The antigenic shift is characteristic of the influenza virus type. Antigenic structure

R. G. WEBSTER and W. G. LEIVER i(R. G. WEBSTER and W. G. LAYER)

I. INTRODUCTION

The influenza virus type A1 is unique among the causative agents of human infectious diseases due to its ability to change its own antigenic structure so strongly that the specific immunity acquired in response to infection with one strain provides very little or no protection against the next emerging virus. Due to this Due to the variability of the virus, influenza continues to be one of the main epidemic human diseases.

Two types of antigenic variation have been found in influenza viruses: antigenic drift (Burnet, 1955) and significant antigenic shift. Antigenic drift is characterized by relatively small changes that occur mainly within a certain family of strains, each of which can be easily correlated with all other strains of this family in relation to both internal and surface antigens. Among the influenza A virus strains that infect humans, each subsequent variant replaces the previous one. This is possibly due to the selective advantage that new antigenic variants have in overcoming host immunological barriers. Antigenic drift is characteristic of influenza viruses not only A, IAO and B.

The second type of antigenic variation, which has only been described for virus A, involves more unexpected and dramatic changes. These are called significant antigenic shifts2. These shifts occur at intervals of 10-15 years (see Chapter 15) and are marked by the emergence of antigenically “new” viruses to which the population has no immunity, and these are precisely the “viruses” that cause significant influenza pandemics.

These “new” viruses have HA1 and NA subunits that are completely different from those circulating among humans before the emergence of the new virus. A significant shift may occur in one or both surface antigens; Two influenza pandemics have been described, caused by viruses belonging to each of these two categories (see Chapter 15).

Influenza is also a natural infection of some animals and birds. Viruses, exclusively type A, have been isolated from pigs, horses, and a variety of birds, including chickens, ducks, turkeys, quail, pheasants, and terns (McQueen et al., 1968; Pereira, 1969; World Health Organization, 1972). Previously, it was believed that the surface of an influenza virus particle consists of a mosaic of antigens that are part of all strains of this type, and that antigenic variation results from the movement of these antigens from prominent to recessed positions and vice versa. Later, another mechanism of antigenic drift was proposed. It is currently believed that changes are constantly occurring in the amino acids that make up the antigenic determinants of the HA and NA subunits. They are the result of the selection of mutants that exhibit changes in the amino acid sequence of polyeltide subunits, caused in turn by mutation of the viral RNA. Significant antigenic shifts, as a result of which “new” viruses arise, are probably due to a different mechanism. The hemagglutinating and neuraminidase subunits of these “new” viruses are antigenically completely different from the subunits of viruses circulating among humans before the emergence of new strains. We believe that the “new” virus is not the result of a mutation of a previous human influenza virus, but arises from genetic recombination between a human virus and one of the many strains of influenza A virus whose natural hosts are animals or birds. Having emerged, the “new” virus replaces the “old” one, which completely disappears from the human population.

Significant antigenic shifts in influenza B viruses have not yet been identified. Pereira (1969) suggested that the lack of significant antigenic changes in influenza B viruses may be a consequence of the absence of such influenza viruses among lower animals and birds.

Antigenic variation involves only the HA and NA subunits; The internal proteins of the virus (nucleoirotein antigen and matrix or membrane M protein) are largely constant. Of the two surface antigens, HA is the more important because antibodies to this antigen neutralize the infectivity of the virus.

II. FLU IN HISTORICAL ASPECT (see also Chapter 15)

A. EVIDENCE OF ANTIGENIC CHANGES

Influenza-like illness has been frequently reported in past centuries (Hirsch, 1883); the disease occurred either in the form of pandemics, affecting a very large part of the population and spreading almost throughout the world, or as local outbreaks. Until 1933, when the influenza virus was first isolated (from humans. Ed.)1, it was impossible to say with certainty whether a given pandemic was actually caused by an influenza virus. However, the characteristics of the epidemics described in historical documents indicate that these epidemics could well be caused by influenza viruses. Although other infectious diseases may have symptoms characteristic of influenza, only influenza causes sudden epidemics that last several weeks and disappear just as suddenly (Burnet and White, 1972). Serological studies conducted on elderly people also indicate about previous influenza epidemics that occurred in not so distant times (Mulder, Mazurel, 1958).

