the development of complications - for this you just need to create all the conditions for the body to fight the virus.
- Vaccination remains the best way to fight the virus. Even if a person has been vaccinated against one strain and picked up another, existing antibodies can provide at least minimal protection and ease the course of the disease.
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K. SCHOLTISSEK and H.-D. KLENK (CH. SCHOLTISSEK, H.-D. KLENK)
I. INTRODUCTION
There are a number of reviews on the problem of influenza virus replication. The literature prior to 1968 is summarized in Hoyle (1968) and Scholtissek (1969); more recent reviews are White (1973) and Compans and Choppin (1974).
Most of the data on replication was obtained from the study of type A influenza virus. Significant differences in (Mechanisms of replication of other types of influenza virus have not yet been found.
Several cell culture systems suitable for studying replication have become popular, such as propagation of the WSN strain of influenza virus in MDBK cells (Choppin, 1969) or propagation of bird distemper virus (FPV) in chick embryo fibroblast cells. An example of the growth curve of the last virus in a single cycle is proven at 30, where the latency period is approximately 3 hours and the production of the virus reaches a plateau between 8 and 12 hours. infections. Therefore, such systems are very convenient for biochemical studies.
II. ADSORPTION, PENETRATION, "STRIPING" OF THE VIRUS
Infection of a cell with a virus begins with adsorption, i.e., attachment of a viral particle to the cell surface. Attachment requires two complementary structures, namely: receptor sites on the cell surface and a viral component responsible for recognizing these receptor sites. The ability of the influenza virus to interact with erythrocytes of various origins and agglutinate them has been known for many years (Hirst, 1941; McClelland, Hare, 1941.) Hemagglutination has been used as a model for the interaction of influenza virus with a cell surface, and "most of your knowledge of this phenomenon comes from such studies. However, one should be very careful in generalizations, since the structures of the surface of erythrocytes and the surfaces of infected cells can be completely different (see also Chapter 3).
A. ROLE OF HEMAGGLUTININ IN ADSORPTION
The virion component involved in binding is the HA* spike. The role of viral β-proteins in infection initiation has been studied using antibodies specific for two surface proteins: HA* and NA*. These antibodies can be obtained using recombinant viruses. For example, crossing between AO and A2 viruses results in the formation of the X7F1 recombinant, which carries HA*, AO, and NA* A2 (Kilbourne et al., 1968). Antiserum against the X7F1 virus does not inhibit NA* of AO-type influenza viruses, but inhibits hemagglutination and neutralizes the infectivity of this type of viruses. The interaction of the same serum with A2 influenza viruses does not inhibit hemagglutination or infectivity, although the neutralization of NA* activity is complete. Thus, NA* is not involved in the infection initiation process, and only NA* appears to be responsible for adsorption. This concept is supported by evidence that viral particles from which only neuraminidase spikes were removed by proteolytic enzymes remained infectious (Schulze, 1970).
There is evidence that the hemagglutinating part is localized on the outer part of the "spike" of HA*, rich in carbohydrates (see Chap. 3). Carbohydrates appear to be essential for HA* to function, since non-glycosylated HA* proteins are unable to bind to erythrocytes (Klenk et al., 1972b).
B. INFLUENZA VIRUS RECEPTOR
Carbohydrates are an essential (component not only of hemagglutishin, but also of the viral receptor on the cell surface. Hirst (1942) observed that the virus-erythrocyte complex is unstable and that the receptor on the cell surface is destroyed by the enzyme of the virus. As was shown later, this enzyme is neuraminidase , which cleaves neuraschic acid from glycoproteins (Klenk et al., 1955; Klenk, Stoffel, 1956; Gottschalk, 1957). This was the first demonstration of an enzyme that is an integral part of a viral particle. , 1947. Thus, it was found that the receptor for the influenza virus is a glycoprotein containing neuraminic acid.
Since then, a lot of information has been accumulated
about the myxovirus receptor, summarized recently in a review
Hughes (1973). Briefly, the obtained data are reduced to the following
blowing. Receptor sites contain remnants of neuromyo-
ioic acid, which are present in carbohydrate chains
glycoproteins. Non-oxidizing terminal residues of neurams
noznla are necessary for the interaction of glycoproteins with vi
influenza virus. Treatment with neuraminidase completely removes
binding activity. Degradation studies with
using periodat suggest that for the linking
activity requires an intact neuraminic molecule
acids (Suttajit and Winzler, 1971). carboxyl group,
probably also plays an important role, since it is necessary,
apparently also electrostatic forces (Huang, 1974).
It seems likely that there is only a weak special
feature with respect to the structure with which it is associated
neuraminic acid, since, as has been shown, the whole
a set of glycoproteins containing yairamic acid,
associated with myxoviruses. Moreover, gangliosides (gli-
are active in this regard (Haywood, 1975).
It is to be hoped that the issue of influenza virus binding will become clearer once the molecular structure of the receptor has been established. To some extent, this has already been achieved in the case of erythrocytes (Marchesi et al., 1973). Additional information may also be provided by the study of the attachment of myxoviruses to artificial membranes (Tiffany and Blough, 1971).
C. POSSIBLE MECHANISMS OF PENETRATION AND "STRIPING"
Two different "Mechanisms for the penetration and" stripping "of not only the influenza virus, but also viruses in general, have been proposed. Both points of view are based mainly on studies in the electron microscope. One of these mechanisms is vironexis, which is believed to is a process of ppnocytosis when viral particles are included in pinosomes, which subsequently merge with lysosomes, and the enzymes of lysosomes cause the virus to “undress” (Fazekas de St. Grot, 1948) Electron microscopic confirmation of this point of view was obtained in the works of Dales and Choppin (1962) and Dourmashkin and Tyrrell (1970) At 10 minutes after infection, viral particles were seen in direct contact with the cell surface, and by 20 minutes the particles were found within cytoplasmic vacuoles.In contrast to this study, Morgan and Rose (1968) suggest that entry may be due to the fusion of the viral envelope with the host cell membrane. Thus, at present there is no consensus regarding the mechanism of penetration of the influenza virus.
As described in ch. 5, influenza virus virions contain an RNA polymerase associated with their ribonucleoprotein components. Therefore, it is unlikely that the process of "undressing" is spatially dissociated from the process of releasing ribonucleolrotein. And this stage occurs on the cell surface, according to the mechanism of membrane fusion, and in phagocytic vesicles, according to the mechanism of virohexis.
III. TRANSCRIPTION A. SEQUENCE OF RNA SYNTHESIS
After "undressing" the sRNA of the virion must be transcribed into complementary RNA. The RNA polymerase introduced with the infecting particle must function in the first stage of virus reproduction (see Chapter 5). The influenza virus genome can be isolated from virions only in the form
individual fragments (see Ch. 6). Moreover, it functions in the form of - separate fragments, as was shown by genetic analysis (om. Ch. 7) and stepwise inactivation of the infectious virus (Scholtissek, Rott, 1964). In this regard, it should be assumed that - polymerase initiates the synthesis RNA in each individual fragment Since RNA does not exist in the cell as a free molecule, but is always dressed with a protein, the question arises: what protein is the viral RNA associated with during its replication?
Experiments conducted on the synthesis of influenza virus RNA present great difficulties, since actinomycin D cannot be used to detect viral RNA production with specific inhibition of cellular RNA synthesis, since this antibiotic inhibits the reproduction of the influenza virus (Barry et al., 1962 ; Rott and Scholtissek, 1964; Barry et al., 1965; Pons, 1967). For this reason, to determine the sequence of RNA synthesis over time, specific hybridization of pulse-labeled RNA was used at various points after infection with an excess of either unlabeled vRNA or ecRNA, followed by treatment with RNase (Scholtissek and Rott, 1970). In the early stages of the infectious cycle, ecRNA synthesis prevailed, reaching a maximum about 2 hours after infection, while in the later stages most of the produced virus-specific RNA was vRNA. Krug (1972), using a different method, also showed that 4 hours after infection, BIKPHK synthesis almost completely stops. After extraction with phenol, a relatively small amount of lncRNA is found (Scholtissek and Rott, 1970).
Due to the fact that one or another type of viral RNA has information1!! functions and is used as a template for the synthesis of viral proteins (see section IVA), there may be some translational control at the level of differential viral RNA catabolism. Therefore, pulse-chase experiments were carried out to study the stability of influenza virus RNA in vivo. It has been found that, in contrast to cellular RNA, both types of viral RNA are completely stable during the 90-minute chenza period (Scholtissek et al., 1972).
Previously, when studying the synthesis of viral RNA in vivo, when actinomycin D was added at the late stages of the infectious cycle (Duesberg and Robinson, 1967; Nayak, 1970; Ma-hy, 1970), it was not taken into account that the antibiotic specifically inhibits the synthesis of complementary RNA in vivo (Scholtissek and Rott, 1970; Pons, 1973). Since lncRNA is isolated from infected cells as at least five separate fragments, it was concluded that viral RNA is also synthesized as fragments (Pons and Hirst, 1968).
B. LOCALIZATION OF VIRAL RNA SYNTHESIS INSIDE THE HOST CELL
From the data obtained by autoradiography, it was concluded that the site of viral RNA synthesis is, apparently, cell nuclei (Scholtissek et al., 1962; Barry et al., 1974). Since the pulse periods used in these studies were still too long, it cannot be ruled out that the viral RNA is synthesized in the cytoplasm of the cell and then transported to the nuclei where it can accumulate. In addition, vRNA and scRNA can be synthesized in a cell in different places.
C. INHIBITION OF VIRAL RNA SYNTHESIS 1. Actimomycin D, Mithramycin and α-amanitin
When actinomycin D or mithramycin, which interfere with DNA template function, is added to infected cells at a time when viral RNA-dependent RNA iopolymerase is already present (for example, 2 hours after infection), vRNA continues to be synthesized for about 2 hours more. scRNA production, however, stops immediately. Later, vRNA synthesis also decreases, indicating that it requires continuous production of scRNA (Rott et al., 1965; Scholtissek and Rott, 1970; Scholtissek et al., 1970; Pons, 1973). Gregoriades (1970) showed that actinomycin D also has a strong effect on vRNA synthesis when added late in the infectious cycle. In these experiments, the synthesis of viral RNA was determined by the increase in the incorporation of labeled uridine into the total RNA of infected cells. This increase can be abolished by the addition of actinomycin D. However, it should be kept in mind that influenza virus infection causes an increase in the incorporation of labeled uridine into the cell after infection (Scholtissek et al., 1967) and that actinomycin D has an inhibitory effect on this incorporation (Scholtissek et al. ., 1969). siAmanitin, which has no affinity for DNA, affects the activity of one of the cellular RNA polymerases (RNA podimerase II), also inhibits the synthesis of escRNA when added to the culture fluid immediately after infection (Rott and Scholtissek, 1970; Mahy et al., 1972).
The mechanism by which these antibiotics specifically inhibit ssRNA synthesis is not entirely understood, as they do not interfere with ssRNA formation in vitro. Thus, these antibiotics only act in vivo, although the ssRNA-synthesizing enzyme can be isolated from cells that reach after 2 h after infection, actinomycin D was added (Scholtissek and Rott, 1969a).
Reproduction of "influenza viruses can also be suppressed by other actions on the DNA of the host cell - the introduction of mitomcin C, pretreatment with ultraviolet radiation or removal of cell nuclei before infection (Barry, 1964; Rott et al., 1965; Nayak, Rasmussen, 1966; Fol- .lett et al., 1974; Kelly et al., 1974). The mechanism by which these effects affect influenza virus replication may "be the same as that of other antibiotics. The only suggestion" that can be made from these studies is that there is a need for "functionally" active cell nuclei and (or) in the DNA-dependent function of the cell for reproduction of influenza viruses. It is impossible to say what these functions are.
2. Cycloheximide
When cycloheximide, which specifically inhibits protein synthesis in animal cells, is added, 2 h after infection with the influenza virus, the formation of vRNA immediately stops, while the formation of vzhRNA still continues for at least 2 h (Scholtissek and Rott, 1970; Pons, 1973). It is not yet known whether continuous synthesis of a viral or "Cellular" protein used as a "Quality/cofactor for the polymerase that synthesizes vRNA" is necessary, or if some "protein (for example, NP protein) is necessary to stabilize the newly synthesized vRNA, or suppress the synthesis of a certain viral protein leads to the continuous formation of ecRNA, the synthesis of which is normally turned off 3 hours after infection. This shutdown may be necessary to trigger vRNA synthesis. Studies with temperature-sensitive mutants should answer some of these questions.
Experiments by Bean and Simpson (1973) showed that in vivo primary transcription (synthesis of ecRNA on the mRNA template using the polymerase of the infecting particle) is not suppressed by cycloheximide, whereas actinomycin D completely suppresses transcription. Thus, cycloheximide does not affect in vivo the activity of the polymerase introduced with the infecting particle and synthesizing ecRNA. However, it inhibits the synthesis of a new polymerase necessary for the production of ecRNA.
3. Glucosamine
Glucosamine is known to deplete the UTP pool in chick embryo cells by generating UTP-]M-acetylglucosamine (Scholtissek, 1971). When Earle's solution containing glucose is used as a culture medium, it
affects only the synthesis of viral glycoproteins (see section V). If, however, glucose as an energy source is replaced by lyruvate or fucose, then the depletion of the UTP pool by these amino sugars occurs approximately 10 times more actively. Under these conditions, the UTP pool of the host cell becomes a specific limiter on the rate of vRNA synthesis, while the synthesis of (cellular RNA) is not yet affected (Scholtissek, 1975). As a result of the suppression of viral RNA synthesis, the formation of viral proteins is also absent.
These data can be interpreted in two ways: either the viral RNA-dependent RNA polymerase has a low affinity for UTP compared to cellular DNA-dependent "and RIC polymerases, or there are two more or less independent pools of UTP in the cell, one of which can " be used for the synthesis of viral RNA and is more affected by glucosamine than the other pool, which can be used as a substrate by RNA polymerases.
D. SYNTHESIS OF VIRAL RNA IN VITRO
In cells infected with the influenza virus, several researchers have found RNA-dependent RNA polymerase (Ho, Walters, 1966; Scholtissek, Rott, 1969a; Skehel, Burke, 1969; Ruck et al., 1969; Mahy, Bromley, 1970; Compans, Caliguiri, 1973). Most of the enzyme activity is found in the microsomal fraction of infected cells. In the in vitro system, this activity is removed by RNase, but not by DNase. This means that the internal template is RNA. The reaction requires the presence of all four nucleoside triphosphates and is sensitive to actinom-icin D. Most of the production of the reaction in vitro has a low relative molecular weight. The data of Horisberger and Guskey (1973) suggest that two different enzyme activities are present in the cytoplasm: one is Mg++ dependent and inhibited by relatively high salt concentrations, the other is Mn++ dependent and more resistant to salts. The last activity of the enzyme is also found inside the viral particle (see Chapter 5).
Conflicting results have been obtained with respect to the cytoplasmic enzyme product in the in vitro system. Ruck ■ et al. (1969) reported that in their hands this enzyme synthesizes at least "some of the virion-type RNAs (from 14 to 19S). The authors came to this conclusion when determining the base composition of the product in an in vitro system after incubation of the microsomal fraction with all four -labeled nucleoside triphosphates of known specific radioactivity.
to the adenylic acid of the neighbors obtained in the same work using [(a-32P]ATP) are consistent with the data of the nearest neighbor analysis obtained by Scholtissek (1969), as a result of which it was concluded that the product in the in vitro system has the structure of an escRNA Mahy and Bromley (1970) in their original publication also stated that some of the product in an in vitro system produced by a cytoplasmic enzyme must "be an esRNA. However, recently Hastie and Mahy (1973) in their nearest neighbor analysis and specific hybridization confirmed the production of almost exclusively scRNA by the cytoplasmic enzyme, as was first shown by Scholtissek (1969). less than 90% of which has a base sequence complementary to svRNA Hastie and Mahy (1973) "found that a significant percentage of the product in the system ieme in vitro, synthesized by a nuclear enzyme in the presence of actinomycin D, was not able to hybridize with unlabeled vRNA. It is not yet clear which type of RNA is not capable of such hybridization. A very small proportion of the RNA synthesized under these conditions hybridizes with unlabeled acRNA (Scholtissek, unpublished data).
The kinetics of labeled GTP incorporation into viral RNA can be interpreted as indicating that there is no reinitiation of RNA synthesis in the in vitro system. If the crude enzyme preparation is incubated at low salt concentrations, almost all newly synthesized RNA is initially single-stranded. However, after extraction with phenol, "a large percentage of RNA becomes RNAase resistant. Phenol converts the intermediate replication structure, consisting of a single-stranded template and newly synthesized scRNA held together at the site of replication by the polymerase molecule, into a partially double-stranded structure (Feix et al., 1967; Oberg and Philipson, 1971). These data on the product of the influenza virus enzyme in the in vitro system can be interpreted in such a way that the polymerase not only initiates and continues polymerization, but it separates the newly synthesized chain from its template. Otherwise, a double-stranded RNA structure is formed, which does not have "biological functions" (Paffenholz, Scholtissek, 1973). ).
This property of influenza virus RNA polymerase to synthesize exclusively scRNA in an in vitro system has been used
Called to establish the genetic relationship of various strains of the influenza virus by determining the homology in the base sequence between them (Scholtissek, Rott, 1969b; Hobson, Scholtissek, 1970; Anschutz et al., 1972).
IV. SYNTHESIS OF VIRAL PROTEINS
A. IN VITRO TRANSLATION
The problem of which type of RNA - whether vir ionic or complementary - is "informative for the synthesis of viral proteins, has not yet been resolved. Conflicting results have been obtained regarding the type of virus-specific RNA associated with polysomes. Nayak (1970) found in the poly-somal region of the sucrose gradient, mainly vRNA, while Pons (1972) isolated only scRNA from polysam.The latter was confirmed by the observation that after addition 2 h after infection, actinomycin D, which preferentially affects the synthesis of scRNA (see section III, B, 1), in polysomes of infected cells, ecRNA is not detected (Pons, 1973).
Using a "protein synthesizing system from E. coli and influenza virus vRNA as a template, Siegert et al. (1973) observed the production of a viral NP protein under in vitro conditions. This labeled NP protein was characterized by the Ouchterlony gel precipitation method. In in contrast, Kingsbury and Webster (1973) did not observe any viral protein synthesis from vRNA using a protein-synthesizing system derived from rabbit reticulocytes In the same system, however, they found viral M-protein synthesis (Vol. Ch. 2) on an RNA template isolated from infected cells.Thus, at the moment it is impossible to answer the question whether only viriope or only complementary, or some RNA fragments of one type and some RNA fragments of another type are used as templates for protein synthesis. it is difficult for influenza viruses to apply the definition of "negative" or "positive" viral chain, as proposed by Baltimore (1971).