The earliest known influenza epidemic was recorded in Germany in 1170 (Hirsch, 1883), and from other historical sources it is possible to compile a fairly complete list of epidemics in Europe since 1500. Only the most severe epidemics will be mentioned here. More details can be found in Hirsch (1883), Creighton (1891, 1894), Burnet and Clarke (1942), and Burnet and White (1972).

Epidemic of 1781-1782 began in Asia in 1781, and then by early 1782 spread through Russia to Europe. This epidemic caused relatively few deaths, but its peculiarity was that the disease more often affected middle-aged people than children and the elderly. Quite severe epidemics also occurred in 1803, 1833, 1837 and 1847. Epidemic of 1847-1848 began in Eastern Russia in March 1-847 and reached Europe and England in the winter of 1847-1848. This epidemic has caused many deaths, especially among the elderly.

The 1889 pandemic also came to Europe from Russia, reaching England and America in early 1890. The disease spread at the speed of travellers. After the virus emerged in 1889, there were four further waves of infection in each of the following years. The second and third outbreaks caused many deaths, especially among children and the elderly. Serological (Mulder and Mazurel, 1958) and other studies (Pereira, 1969) suggest that viruses related to influenza viruses of Asia, Hong Kong and equine serotype 2 were present at that time.

The most severe influenza pandemic occurred in 1918-1919. The exact location of this pandemic is not known, but Burnet and Clarke (1942) believe that the virus could have developed independently in Asia and Europe or could have been introduced to Europe (by Chinese workers. The pandemic occurred in waves and killed an average of 20 to 50 million people. "human lives, mainly young people. The 1918-1919 pandemic was probably caused by a strain of influenza A virus related to the swine influenza virus. This was first suggested by Laidlaw (1935) and Shope (1936), but it is possible that this virus was transferred from humans to pigs, and not in the opposite direction.Intensive studies of the decline with age of antibodies to the swine influenza virus in human sera, carried out by Davenport et al. (1953-1964), Hennessy et al. (1965), give reason to believe that the virus that caused epidemic of 1918-1919 1pg., serologically related to the swine influenza virus.

The large number of deaths led Burnet and Clarke (1942) to suggest that the virus may have had unusual virulence. According to other researchers (Zhdanov et al., 1958; Kilbourne, 1960), the reasons for the high mortality from secondary bacterial infections could be war conditions and the lack of antibiotics. It seems likely, however, that some virus mutants were highly virulent, because the pandemic virus of 1781 “g., which also affected young people, did not cause such a high mortality rate.

B. ANTIGENIC CHANGES IN THE VIRUS IN THE PERIOD AFTER 1933

After the identification of the first influenza virus, which was designated H0N1 (World Health Organization, 1971), antigenic shifts occurred in 1947, when the H1N1 virus appeared (for example, A/FM/1/47), in 1957, when the H2N2 viruses (for example, A/Singapore/1/57), and in 1968, when the Hong Kong virus (A/Hong Kong/1/68) appeared. The antigenic shift in 1947 consisted of a change in the hemagglutinating antigen (from H0N1 to H1N1); in 1957, both HA and NA were antigenically completely different from the antigens of viruses of previous years (from H1N1 to H2N2), and in 1968 a variant Hong Kong showed significant antigenic difference in HA (from H2N2 to H3N2).

The Asian strain of influenza virus (H2N2), which first appeared in a Chinese province in 1957, contained HA and NA subunits that were completely antigenically different from the H0N1 and H1N1 subunits of influenza viruses previously circulating in humans. This strain of influenza virus caused a pandemic unprecedented in history (Burnet and White, 1972), but the number of deaths was small. The next and so far the last influenza pandemic was caused by the A/Hong Kong/68 virus, in which the NA subunits were similar to those of the “old” Asian A2 virus, and the HA subunits were completely antigenically different from those of the “old” Asian strain (Coleman et al., 1968; Schulman, Kilbourne, 1969; Webster, Laver, 1972).