B. SYNTHESIS OF VIRAL PROTEINS IN VIVO
The study of the synthesis of viral "proteins" is favored by the fact that in the infected cell the synthesis of cell polypeptides is replaced by virus-specific synthesis. In chicken embryo fibroblast cells infected with HPV (Joss et al., 1969; Skehel, 1972; Klenk, Rott, 1973), and in BHK 2IF cells infected with the WSN strain of influenza virus (Lasarowitz et al., 1971), 4 hours after infection, almost
only one viral proteins (31). Somewhat earlier researchers observed the synthesis of three or four polypeptides in infected mites (Taylor et al., 1969; Joss et al., 1969; Holland and Kiehn, 1970; White et al., 1970). Other polypeptides have subsequently been discovered (Lazorowitz et al., 1971; Skehel, 1972; Klenk et al., 1972b; Krug and Etkind, 1973). In general, all structural α-proteins were found in infected cells: one or two P-proteins, the NP nucleocapoid subunit, the M membrane protein, the uncleaved (HA) and cleaved (HA1 and HA2) hemagglutinin glycoprotein, and the NA subunit.
In addition to virion proteins, one or two non-structural proteins (NS) have been described.
There are notable differences<в уровнях синтеза отдельных вирусных полипептидов. NP- и NS-полипептиды обычно первыми обнаруживаются в зараженных «летках. Skehel (1973) предположил, что полипептиды Р2, NP и NS, которые первыми обнаруживаются в «клетках, зараженных ВЧП, являются
products of RNA fragments formed during selective transcription by virion polymerase of three fragments of the viral genome. When cells were infected in the presence of cycloheximide and the pulse tag was added after removal of the antibiotic, only these three polypeptides were found. Based on this, it was assumed that the RNA molecules for these components were formed with the help of the introduced virion lolimerase in the process of primary transcription. From the 4th to the 6th hour after infection of chicken fibroblasts with the MPS, the level of M-protein synthesis increases, while the synthesis of NS-lolileptide decreases (Skehel, 1972, 1973). Thus, levels of iolipid synthesis can be individually controlled and can vary during the growth cycle.
In addition to cleavage of the HA polypeptide into HA1 and HA2, there is no evidence that virus-specific influenza polypeptides result from the cleavage of large precursors (Taylor et al., 1969; Lazarowitz et al., 1971, Skehel, 1972; Klenk, Rott, 1973 ).
Recently, new information has been obtained on the localization of viral components in infected cells using autoradiography (Becht, 1971) or cell fractionation and gel electrophoresis techniques (Taylor et al., 1969, 1970). According to these studies, the synthesis of all viral proteins apparently takes place in the cytoplasm. Previous localization studies of the nucleoprotein antigen by immunofluorescence have been interpreted as indicating that synthesis occurs in the nucleus with subsequent release of the antigen into the cytoplasm (Liu, 1955; Breitenfeld and Schafer, 1957; Holtermann et al., 1960). However, it is clear that immunofluorescence determines the accumulation of the antigen, and not its synthesis (see section IV, B, 2).
1. RNA polymerase
Virus-specific RNA-dependent RNA polymerase activity can be detected in influenza virus-infected cells between 13D and 3h post-infection, depending on the cell system used (Scholtissek and Rott, 1969a; Skechel and Burke, 1969; Ruck et al. al., 1969; Mahy and Bromley, 1970). This is the first detected virus-specific activity after infection. Most of the viral activity of lolimerase is detected in the microsomal fraction; some part of this activity remains in the nuclei and cannot be removed from there even by intensive washing. There are no fundamental differences in the kinetics of manifestation or in the required cofactors between nuclear and microsomal enzymes (Scholtissek and Rott, 1969a; Mahy et al., 1975).
Upon further fractionation of the cytoplasm in a stepwise sucrose gradient by the method of Caliguiri and Tamm (1970), polymerase activity is found in rough membranes (Compans and Caliguiri, 1973; Klenk et al., 1974a).
Since the activity of viral polymerase was found in purified viral particles (see Chapter 5), the question arises with which of the viral "proteins" it can be associated. The only virus-specific product associated with polymerase activity was the RNP antigen (NP protein plus viral vRNA, determined by complement fixation). All attempts to remove RNA from this complex resulted in a complete loss of enzyme activity (Schwarz and Scholtissek, 1973). The P-protein was put forward as a candidate for the role of viral polymerase (Kilbourne et al., 1972). When the enzymatic "complex labeled in vivo with amino acids" was isolated from influenza virus-infected cells and purified by about 35 times, electrophoretic analysis initially revealed only Np-whitea in this complex.< (Compans, Caliguiri, 1973). Впоследствии, однако, при других условиях введения;метки удалось обнаружить и Р-белок (Caliguiri, Compans, 1974). С другой стороны, Klenk и соавт. (1974) обнаружили Р-белок в цитоплазматическом золе, "который не обладает полимеразной активностью (Scholtissek, Rott, 1969a; Skehel, Burke, 1969). Эти наблюдения могут означать, что Р-белок осуществляет свою (Предполагаемую активность ферментов только при связывании с РНП-антигеном.
That the RNP antigen itself has polymerase activity is unlikely, since hyperimmune serum against RNP antigen does not inhibit polymerase activity, while convalescent serum, which may contain antibodies to polymerase, inhibits it (Scholtissek et al., 1971) . This convalescent serum (which was obtained from animals that had been infected with influenza A viruses) inhibited the polymerase activity of all influenza A virus strains studied, but did not inhibit the activity of influenza B virus polymerase. All these observations are consistent with the idea that RNP- antigen (BPHK + NP = = protein), can serve as a template for the synthesis of ecRNA-
2. Nucleocapsid protein
The NP protein binds to the viral RNA, forming the RNP antigen. This is true for the NP protein isolated not only from the virion, but also from the infected cell (Schafer, 1957).
approximately 3 hours after infection, one hour before the appearance of hemagglutinin (Breitenfeld, Schafer, 1957). After this time, the RNP antigen titer does not increase significantly. This may be due to the balance between new synthesis and incorporation into mature particles. According to the label, the NP protein can be detected in infected cells within 2 hours after infection (Scholtissek and Rott, 1961; Krug, 1972).
With the help of fluorescent antibodies, the RNP antigen is first detected in the nuclei. Later, it also appears in the cytoplasm (Breitenfeld and Schafer, 1957). Under certain conditions, such as abortive infection (Franklin and Brietenfeld, 1959), in the presence of p-fluorophenylalanine (Zimmermann and Schafer, 1960), or under the conditions of the von Magnus phenomenon (Rott and Scholtissek, 1963), RNP -antigen remains in the nuclei.
The early accumulation of the RNP antigen in the nuclei of infected cells does not mean that the NP protein is also synthesized inside the nuclei. Autoradiographic studies, as well as the use of cell fractionation methods, indicate cytoplasmic synthesis of this and another arginine-rich protein and their rapid transport from the cytoplasm to the nuclei (Taylor et al., 1969, 1970; Becht, 1971).
In extracts of infected cells, a certain fraction of the RNP antigen contains ecRNA (Pons, 1971; Krug, 1972; Krug and Etkind, 1973), although only one type of RNA is found in viral particles (which follows from the absence of any self-hybridization of vRNA) (Scholtissek, Rott, 1971; Pons, 1971). It is not possible to decide whether the RNP antigen containing ecRNA has a specific significance in the process of viral reproduction or is it just an artifact that appears in the process of cell fractionation. Both RNA strands have been shown to bind equally well to the NP protein in vitro (Scholtissek and Becht, 1971). Thus, if there is any free NP protein and free scRNA, the corresponding RNP antigen is immediately formed during the homogenization process. Virion RNA can be displaced from the RNP antigen polyvin "Ilsulfate O" M (Pons et al., 1969). Therefore, it can be tested whether the substitution of various viral RNAs in the RNP antigen in cell homogenates is feasible. From changes in the composition of the bases of viral RNA labeled for various periods of time with 32P and isolated from the cytoplasmic RNP antigen, Krug (1972) concludes that some part of the ecRNA, before it is included in the RNP antigen, exists in a form free from NP- squirrel. The incorporation of 32P into animal cell RNA occurs with a significant lag phase due to the rather slow incorporation of labeled phosphorus "in" (x-position of nucleoside triphosphates (Scholtissek, 1965). Until appropriate adjustments are made to calculate changes in composition
reasons are not made, the data of Krug (1972) should be interpreted with caution.
Krug's (1972) kinetic analysis of the "appearance of RNP antigen in nuclei and cytoplasm" suggests that RNP antigen accumulating in nuclei is not a precursor of RNP antigen found in the cytoplasm.
3. Non-structural proteins
Several non-structural virus-specific proteins of unknown function have been described for infected cells. One of them, with a relative molecular weight of 25,000, accumulating in "large amounts, was designated as NS (Lazarowitz et al., 1971). In polyacrylamide gels, it has a migration rate close to the mobility of the M protein. However, both proteins, appear to be independent of each other, as seen from the differences in their peptide maps. Large "amounts of NS-" protein have been found in nuclei (Lazarowitz et al.,. 1971; Krug, Etkind, 1973). These findings are consistent with the data previous immunofluorescent studies Dimock (1969), who observed "bright staining of nuclei with antiserum specific for non-structural viral antigens, and this staining probably reflected the presence of the NS-" protein. As "it was found that this protein is also the main virus-specific protein in fractions of free and membrane-bound ribosomes isolated from infected cells (Pons, 1972; Compans, 1973; Klenk et al., 1974a). The association of NS with ribosomes seems to depend on the tonnage (Krug and Etkind, 1973). In buffers with low ionic strength, THIS nolipaptide -adsorbed on both ribosomal subunits, while when salt was added, it was removed from them.
A recent study (Gregoriades, 1973) raised some doubts about the identification of NS as a nonstructural polypeptide distinct from the virion M polypeptide. from whole infected cells, α-nuclei or polysomes. Analysis of tryptic processing products of the M-protein, as well as nuclear and ribosome-associated β-proteins, led to many coincidences, suggesting that the M- and NS-proteins are identical. Necessary, however , further information to convincingly explain these results.
In addition to NS, there may be other non-structural virus-specific components, although none of them. has not been sufficiently characterized. Using antiserum directed against non-structural viral antigens,
Dimmock and Watson (1969) precipitated radioactively labeled polypeptides from infected cells. Electrophoretic analysis in polyacrylamide gel suggested the presence of several non-structural polypeptides with the main component corresponding to NS. One of the remaining non-structural components migrates more rapidly and may correspond to the 10,000 to 15,000 relative molecular weight component described by Skehel (1972), Krug and Etkind (1973).
4. Membrane M-protein
The M-protein, which "lines the inner surface of the lipid bilayer of the envelope and is rich in the virion, is found in infected cells in relatively small amounts. This suggests not only the controllability of the M-belm synthesis, but also the possibility that this synthesis is the rate-limiting stage of virus reproduction (Lazarowitz et al., 1971) This concept is supported by the data that at a temperature of 29 °C, at which the formation of the virus is suppressed, the M-protein is the only (virusopedic) protein that cannot be detected in infected cells (Klenk, Rott , 1973).
M-protein (can be found on the smooth and plasma membranes of infected cells (Lazarowitz et al., 1971; Corn-pans, 1973a; Klenk et al., 1974a). These data indicate the affinity of this protein for membranes.
5. Hemagglutinin
Hemagglutinin is synthesized as a large glycoprotein - a precursor of HA, which is subsequently cleaved into two smaller glycoproteins: HAi and HA2 "(Lazarowitz" t al., 1971). Cleavage, which can be suppressed by protease inhibitors (Klenk, Rott, 1973), is carried out, apparently proteolytic enzymes of the host cell (Lazarowitz et al., 1973) The degree of cleavage depends on the virus strain, the host cell, the level of cytoiatic effect and the presence or absence of serum in the medium (Lazarowitz et al., 1971, 1973a, b Klenk and Rott 1973 Stanley et al. serum In the presence of serum, however, WSN hemagglutinin is also cleaved.
The system takes place on the plasma membrane (Lazaro-witz et al., 1973a). In the VChP system, the mechanism of such splitting, apparently, is different. Cleavage "occurs on intracellular membranes, and plasminogen is not necessary in this case (Shchepk et al., 1974a). The degree of splitting sharply decreases three 25 °C (Klenk, Rott, 1973).
Cleavage of HA is not a necessary condition for hemagglutination activity and for virion assembly (Lazarowitz et al., 1973a; Stanley et al., 1973), but recent studies have "found that it is necessary for infectivity (Klenk et al. , 1975b) These data are consistent with the hypothesis that, in addition to its role in adsorption, HA* has another function in the infectious process and that cleavage is necessary for this function. host cells and that particles containing uncleaved HA are of low infectivity suggest that host range and the spread of influenza virus infection are dependent on the presence of the host cell protease as an activating enzyme.
In experiments on the fractionation of "cells infected with
influenza virus, it was found that HA glycoproteins are always ac
associated with membranes (Compans, 1973a; Klenk et al.,.
1974a). Intracellular localization of these proteins and their moment
walkie-talkie from rough membranes to smooth endolasmatic
reticulum and to plasma membranes will be de
are described in detail in section VII, B. . .,
6. Neuraminidase
Viral NA* as an active enzyme was found,
3 hours after infection in the chorion-allantoic membrane
nah, and by extrapolation it was found that the beginning of her syn
thesis occurs 1-2 hours after infection (Noll et al.,
1961). The intracellular localization of NA* was studied by
cell fractionation, and found that it apparently
mu, similar to the localization of HA* (Compans, 1973a; Klenk et al.,
1974a). NA* has been found in association with membranes,
derived from smooth endoplasmic reticulum
when determining it by biological activity and by analysis
zu in polyagrylamide gel. The activity of the enzyme was found
den also in fractions containing rough membranes
us. These data are consistent with those obtained with
immunofluorescence (Maeno, Kilbourne, 1970). After 4 hours
after infection, yairaminidase can be detected in the cyto
plasma; later she seems to concentrate on the peri
cell feria.
V. SYNTHESIS OF CARBOHYDRATES
Carbohydrates are involved in the formation of glycoproteins and glycolylides of the influenza virus envelope (Klenk et al., 1972a). Glycolipids of myxoviruses (derived from the plasma membranes of the host cell (Klenk, Choppin, 1970), “it has not been determined what is predominantly (included in the virion: already existing or newly synthesized glycolipids.
The use of radioactive precursors such as glucosamine, mannose, galactose and fucose, which are specifically incorporated into viral glmkopeltides, has shown that the carbohydrate side chains of these glycopeptides are resynthesized during infection (Haslam et al., 1970; Cornpans et al., 1970a; Schwarz and Klenk, 1974). Cell fractionation experiments have provided additional information on the sites of glycosylation of viral glycoproteins. Glucosamine is associated with the HA polypeptide in fractions of both smooth and rough cytoplaemic membranes; however, fucose is associated with HA in smooth but not rough membranes (Compans, 1973b). Suppression of protein synthesis by puromycin stops the incorporation of glucosamine almost immediately, while fucose continues to be incorporated for about 10–15 min (Stanley et al., 1973).<и НА2 содержат, по-видимому, полный состав маннозы « глюкозамина, тогда как содержание фукозы и галактозы значительно выше в продуктах расщепления (Klenk et al., 1975a; Schwarz, Klenk, 1974). Эта наблюдения предполагают, что биосинтез углеводных боковых целей гликопротеинов НА осуществляется по стадиям с различными остатками Сахаров, добавляемыми в разных участках клетки. Глюкозамин и манноза (присоединяются, по-видимому, к полипептидам НА на шероховатых мембранах вскоре после или даже в процессе синтеза поляпептида, в то время как фукоза, вероятно, прикрепляется позже с помощью трансфераз, присутствующих в гладких мембранах.
These glycosyltransferases are probably cellular enzymes. Consequently, the carbohydrate (part of glycoproteins is apparently determined by the host cell. However, there is evidence that, in addition to these enzymes of the host cell, viral NA* plays a significant role in the formation of carbohydrate side chains. It has been established that the surface of the myceovirus envelopes lacks neuraminic acid (Klenk and Choppin, 1970b; Klenk et al., 1970), while in viral envelopes that do not contain this enzyme, this carbohydrate is a common constituent (Klenk and Choppin, 1971; McSharry and Wagner, 1971 ; Renkonen et al., 1971) These data suggest that
The action of neuraminic acid is an essential feature of myxoviruses. It has recently been shown that NA* is responsible for removing neuraminic acid from the influenza virus envelope, thereby preventing the formation of receptors on the viral envelope that would otherwise lead to the formation of large aggregates of viral particles (Palese et al., 1974). These data support the concept that the carbohydrate moiety of HA* as the main surface glycoprotein is a product of the combined action of (Cell transferases and viral NA*. Through its action, the virus is able to introduce a virus-specific modification into the (Initially host-specific, complex carbohydrate-modification structure, which, according to apparently essential for the -biological activity of the virus.
D-glucosamine and 2-deoxy-0-glk>goat inhibit the formation of biologically active HA*, NA* and infectious virus (Kilbourne, 1959; Kaluza et al., 1972). Biochemical studies have shown that these sugars compete with the biosynthesis of viral glycogroteins (Ghandi et al., 1972; Klenk et al., 1972b). In the presence of these inhibitors, the size of glycoprotein HA decreases. The degree of reduction depends on the dose. Thus, with an increase in sugar concentration, glycoprotein HA with a relative molecular weight of 76,000 gradually turns into a compound with a molecular weight of 64,000, which was designated as HA0 (Klenk et al., 1972b; Schwarz, Klenk, 1974) The molecular weight shift parallels the decrease in carbohydrate content and the HA0 protein has been shown to be almost free of carbohydrates (Schwarz, Klenk, 1974).These results show that HA0 is an incompletely glycosylated or non-glycosylated polypeptide chain of the HA glycoprotein and that "the inhibitory effect of D-glucose amine a and 2-deoxy-0-glucose is due to damage to glycosylation. The HA0 polypeptide is associated with membranes, like normal HA It also migrates from the rough to the smooth ejadoplastic reticulum, where it is cleaved into the polypeptides HA01 and HA02. probably not essential to the affinity of this polypeptide for the membrane. However, the lack of hemagglutination activity in infected cells suggests that the non-glycosylated protein is unable to bind to the receptors.
VI. SYNTHESIS OF LIPID
Like all enveloped viruses, the influenza virus acquires its lipids by utilizing host cell lipids. This position is confirmed by the following observations.
Denia. The lipid composition of the influenza virus has been found to be similar to that of the host cell (Ambruster and Beiss, 1958; Frommhagen et al., 1959). Host cell lipids, radioactively labeled before infection, are incorporated into viral particles (Wecker, 1957). When growing the virus in various host cells, modifications of viral lipids are detected (Kates et al., 1961, 1962). In general, the lipids of viruses (which bud off the cell surface closely mirror the lipid composition of the host cell's plasma membrane (Klenk and Choppin, 1969; 1970a, b; Renko-nen et al., 1971). ospholilides in chick fibroblast cells remained unchanged for 7 hours after infection with influenza virus, after which all lipid synthesis was suppressed (Blough et al., 1973).This suppression is probably not a primary effect, and may be secondary to in relation to the inhibition of RNA or protein synthesis, or to ".other cytolytic effects.
Thus, based on the results obtained so far, it can be assumed that the synthesis of viral lipids is carried out through normal cellular processes of lipid biosynthesis, and the viral envelope is formed by incorporation of lipids from the plasma membrane of the host cell.