B. GENERAL PROPERTIES OF PREVIOUS PANDEMICS

The pandemic nature of influenza in humans indicates that, at irregular intervals, humanity is affected by viruses that have new antigenic determinants. The above information indicates that these pandemics often originate in Southeast Asia and spread at the speed of travellers. Most pandemics caused increased mortality among children and the elderly, but at least two pandemics (1781 and 1918) caused increased mortality among young people.

III. PROPERTIES OF THE FLU VIRUS GENOME

The influenza virus has a fragmented genome, consisting of at least seven single-stranded RNA fragments. This fragmentation allows the genome to rearrange (“recombine”) during mixed infections with different strains (see Chapter 7) and may be fundamental to antigenic variation influenza virus. After mixed infection of cells with two different influenza A viruses, viral recombinants are formed with a high frequency. The high frequency of recombinations between influenza A viruses was first demonstrated by Burnet (Burnet, Lind, 1949, 1951) and repeatedly confirmed by other researchers working in this area (Hirst , Gotlieb, 1953, 1955; Simpson, Hirst, 1961; Simpson, 1964; Sugiura, Kilbourne, 1966) It was found that the recombination frequency can reach up to 97%.

The high frequency of recombination between influenza viruses allows the formation of antigen-hybrid viruses during a mixed infection in experiments both in vitro and in vivo. For the first time, biochemical confirmation of this was given by Laver and Kilbourne (1966), who discovered that the genetically stable recombinant X7 virus, isolated from cells mixed with influenza virus strains NW-S (H0N1) and RI/5+ (H2N2), possesses HA subunits of the virus H0N1, and the NA subunits of the H2N2 virus. Many other such recombinant influenza A viruses have subsequently been isolated and, in fact, they can be created “in the right order” (Webster, 1970) (see also 39). The formation of new influenza strains by recombination between animal (or avian) and human viruses is discussed in section VII. Evidence has been obtained that the strains of viruses that cause influenza pandemics can arise in nature in this way. Recombinations between influenza B viruses are also possible (Perry, Burner, 1953; Perry et al., 1954; Ledinko, 1955; Tobita, Kilbourne, 1974), but recombination between influenza viruses of types A and B has never been discovered.

IV. HEMAGGLUTININ SUBUNITS

AND NEURAMINIDASES AS HIGHLY VARIABLE

ANTIGENS

The hemagglutinating and neuraminidase activities of the influenza virus are associated with various subunits (Laver and Valentine, 1969; Laver, 1973), which form a layer of “spikes” on the surface of viral particles (32).

Hemagglutiin is the main surface antigen. It is responsible for the interaction of the virus with the cell surface and for the induction of neutralizing antibodies. Variability of the hemagglutinating antigen contributes to the emergence of new influenza viruses.

The NA enzyme is the second virus-specific surface antigen of the influenza virus particle. Antigenically, NA is completely different from HA (Seto, Rott, 1966; Webster, Laver, 1967). NA antibodies do not neutralize the infectivity of the virus (except at very high concentrations). but they greatly slow down the release of the virus from infected cells (Seto and Rott, 1966; Webster and Laver, 1967; Kilbourne et al., 1968; Becht et al., 1971; Dowdle et al., 1974), and these antibodies may play an important role role in reducing viral replication in vivo and preventing spread

infections (Schulman et al., 1968). While normal variability is also inherent in NA, variations in this antigen are perhaps less significant for the epidemiology of influenza.

Hemagglutinating subunits are glycoprotein rod-shaped structures, triangular in cross-section, with a relative molecular weight of approximately 215,000 (33). They are "monovalent" and (interact with

cell receptors at only one end (Laver and Valentine, 1969). Isolated subunits are highly immunogenic when administered to animals in the presence of an adjuvant. Each virus particle contains approximately 400 HA subunits (Tiffany Blough, 1970; Schulze, 1973; Layer, 1973).