VII. ASSEMBLY (see also chapter 2)
A. FORMATION OF THE NUCLEOCAPSID
As already mentioned, it is most likely that the nucleocapsid protein is synthesized in the cytoplasm. Apparently, it is present there for a short time in free form and then associates with viral RNA, forming nucleocapsids (Klenk et al., 1974; Compans and Caliguiri, 1973). Because the NP protein is rapidly incorporated into the nucleocapsids, RNA can be selected from the n-reformed pool (Krug, 1972). Due to the small size of influenza virus nucleocapsids, they cannot be accurately identified in infected cells using electron microscopy. Clusters of threads or fibers with a diameter of about 5 nm, observed in the cytoplasm, possibly represent viral ribo-nucleoproteins (Apostolov et al., 1970; Compans et al., 1970b).
Available data indicate that the RNA genome of influenza virus virions consists of 5-7 fragments (see Chapter 6).
Therefore, any infectious particle needs at least one copy of each fragment. Hirst (1962) pre-
suggested that nucleocapsids from the intracellular pool can be included in (virions by chance. The proportion of "infectious virions in the E population can be increased by including additional RNA fragments in the average viral (Compans et al., 1970). For example, if five of different RNA fragments, each virna contains a total of 7 fragments included in a viral case, then approximately 22% of the virions should be infectious.Evidence for the random inclusion of RNA fragments was supported by recent observations by Hirst (1973) that in the viral population, recombination occurs between non-plaque-forming particles.The ability of such particles to participate in recombination can be explained by the absence of one or more fragments in the particles, while varying the missing fragments from one particle to another, thus, suitable nests of a defective virus can form recombinants.
B. PROCESS OF VIRUS BUDDING
Like most enveloped viruses, the influenza virus assembles on preformed cell membranes; assembly is carried out by budding from the plasma membrane. The first demonstration of the release of a virus from a "cell by a process that did not involve lysis" was presented by Murphy and Bang (1952) in early electron microscopy studies of cells infected with influenza virus. ■ spherical structures. No viral particles were seen within the cells during the formation of the "infectious virus" and hence it was clear that the viral particles were forming on the surface of the cell. Using ferritin-labeled antibodies, Morgan et al. viral antigen in those areas where the virus is formed.Later electron microscopy studies have shown that the surface of the budding virus contains the same membrane as the host cell with a layer of protrusions corresponding to viral "spikes" on the outer surface. On the surface of the viral membrane, there is an additional electron-dense layer missing on the cell surface, which probably consists of M-iolypeptides (Bachi et al., 1969; Compans and Dimmock, 1969; Apostolov et al., 1970).
Electron microscope studies gave grounds to ■ suggest the order in which the viral components of the as-
socialize on the cell membrane (Bachi et al., 1961; Compans and Dimmock, 1969; Compans et al., 1970b).
The viral envelope proteins appear first, being included in some regions of the membrane, which should have a normal morphology; however, the observed specific adsorption of erythrocytes to these regions of the membrane indicates the presence of the HA protein here. Then, apparently, the M-protein (associates with the inner surface of such areas of the membranes, forming an electron-dense layer. Further, the ribo-nucleoprotein specifically binds to the membrane in these areas and the process of budding occurs by bending and protrusion of the membrane segment and surrounding the associated ribonucleoprotein. Polyacrylamide gel electrophoresis data also support the idea that envelope proteins associate with the plasma membrane faster than RNPs (Lazarowitz et al., 1971). are found in purified virions.As already mentioned, neuraminic acid residues are absent in the shell of budding influenza virus particles, but are present in neighboring regions of the cell membrane (Klenk et al., 1970).
These data prove a sharp transition in chemical composition between the shell of a budding viral particle and the adjacent cell membrane.
However, on the other hand, an important feature of the budding process is that the viral envelope is continuous with the plasma membrane of the host cell and is morphologically similar to it (Compans and Dimmock, 1969). As mentioned, the lipids in these membranes bear a very close resemblance to the membrane lipids of the host cell. These observations suggest that lipids in the intact plasma membrane readily exchange with lipids in budding viral particles by radial diffusion.
Therefore, the envelope of the budding virion is formed from a small portion of the cell membrane modified to include the proteins of the virion envelope. This concept, of course, does not imply the need to synthesize all the envelope components on the plasma membrane.
Indeed, it has been known for a long time that the components of the envelope must migrate considerable distances from one cell site to another in order to get from the site of their biosynthesis to the site of membrane assembly. Breitenield and Schafer (1957) showed that in cells infected with the influenza virus, HA* can first be seen
to put in all parts of the cell and that it is localized in the perinuclear zone in an increased -concentration. Later, HA* accumulates in the "peripheral region" of the notch and can also be demonstrated in thin filaments that protrude from the flare membrane.
The notion that envelope components migrate from inside the cell to the surface has recently been confirmed and extended by a series of studies using cell fractionation and analysis of viral proteins in various cell fractions. These works also suggest that the HA glycoprotein, and possibly other envelope proteins, are synthesized on the rough endoplasmic reticulum (Compans, 1973a; Klenk et al., 1974). As detected in pulse-chase experiments, after a few minutes, HA is found already in the membranes of the smooth endoplasmic reticulum (Compans, 1973a; Stanley et al.,. 1973; Klenk et al., 1974) and in the plasma membrane (Stanley et al. ., 1973). Although experiments to chase from the smooth endoplasmic reticulum to the plasma membrane have not been performed, it seems plausible that HA migrates from the rough endoplasmic reticulum to the plasma membrane, bypassing the smooth endoplasmic reticulum. It should be noted that during the entire time of such migration, HA and other envelope proteins are components of the membranes along which they move; OBIs are never found as dissolved proteins.
Fractions of smooth membranes, which are believed to be derived primarily from the endoplasmic reticulum, contain all of the major envelope proteins (Compans 1973a; Klenk et al., 1974). However, their relative amounts here differ from those in the plasma membrane and virion (Stanley et al., 1973; Klenk et al., 1974). The ratio of M-protein to NA lycoprotectors is higher in the envelope of the mature virion than in the membranes of the endolasmatic reticulum. These data suggest that only a small number of membranes carrying the HA glycoprotein are converted into the viral envelope, namely, the fractions of membranes containing carbohydrate-free proteins. As already mentioned, the synthesis of the M-protein may be the stage that limits the assembly process of the virus.
The difference in the rate of synthesis of different envelope proteins supports the “hypothesis that the assembly of the envelope is a multi-stage process. The question of the process of formation of HA *, including the sequential addition of the carbohydrate moiety and proteolytic cleavage during the migration of the primary gene product, is consistent with this concept.
VIII. RELEASE OF THE FLU VIRUS
The problem of releasing the influenza virus from the host cell,
seems to be closely related to the problem of the function of the viral
NA*, which has already been discussed in detail earlier (see
cases V and ch. four). That this enzyme plays an essential
role in the release of the virus, stemmed from the ability of anti
bodies specific to NA* to suppress such release (Se-
to, Rott, 1966; Webster et al. G968). Moreover, these antibodies
prevent virus elution from erythrocytes (Brown, Laver,
1968). Bacterial NA* that is not inhibited by anti-antibody
lamy to viral NA*, is able to release the virus from cells,
treated with such antibodies (Compans et al., 1969;
Webster, 1970). On the other hand, bivalent antibodies
to NA also cause virus aggregation (Seto, Chang, 1969;
Compans et al., 1969; Webster, 1970), while monovalent anti
bodies do not prevent the release of the virus, although they inhibit
over 90% neuraminidase activity (Becht et al., 1971).
All these data taken together suggest that
bivalent antibodies prevent the release of the virus
by binding it to antigens present on the
cell surface, and by inhibiting the activity of fer
cop. The role of neuraminidase in the release of the screening virus
or also Palese et al. (G974), who found that this fer
ment is necessary to remove neuroamylic acid from vi
Russian surface to avoid aggregation of virions - then -
coves on the surface of the cell.
IX. INCORRECT FORMS OF BREEDING
A. HOST CELL DEPENDENT ABORTIVE REPRODUCTION
Influenza viruses can infect a wide variety of host cells. However, in many of the infected cells, the yield of infectious progeny is either very low or non-detectable, although viral components can be detected in normal titers. This type of host cell-dependent interruption of the infectious cycle is called abortive infection. infected in the brain with non-neurotropic strains of the influenza virus (Schlesinger, 1953).The higher the dose of virus administered, the greater the amount of newly synthesized hemagglutinin.Several other cell-host-influenza A virus systems have been described in which only RNP-I was produced. antigen and hemagglutinin, but not infectious virus.In all of these systems studied so far, the RNP-antigen accumulated in the nucleus and was not detected.
was detected by fluorescent antibodies in the cytoplasm (Henle et al., 1955; Franklin and Breitenfeld, 1959; Ter Meulen and Love, 1967; Fraser, 1967).
B. VON MAGNUS PHENOMENON
In the course of serial passages of "influenza viruses at a multiplicity higher than 1 (Barry, 1961), increasing amounts of incomplete virus are formed that emerge from the host cell (von Magnus, 1951, 1952). These viral particles have a surface structure very similar to on the structure of an infectious virus, are immunogenic and cause homologous interference, contain less RNA and RNP antigen, exhibit a lower ratio of infectivity to hemagglutination activity, and contain more lipids than complete viral particles (von Magnus, 1954; Isaacs, 1959; Pauker et al., 1959; Rott and Schafer, 1961; Rott and Scholtissek, 1963).
In the analysis of RNA of an incomplete virus, it was found that viral RNA with a relatively high [molecular weight is either absent or present in them in reduced quantities, while the amount of RNA with a low molecular weight increases (Duesberg, 1968; Pons, Hirst, 1969; Nayak, 1969) .