HA subunits consist of two polyleptides with relative molecular weights of about 25,000 and 55,000 (Compans et al., 1970; Schulze, 1970; Laver, 1971; Skehel and Schild, 1971; Stanley and Haslam, 1971; Skehel, 1971, 1972; Klenk et al., 1972). They are designated as heavy and light cholypeptides HA1 and HA2. Oi6e, these chains are synthesized as a single peltide precursor with a molecular weight of about 80,000, which in some cells is cleaved into light and heavy polypeptides (Lazarowitz et al., 1971, 1973; Skehel, 1972; Klenk et al. ., 1972). In intact subunits, the heavy and light chains are connected by disulfide bonds, forming a dimer, and each HA subunit consists of two or three such dimers (Laver, 1971).

The HA subunit has hydrophobic and hydrophilic ends (34). The hydrophilic end is responsible for the biological activity of the subunit, while the hydrophobic end communicates with the lipids of the viral envelope. The hydrophobic properties of the subunit are apparently associated with the C-terminus by molding polypeptide chain (HA2) (Skehel, Waterfield, 1975) (OM. Chapter 3).

The neuraminidase subunit is a sglycoprotein structure with a relative molecular weight of about 240,000. It consists of square, box-shaped heads measuring 8-8-4 wells, to the center of which is attached a thread with a diffuse tail or with a small head at the end (, 35) (Laver and Valentine, 1969; Wrigley et al., 1973). The isolated subunits have full enzymatic activity and are highly immunogenic when administered to animals with an adjuvant. Each virus particle contains approximately 80 NA subunits (Schulze, 1973; Laver, 1973). However, the number of NA subunits in a viral particle can vary depending on the strain (Webster et al., 1968; Webster and Laver, 1972; Palese and Schulman, 1974), as well as on the type of host cell on which the virus was grown

NA subunits consist of four glycosylated lolipeptides with a relative molecular weight of about 60,000, linked to each other by disulfide bonds located in the filament or in its tail (see also Chapter 4). In most strains, these 4 polypeptides appear to be identical. However, in some strains, NA may consist of two types of polypeptides slightly different in size (Webster, 1970a; Skehel, Schield, 1971; Bucher, Kilbourne, 1972; Laver, Baker, 1972; Lazdins et al., 1972; Downie, Laver, 1973; Wrigley et al., 1973).

The active site of the enzyme and antigenic determinants are localized in various regions of the head of the NA subunit (Ada et al., 1963; Fazekas de St. Groth, 1963), and these heads have hydrophilic properties. The “tail” of NA is hydrophobic and serves to attach the subunit to the lipid shell of the virus (Laver, Valentine, 1969) (see "29).

A. ISOLATION AND SEPARATION OF SUBUNITS ON AND NA FROM EACH OTHER

For some influenza virus strains, pure, intact HA and NA subunits can be obtained by electrophoresis on cellulose acetate strips after destruction of viral particles with SDS (Laver, 1964, 1971; Laver and Valentine, 1969; Downie, 1973). The success of isolating any of these subunits using this technique depends on their resistance to denaturation by SDS at room temperature. According to this criterion, influenza viruses can be divided into four groups.

1. Viruses with HA subunits resistant to denatured alcohol

tions SDS. When this type of virus is destroyed by SDS and elec

trophoresis on cellulose acetate strips. all viral proteins,

“besides HA subunits, they migrate as anions. Hemaggluti-

nin migrating as a cation can be isolated in pure

form with complete restoration of biological activity

under conditions that do not destroy covalent bonds [for example

measures: A/Bel/42 (H0N1)].

2. Viruses with NA subunits that are resistant to denature

tions SDS. Pure, active NA subunits can be you

separated from these viruses by the method described above (eg

measures: B/LEE/40).

3. Viruses in which neither HA nor NA are resistant to dena

turation SDS. In this case, all viral proteins migrate

as anions and none of the surface subunits can

can be isolated using the described methods [for example:

A/NWS/33 (H0N1)].

4. Viruses that have both HA and NA subunits

resistant to SDS denaturation. For these viruses, both sub

units during electrophoresis - migrate as cations

and cannot be divided in this way [for example:

A/Singapore? 1/57 (H2N2)].

The HA and NA subunits of the latter group of viruses can be isolated, as shown in 36. An avian influenza virus (A/petrel/Australia/1/72(Hay6Mau5)) was isolated, which was stable to SDSHAHNA (Downie and Laver, 1973). In progress cellulose acetate electrophoresis, they moved together as cations (see 31, top) and could not be separated in this way. In this regard, the two types of these subunits were separated genetically using recombination (Webster, 1970b). To obtain recombinants, parental viruses with HA or NA subunits sensitive to SDS denaturation. SDS-stable avian virus HA and NA subunits were then isolated from SDS-degraded recombinant virus particles by electrophoresis on cellulose acetate strips (Em. 31, IB middle and bottom). pure subunits needed for chemical analysis and preparation of “monospecific” antisera.

HA and NA subunits can also be isolated from certain strains of influenza virus by treating viral particles with schroteolytic enzymes (Noll et al., 1962; Seto et al., 1966; Compans et al., 1970; Brand and Skehel, 1972; Wrigley et al. al., 1973). With this method, the separation of surface subunits from viral particles occurs, apparently, as a result of digestion of the hydrophobic (ends of the polypentide chain, which are responsible for attaching the subunits to the lipid layer of the viral envelope. However, partial digestion should also occur other regions of the HA subunit, as a result of which hemagglutinating activity is disrupted and some antigenic determinants are lost.

B. SEPARATION OF HEMAGLUTININ POLYPEPTIDES (HA1 AND HA2)

The light and heavy chains of hemagglutinating subunits can be separated by SDS-polyacrylamide body electrophoresis. However, for preparative purposes, the best separation is achieved by density gradient centrifugation of guanidine hydrochloride-dithiothriethol (Laver, 1971), carried out under conditions in which disulfide bonds are broken, or by tel filtration in a solution of guanidine hydrochloride-dithiothriethol (Webster, 1970a). This separation is apparently based on the significant hydrophobicity of the light polypeptide chain. During centrifugation in a concentrated solution of guanidine hydrochloride - dithiothriethol, this light polypeptide digests it faster than the heavy chain, and during gel filtration the light chain comes out first, apparently due to the fact that even in such a strongly dissociating environment the light chain does not exists iB as a monomer.

These remarks apply only to “HA subunits obtained from a virus grown on cells in which complete proteolytic cleavage of the precursor occurs.”

of the HA polypeptide into NAL and HA2. Moreover, the heavy and light polypeptides (HA1 and HA2) of the HA subunits produced by proteolytic digestion cannot be separated in this manner, possibly because digestion destroys the hydrophobic regions of the light chain (Skehel, Laver, unpublished data ).

B. PROPERTIES OF NA1 AND NA2

The light and heavy polypeptide chains of influenza A virus strain BEL (H0N1) had a similar polypeptide composition, except that the heavy polypeptide contained significantly more proline than the light chain (Laver and Raker, 1972). However, the peptide maps of the tryptic cleavage products of these two chains were completely different, indicating different amino acid sequences in these chains (Laver, 1971). Both polypeptide chains contain carbohydrates, but analysis of glucosamine suggests that the heavy polypeptide contains many more carbohydrates than the light chain. The heavy chain was found to contain 9.4% N-acetylglucosamine, as well as neutral sugars; so it probably contains about 20% carbohydrates.

D. NUMBER OF DIFFERENT SPECIFIC VIRUSES

ANTIGENIC DETERMINANTS ON THE SURFACE

SUBUNITS PER

Number of different virus-specific antigens

determinants on hemalglutinating subunits of the virus

influenza unknown (on the surface of hemagglutinating

subunits there are also determinants specific

to the host cell). Recent experiments have shown

however, that the hemagglutinating subunits of the Gon strain

Kong (H3N2) human influenza virus have at least

at least two, and possibly more, different virus-specific

ical antigenic determinants (Laver et al., 1974).