In cells infected with the second undiluted passage of the influenza virus, all the genetic information of the virus is present, since the ecRNA isolated from these "cells" is able to convert, after hybridization, the labeled RNA isolated from the infectious virus into a fully RNA-azoresistive form (Scholtissek, Rott , 1969b). Thus, the increase in the amount of low molecular weight RNA in incomplete particles may be due to high molecular weight RNA, which can be included in these particles in the form of destroyed and, therefore, non-functional molecules. This idea is supported by evidence that with thirsty undiluted passage, the ability to produce infectious virus first decreases, after which the synthesis of hemagglutinin, neuraminidase, and finally RNP antigen decreases (Scholtissek et al., 1966).
Nayak (1972) found that during the first passage at high multiplicity, the virus emerging at the beginning was completely infectious and gave a normal pattern of infectious virus RNA fragments in the sucrose gradient, while the virus emerging later had an RNA profile typical of an incomplete (background -magnus) virus1.
They are obligate intracellular parasites, which means they cannot replicate or pass on their genes without assistance. The single viral particle (virion) itself is inert. When a virus infects a cell, it uses enzymes and the bulk of the cell structure to replicate.
Unlike what we see in cell division processes such as , viral replication produces many offspring that destroy the host cell and then infect other cells in the body.
Viral genetic material
Viruses may contain single/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 the host is infected varies depending on the nature of the virus.
The replication process for viruses with double-stranded DNA, single-stranded DNA, double-stranded RNA and single-stranded RNA will be different. For example, double-stranded DNA viruses typically must enter host cells before they can replicate. However, single-stranded RNA viruses replicate mainly in host cells.
Once the virus infects the host, the components of the viral progeny are produced by cellular machinery and the 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 viral particle must correspond to certain receptor proteins on the cell surface of a particular host.
How do viruses infect cells?
The main process of infection and virus replication 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, which begin to assemble.
- Maturation - viruses develop from the assembled components.
- Exit - new viruses break out of the host cell in search of new victims for infection.
Viruses can infect any type of cell, including
IN FLU IRIS: EVENTS AND PREDICTIONS
D.K. Lvov, A.D. Zaberezhny, T.I. Aliper
Dmitry Konstantinovich Lvov, Academician of the Russian Academy of Medical Sciences, Director of the Research Institute of Virology named after A.I. DI. Ivanovsky RAMS, Head of the Department of Virology, Moscow Medical Academy. THEM. Sechenov. Project Manager 05-04-52136, 06-04-48822.
Aleksey Dmitrievich Zaberezhny, Doctor of Biological Sciences, Head of the Laboratory of Molecular Diagnostics of the same Institute, Head of the Department of Molecular Biology at NPO Narvak.
Taras Ivanovich Aliper, Doctor of Biological Sciences, Head of the Laboratory of Means of Specific Prevention of Viral Diseases of the same Institute, Director of NPO Narvak.
See the first publication of the article: Nature. 2006. No. 6. pp. 3–13.
Influenza epidemics occur annually and are not perceived as something extraordinary. Since the disease is caused by viruses already familiar to the immune system, it
they usually get along well. A pandemic is another matter: in this case, the causative agent of influenza is a virus with new antigenic and biological properties, spreading at lightning speed in the world, affecting up to a quarter of the planet's population and claiming tens of millions of lives. This "famous" pandemic of the last century. It was impossible to foresee them, just as, however, it is impossible to name the exact time of the onset of a new one.
However, now, thanks to constant monitoring of viruses circulating among humans, domestic and wild animals, as well as knowledge obtained using molecular genetic methods, it is already possible to predict the emergence of new variants of the virus with pandemic tendencies. In recent years, a contender for this role has been identified: at the end of 2003, the initially non-pathogenic H5N1 avian virus caused an influenza epizootic among domestic birds, which turned into a panzootic this year. This virus has begun to infect other animals, including humans, but so far cannot be transmitted from person to person. In order for it to acquire this ability, it is enough to replace just one amino acid in one of the viral proteins.
Virion structures
The influenza virus has a rather simple structure: it is a spherical particle (virion) with a diameter of about 0.13 microns, in the core, which contains the nucleocapsid (an RNA molecule packaged in a shell of M1 protein), surrounded by a lipid membrane (Fig. 1). Three proteins are immersed in this membrane - hemagglutinin, neuraminidase and an ion channel (M2 protein), which play a major role in the infectious process.
Hemagglutinin is the first to come into contact with host cell receptors. On the surface of the viral envelope, it is presented in the form of very complex trimers. Each of their monomers is firmly anchored in the membrane and contains two subunits - one of them provides primary contact with the target cell, the other is responsible for the fusion of the viral and cellular membranes. In the upper part of the protein, there are sites that bind to sialic acid, which is part of the host cell receptor.
The enzyme neuraminidase cleaves off the terminal groups of sialic acid of cell receptors, as a result of which the cell loses the ability to recognize the antigen, and the virus penetrates
BIOLOGY AND MEDICAL SCIENCE | Influenza viruses: events and forecasts |
enters it by endocytosis, the usual way of delivering substances into the cell. The acidic environment of the endosome budded from the cell membrane activates the M2 ion channel, which lowers the pH inside the viral particle, which leads to the destruction of the M1 protein coat. At the same time, hemagglutinin is activated. It is synthesized in the form of a precursor, which in an acidic environment passes into a mature state - it is cleaved by proteolytic enzymes into two subunits, while the fusion peptide hidden inside the trimer changes conformation, is released, moves to the upper end of the molecule and is introduced into the membrane. The viral envelope merges with the endosomal one, a fusion pore is formed, through which a path for foreign genetic material opens into the cytoplasm. The viral RNA then enters the cell nucleus. As a result, vital processes are disrupted in the cell, and it itself, using its own resources, begins to produce viral proteins. Viral RNA is immediately replicated and new viral particles are assembled, which are released from damaged cells with the help of neuraminidase (the products of their decay cause intoxication of the body and a feverish state) and are carried throughout the body with the bloodstream.
Fig. 1. Scheme of the influenza virus virion. Its lipoprotein membrane is covered with spines of two glycoproteins - hemagglutinin and neuraminidase. Inside is the nucleocapsid, an RNA molecule wrapped in a shell of the M1 protein. The genome consists of eight fragments, of which the first six encode one protein each (hemagglutinin - HA, neuraminidase - NA, RNA polymerase subunits - PB1, PB2, PA, nucleoprotein
NP), and the last two genes - two proteins with unique amino sequences.
no acids (matrix proteins - M1, M2 and non-structural proteins - HS1, HS2).
The multiplying virus depresses the hematopoietic and immune systems, damages the capillary endothelium, which leads to increased vascular permeability and hemorrhages up to cerebral edema with a fatal outcome. But this happens quite rarely, the immune system usually turns on - first, factors of innate (non-specific) immunity are involved, and after a while, specific antibodies begin to be produced that release and, upon re-infection, protect the body from the virus.
A distinctive feature of influenza viruses is the high variability of antigenic properties. Internal proteins are constant in structure and determine the type of virus (A, B and C). Surface antigens, on the contrary, are heterogeneous and variable, and to a greater extent
BIOLOGY AND MEDICAL SCIENCE | Influenza viruses: events and forecasts |
penis, this concerns hemagglutinin (H), which, along with neuraminidase (N), determines the subtype of the virus (H1N1, H2N2, H3N2, etc.). Antigenic variability of surface proteins is due to two genetic processes - drift and shift changes in the viral genome.
Drift changes are caused by point mutations in the genes encoding hemagglutinin and neuraminidase, and lead to minor changes in the structure of these proteins. As a rule, such changes occur between pandemics in all types of viruses (A, B and C). As a result, epidemics, rather than pandemics, occur annually, as protection from previous exposure to the virus is maintained, although it is insufficient.
Shift changes occur after a complete gene replacement. This is possible because the influenza virus genome is segmented - it consists of eight fragments of single-stranded linear RNA, encoding, in addition to hemagglutinin and neuraminidase, a virus-specific enzyme (RNA polymerase, or transcriptase, consisting of three subunits - proteins PB1, PB2, PA), as well as a nucleoprotein (NP ), matrix (M1 and M2) and non-structural (NS1 and NS2) proteins. When a cell is simultaneously infected with two different strains, segments of their replicating genomes are mixed in any combination, so new virions contain different sets of genes borrowed from each of the original viruses. Such a combination of viral RNA segments is called genetic shuffling, or reassortment, not to be confused with the already existing term - recombination, during which the genetic material is rearranged either by the crossover mechanism or as a result of a template change. Shift changes, as a rule, affect the antigenic structure of hemagglutinin, less often - neuraminidase. Thus, at irregular intervals (10-40 years), viruses with new antigenic and biological properties appear, including new pandemic variants.
Species barrier
Among the viruses that can cause extreme epidemic situations, the fight against which at the stage of their occurrence is difficult or even impossible, influenza A viruses are especially dangerous. They are characterized by high antigenic heterogeneity of surface proteins and are represented, according to the nomenclature, by 16 neuraminidase (N1-9). These viruses are widespread in nature and can infect all types of birds and some types of mammals (humans, horses, pigs, whales, seals, etc.). Infection of mammals occurs mainly through the respiratory tract, birds - through the intestines. Their infection is usually asymptomatic or in the form of enteritis, which indicates a high degree of mutual adaptation of influenza viruses and wild birds, which can be considered their natural hosts. The virus persists in water, at +22°C - up to a month, at +4°C and below - for a longer time (6-8 months), therefore, the water-fecal route of infection is the main mechanism for maintaining a constant circulation of the influenza virus in nature.
Despite antigenic heterogeneity, viruses with all known combinations of surface proteins have been isolated only from wild birds of the aquatic and semi-aquatic complexes - ducks, gulls, etc. (Fig. 2).
Among other animals, only viruses with a certain set of surface proteins circulate: for example, viruses of only three subtypes of hemagglutinin (H1-H3) and two neuraminidase (N1-N2) have been isolated from humans until recently. All four pandemics of the 20th century were due to new shift variants of these subtypes: the "Spanish flu" in 1918 was caused by influenza A virus subtype H1N1, "Asian flu" in 1957 - H2N2, "Hong Kong flu" in 1968 - H3N2 and "Russian flu" in 1977 - H1N1. All are reassortants of avian and human influenza viruses.
BIOLOGY AND MEDICAL SCIENCE | Influenza viruses: events and forecasts |
Until recently, it was believed that avian influenza viruses are not pathogenic for humans and, if infected, can only cause conjunctivitis and mild malaise, in rare cases, a mild respiratory syndrome. However, in 1997, H5N1 viruses caused extremely severe disease among people in Hong Kong - out of 18 people who became ill, six died, all of them infected from chickens. The second similar episode occurred in February 2003, also in Hong Kong - out of five infected people, two died. H5N1 viruses have been periodically isolated from chickens and other bird species (including wild birds) in local populations.
Fig.2. Schematic of the circulation of known subtypes of influenza A viruses.
The H9N2 serotype virus, which is widespread among poultry in China and other Asian countries, was detected in five Chinese in August 1998, and a year later in Hong Kong in two girls with a flu-like illness. It is noteworthy that all cases of diseases occurred independently of each other, and transmission of viruses from person to person was not observed. In 1996, the H7N7 avian influenza virus was isolated from a woman with conjunctivitis. One case in the Netherlands ended in death. These are only those cases that are officially recorded, but in reality, avian influenza viruses overcome the species barrier, apparently much more often and begin to infect not only humans, but also other animals (pigs, horses, whales, minks, etc.). Cases of infection of domestic birds (chickens, turkeys) with influenza viruses characteristic of wild birds, especially waterfowl, are known.
It is still difficult to confidently judge all the factors that limit the range of hosts of the virus, and the mechanisms that affect the change of the host. Such a search is being carried out, and has been for quite a long time, in various research groups, including our institute.
Hemagglutinin was the first to be suspected, since it is this glycoprotein that is responsible for recognizing host cell receptors and is involved in the fusion of the viral envelope with the endosomal (in fact, part of the cytoplasmic membrane of the host cell). The structure of these receptors differs depending on the species and tissue origin of the cells. These differences are important in limiting the transfer of viruses between species and have been studied specifically in connection with the emergence of new human pandemic viruses.
It has been established that the receptors of epithelial cells of the human respiratory tract, in addition to protein, contain carbohydrates - sialooligosaccharides, in which the terminal sialic acid (N-acetylneuraminic acid) is connected to galactose by a 2'-6' bond, and cell receptors
BIOLOGY AND MEDICAL SCIENCE | Influenza viruses: events and forecasts |
intestinal epithelium of birds - 2'-3'. Avian influenza viruses do not reproduce well in humans because they simply cannot bind to human receptors. At the same time, mucins (by nature the same complex glycoproteins with sialic acid at the end), which are required to protect the human lungs from microorganisms, contain receptors with a 2'-3' galactose bond. Thus, an avian influenza virus that accidentally enters the human body cannot penetrate the cells, since there are no specific receptors on their surface, and the recognizing activity of virion receptors is blocked by mucin, so a person in this case is only threatened with a slight runny nose.
But in this case, how to explain the emergence of pandemic shift variants of the virus? The situation cleared up a little when it became known that the cells of the respiratory tract of the pig carry both types of receptors and, accordingly, can become infected with both human and avian influenza viruses. This means that pigs can potentially serve as an intermediate host for various viruses and an ideal arena for their reassortment in mixed infections.
With regard to hemagglutinin, its ability to recognize host cell receptors appears to be primarily related to the structure of the receptor-binding site (PCS). Thus, in human influenza viruses, PCC contains the amino acids leucine and serine at positions 226 and 228, respectively, while in avian viruses, these positions are glutamine and glycine. Other amino acid substitutions have been found in PCC in different animals, which means that although PCC is conserved and evolutionarily stable, it still has variable regions that affect receptor binding (affinity) and specificity.
RCC can change after the virus overcomes the interspecies barrier, while avian influenza viruses, for example, can acquire the ability to recognize human cell receptors. Evidence that the bird-human species barrier can be overcome is the 1997 human influenza outbreak in Hong Kong, caused by the avian H5N1 virus.
It is assumed that the "attachment" of the virus to the host is determined not only by the characteristics of hemagglutinin, but also by another surface protein - neuraminidase. In addition, there is reason to believe that the genes of internal and nonstructural proteins of influenza A viruses are involved in limiting the range of hosts. However, it is too early to talk about this, since it is still necessary to study the contribution of each gene and the functions of their products. Be that as it may, it is important to understand that even minimal changes in the structure of viral proteins, especially hemagglutinin, can lead to significant changes not only in the host range of the virus, but also in the degree of its pathogenicity (virulence).
Virulence
Recall that the reproduction of the virus in the host organism requires activation of the precursor of the hemagglutinin molecule; in this case, it is cleaved by the host proteases into two subunits. Proteolysis of hemagglutinins of low pathogenic avian viruses occurs in a limited number of cell types, so the virus is localized only in the respiratory or intestinal tracts. This happens with asymptomatic or moderate infections. The hemagglutinins of highly pathogenic avian viruses are degraded in various cells and are therefore capable of causing lethal systemic infections, especially in poultry.
In various laboratories around the world, they began to study the genome of strains of influenza viruses that are highly pathogenic for humans (H5N1 and H7N7, isolated in 1997-2004). It turned out that these viruses contain several basic amino acids in the cleavage site of the hemagglutinin molecule, which provides them with high infectious activity and pathogenicity. Unlike non-pathogenic or weakly pathogenic viruses, which do not have this amino acid sequence, the hemagglutinin of highly pathogenic viruses is easily cleaved not only by trypsin-like proteases present in the cells of the human respiratory tract and the intestines of birds, but also by furin-like proteases. They operate in combination
BIOLOGY AND MEDICAL SCIENCE | Influenza viruses: events and forecasts |
with ubiquitin, designed to mark proteins for proteases that need to be destroyed. Furin-like proteases are synthesized in different tissues, which gives pathogenic viruses the ability to infect different systems and organs. Insertion of even one basic amino acid at the hemagglutinin proteolytic cleavage site is sufficient to convert a low pathogenic virus into a highly pathogenic one.
Subsequently, this was confirmed in experiments on mice infected with different variants (isolated in different years) of the H5N1 virus. Some of them began to replicate in the brain, liver, spleen, blood cells, causing 100% death of mice on the seventh to eighth day after infection, while other viruses were non-pathogenic for mice and multiplied only in the lungs. Soon, this was explained - in the H5N1 viruses isolated in 2004, in comparison with the viruses obtained in 1997-2003, additional mutations in the hemagglutinin gene were detected, which affected the change in their antigenic properties.
The pathogenicity of the virus can be affected by changes in the structure of not only surface, but also internal proteins. For example, a mutation at position 627 in the PB2 protein was found in a strain of the H5N1 influenza virus that is highly pathogenic for mice. It was this mutation that influenced the difference in the properties of the two H5N1 viruses isolated in Hong Kong, and, as a result, the result of the infectious process. In addition, the virulence of these influenza viruses is associated with structural features of the non-structural NS protein, in particular, with the presence of glutamic acid in position 92 in its molecule, which makes the viruses resistant to the antiviral effect of interferons.
Of course, the use of modern molecular genetic methods in the study of influenza viruses gradually elucidates their biological properties, but no less important are traditional methods that monitor the circulation of influenza viruses among people, domestic and wild animals. Prediction of the emergence of reassortants with pandemic tendencies and the development of effective measures for the prevention and control of influenza can only be developed on the basis of studying the ecology and evolution of the causative agents of this difficult to predict and difficult to control infectious disease. Such studies were started more than 35 years ago by G. Laver in Australia, R. Webster in the USA, D.K. Lvov in our country, and they are being conducted to this day all over the world.
Events and forecasts
A comprehensive study of influenza A viruses was initiated by the World Health Organization (WHO) after the 1968 pandemic caused by the H3N2 virus. Our institute found that the progenitor of H3N2 was a strain similar to the H3N8 virus isolated in Ukraine in 1963 from wild ducks. These and other data served as the basis for the development of the scientific direction - the ecology and evolution of influenza A viruses. The National Center for the Ecology of Influenza A Viruses was created with a network of reference bases, where new data were obtained, confirming the absence of fundamental differences between human and animal influenza A viruses, t .e. the presence of a single protected gene pool. According to these data, in 1980 a classification of influenza viruses was created, regardless of their origin. Since then, each isolated virus has been assigned a name that reflects the type of virus, source of isolation, place and year of isolation, as well as a subtype - for example, A / duck / Ukraine / 63 (H3N8).
The main objective of our research in Russia was to study the evolution of influenza A viruses in the process of interaction of viral populations with populations of wild birds and domestic animals and the formation of strains with epidemic potency. To do this, monitoring is carried out at key points in Northern Eurasia; 14 out of 16 known viruses were isolated
At the end of 2003, i.e. three months before the start of the epizootic caused by the H5N1 avian influenza virus in the countries of Southeast Asia, one of the authors of this article (D.K. Lvov) spoke at the international influenza congress in Japan, reporting on the isolation of these viruses
BIOLOGY AND MEDICAL SCIENCE | Influenza viruses: events and forecasts |
from wild birds in Russia - in Altai and in the south of Primorye. According to molecular genetic data, these strains are classified as low pathogenic. Then, by analogy with earlier observations, the first forecast was made about the possibility of their introduction with migratory birds into the poultry farms of Southeast Asia, where after some time they could turn into highly pathogenic with panzootic and pandemic potencies. Apparently, that is what happened. The outbreak of the epizootic in a short time spanned 10 countries. Since then, more than 150 million chickens and ducks have been killed and slaughtered. According to WHO, by the end of March 2006, 185 people had already become infected, 104 of whom died. The epizootic continues, and the viruses have penetrated into pig populations, which is of particular concern. Perhaps the world is on the verge of an epidemiological catastrophe: reassortants can form at any time when pigs are simultaneously infected with the avian H5 virus and the human H1 or H3 influenza viruses circulating around the world.
A second prediction was also made: in case of infection in the wintering grounds of wild birds with highly pathogenic strains, the risk of their introduction to the territory of Russia, especially to Siberia and the Far East, during spring migrations increases. And then something happened that was supposed to happen. In mid-July 2005, in the settlements of the Novosibirsk region, located within the lake northern forest-steppe of the Baraba lowland, an epizootic among poultry was detected with a mortality rate of over 90% and a rapid spread.
Materials were collected from domestic and wild birds that lived in the immediate vicinity of the epizootic site. Using the SPEV and MDCK cell lines (this non-standard method is currently being patented), we isolated six strains of H5N1 from poultry and grebes (Podiceps cristatus ), with very high tissue concentrations of the virus. With a priority of August 8, 2005, these strains were deposited in the State Collection of Viruses, and the sequencing data of their full-length genome were deposited in GenBank with a priority of September 5, 2005. The site of proteolytic cutting of hemagglutinin of all obtained strains contains the amino acid sequence PQGERRRKKRGLF, which is characteristic of highly pathogenic avian influenza viruses. The nucleotide sequences of the hemagglutinin genes of all analyzed poultry viruses turned out to be completely identical, but differ from the strain isolated from the wild bird (crested grebe), though only by two nucleotide substitutions (Fig. 3). Phylogenetic analysis revealed a high level of homology of hemagglutinins of West Siberian strains with strains isolated in the spring of the same year from mountain goose (Anser indicus) on the lake. Kukunor in the northwestern province of Qinghai (PRC). This was fully confirmed by the analysis of the remaining seven genes.
The identity of the genetic characteristics of the isolated strains proves a direct relationship between the viruses circulating in the populations of wild and domestic birds. At the same time, the strains of the H5N1 influenza virus discovered in 2005 differ significantly from the strains of this virus isolated in previous years, including the A/Vietnam/1194/2004(H5N1) strain obtained from England, which is offered in our country for vaccine production. It is obvious that, at least for a veterinary vaccine, only a strain from the State Collection of Viruses can be used, which corresponds in antigenic properties to the virus circulating in Russia.
The strain we isolated, deposited in the State Virus Collection, is already being used for large-scale vaccine production at the Stavropol Poultry Farm. Domestic birds are vaccinated in the Southern Federal District. By June 15, 2006, it was planned to produce 15 million doses of vaccine with further expansion of production.
By the way, in a pandemic, when it is necessary to quickly establish the production of a vaccine, it is advisable, in our opinion, to use as a substrate cell lines in which the influenza virus rapidly accumulates in high concentrations. This newly developed method has significant advantages over the traditional method, which uses
BIOLOGY AND MEDICAL SCIENCE | Influenza viruses: events and forecasts |
chicken embryos are used: while maintaining all the antigenic domains of hemagglutinin, the cultural vaccine eliminates the occurrence of complications associated with chicken protein. This is especially important in the production of human vaccines. You just need to know which strain should be used to immunize people. This will depend on the antigenic characterization of the resulting pandemic variant. Perhaps it will be different from what it is now. In any case, the use of a live vaccine is absolutely unacceptable. Genetic interaction between vaccine and field viruses can lead to reassortants with unpredictable consequences.
Fig.3. The degree of relatedness of the nucleotide sequences of the hemagglutinin gene of influenza A virus subtype H5 variants isolated from wild and domestic birds in different countries over the past 10 years. Highly pathogenic H5N1 strains of the Qinghai-Novosibirsk group of viruses are highlighted in bold.
Analysis of the genome of the strains we isolated revealed a number of features associated with biological properties. In addition to the amino acid sequence of the hemagglutinin proteolytic cleavage site, which determines the high level of pathogenicity of the virus, deletions were found at position 49-68 in neuraminidase (genotype Z), which indicates an increased tropism of the viruses isolated by us to poultry and potential pathogenicity for humans. Glutamic acid in the 92nd position of the NS1 protein determines the resistance of the virus to the action of interferon and increased virulence for pigs. Lysine at position 627 of the PB2 protein explains the ability of the studied strains to reproduce in various mammalian cell lines. The revealed properties of the virus that penetrated the territory of Russia testify to its high pathogenicity in relation to domestic birds and people.
The presence of serine, rather than asparagine, in the 31st position of M2 indicates the sensitivity of the virus to rimantadine, which fully coincided with the data of a direct study of the effect of antiviral drugs on virus reproduction. For these purposes, we also used cell lines and found that for the prevention and early treatment of influenza, they are equally effective and can be used as expensive foreign drugs - for example, Tamiflu, as well as relatively cheap domestic ones that are available in pharmacies - rimantadine,
BIOLOGY AND MEDICAL SCIENCE | Influenza viruses: events and forecasts |
virazole (with intravenous and aerosol application), arbidol. Unfortunately, there is currently no or insufficient production of these drugs in the country, and it is urgent to create their strategic stocks.
How and when did the highly pathogenic H5N1 influenza virus enter Russia, and how will further events develop?
Fig.4. Pathways of H5N1 influenza virus spread in wild and domestic bird populations. Low pathogenic influenza viruses (LPVV) isolated from wild and domestic birds in the northeastern region of Altai Krai in 1991 and in the south of Primorsky Krai in 2001 were apparently precursors of the 1997 influenza outbreak in Hong Kong and epizootics. in southeast Asia in 2003-2005, as well as in northwest China in 2005. Having become highly pathogenic (HPA), these viruses penetrated during the spring migration of wild birds to Western Siberia, where in the summer of 2005 they caused an outbreak of influenza among poultry. During the period of migration of birds of the aquatic and semi-aquatic complex, HPVH further spread to the north and west of Eurasia, and in the winter of 2006, these viruses were already detected in Africa. Thick arrows indicate infection with the influenza virus from wild
birds to domestic birds and vice versa.
First, low pathogenic strains circulating in Siberia and the Far East among wild birds were introduced during autumn migrations to the countries of Southeast Asia (Fig. 4). Having become highly pathogenic there, they penetrated into Western Siberia with wild birds in the spring of 2005 and sharply intensified during the period of nestlings. Highly pathogenic strains have scattered with birds to nesting sites over 10 million hectares.
km2. After the virus hit the poultry population, an epizootic explosion occurred. This is serious and for a long time.
The third prediction was that when the birds fly back to their wintering grounds through the densely populated areas of Russia and other countries in autumn, they will spread the virus again.
BIOLOGY AND MEDICAL SCIENCE | Influenza viruses: events and forecasts |
And so it happened. In the autumn of 2005, the virus had already reached most European countries; it was also found in Turkey, the Crimea, Iran, Azerbaijan, Georgia, India, and also in Africa. And we got to Tula, Kalmykia and the Volga delta, where an outbreak of influenza in the mute swan population (Cygnusolor) arose in December 2005 after a short stop of flying northern ducks - crested ducks (Aythya fuligula). The strains isolated from swans, according to their molecular genetic analysis, also belong to the Qinghai-West Siberian group of viruses. For half a year of circulation among wild birds, the strains retained their genotype and did not lose their high pathogenicity.
The fourth forecast is the most alarming. The virus has polluted many reservoirs in nesting areas and on migration routes, and it will remain there until spring. Every natural body of water where the feces of infected birds have fallen turns into a “time bomb”. This can be compared with the involvement of peatlands in the taiga fire. In the spring, infected and healthy birds will return and fly through these "minefields", so the events in the summer of 2006 may be much more formidable than in the past season. This is confirmed by the avalanche-like deterioration of the situation in Europe, Asia and Africa already in March. This is panzootic. And when the highly pathogenic strains circulating among wild birds in the spring return to low pathogenic strains, it is impossible to predict how long the process of their reassortment will take - months or years. It is clear that this is a subject of priority study, on which the development of events in the foreseeable future depends.
As for the pandemic virus, it can also arise in us after the infection of pigs with human and bird viruses. But it will most likely come to us from China, where the possibilities for transforming the reassortant are especially great, given the activity of the epizootic process and the huge susceptible contingent among the population. A pandemic virus can appear in our country at any moment - for this, only one amino acid substitution in the RCC of hemagglutinin is enough, as a result, the virus will begin to recognize the receptors of human cells and, accordingly, will begin to be transmitted from person to person.
What, from our point of view, should be done now at the state level, is formulated in the table. We are going to pay special attention to the study of the further evolution of highly pathogenic strains that affected wild bird populations. Ecosystems on the territory of Russia play a key role in this. We plan to continue monitoring in the European part of the country, in Siberia and the Far East, and also, possibly, in some neighboring countries.
In the last five years, our research has been carried out jointly with hunters, ornithologists, employees of the federal services of phytoveterinary and sanitary and epidemiological supervision of the Novosibirsk, Astrakhan, Irkutsk regions, Primorsky Territory, Birobidzhan, the republics of Kalmykia and Buryatia. All this took place within the framework of the federal programs "Protection from pathogens", "Development of means and methods to counter bioterrorism", "Influenza A of pigs and birds: interaction of populations".