This has been demonstrated as follows: hemagglu

tin subunits were derived from influenza virus

Hong Kong (A/Hong Kong/68, H3N2) and its antigenic variant

A/Memphis/102/72, which arose as a result of antigenic

drift. Immunodiffusion tests showed that the subunits

Hong Kong/68 virus variants have at least two

various types antigenic determinants, while va

riant 1972 carries, apparently, at least three times

personal determinants (37).

The hemagglutinating subunits of viruses A/Hong Kong/68 and A/Memphis/102/72 had one common determinant. Antibodies to this determinant cross-reacted with both viruses in immunodiffusion, heme agglutination inhibition, and neutralization tests. Antibodies to other determinants did not show any significant serological cross-reactions between the Hong Kong/68 and Memphis/72 viruses. Thus, it is obvious that in the process of anti-

genetic drift, the Hong Kong influenza virus has undergone significant changes in one of its “specific” determinants. Data from Laver et al. (1974) (suggest that different antigenic determinants are localized on the same HA subunit and that viral particles do not possess a mixture of antigenically distinct subunits.

D. LOCALIZATION OF HOST CELL ANTIGEN

Although the first descriptions of a host cell antigen in an influenza virus (Knight, 1944, 1946) were met with some skepticism, their existence is now firmly established. The presence of such antigens was detected by a number of serological methods, including precipitation reactions (Knight, 1944), immunodiffusion reactions (Howe et al., 1967), complement fixation (Smith et al., 1955), hematglutination inhibition (Knight, 1944; Harboe et al. , 1961; Harboe, 1963a) and the method of blocking the inhibition of hemagglutination (Harboe, 1963b; Laver, Webster, 1966). The host cell antigen consists mainly of carbohydrates and is bound to the polypeptides of the HA and NA subunits. No connections between the host antigen (and carbohydrates) and the internal proteins of the viral particle were detected.

One of the mysterious features of the host antigen of influenza viruses is that it is detected in viruses grown in the allantois cavity of chicken or turkey embryos (Harboe, 1963a), but not in viruses grown, for example, in the allantois cavity of duck embryos or in the lungs of mice or in various cell cultures. Viruses grown on these cells were not at all inhibited in the themagglutination inhibition reaction by antisera obtained against extracts from uninfected host cells. This is probably due to the fact that the virus grown in these cells "Contains carbohydrates" of the host cell, but for some reason they either do not have antigenic properties or antibodies directed against them do not inhibit heme agglutination.

E. ROLE OF THE HOST CELL ANTIGEN

The carbohydrate component may play a very important role in the assembly of the viral envelope. Isolated NA and HA subunits aggregate in the absence of SDS. This gives reason to believe that these subunits have both hydrophobic and hydrophilic ends (Laver and Valentine, 1969) and, perhaps, the carbohydrate component of the host cell determines the hydrophobicity of one end of the HA and NA subunits.

G. ANTIGENIC VARIABILITY OF SUBUNITS

HEMAGGLUTININS AND NEURAMINIDASES DETECTED

MONOSPECIFIC ANTISERUMS

Until recently, it was believed that the V-antigen, or the envelope of the influenza virus particle, was something indivisible, but this is not so. It is now known that the V antigen consists of HA, NA and viral antigen host cells. In none of the previously published works on the antigenic relationships between influenza viruses is this<не принималось во внимание, <в результате чего уровни реакций перекреста ■между данными вирусами зависели от используемых тестов. Так, широко используемая штаммоспецифическая реакция связывания комплемента выявляла перекрестные реакции окзк между нейраминидазными, так и между гемагглютипи-рующими антигенами, :в то время как реакция перекреста между нейраминидазным"и антигенами может выявляться также и в РТГА. Это происходит потому, что в интактном вирусе может возникать «стерическая нейтрализация» нейр-аминидазной активности антителами к гемагглютинину и наоборот (Laver, Kilbourne, 1966; Schulman, Kilbourne, 1969; Easterday et al., 1969; Webster, Darlington, 1969).

Antigenic drift of individual influenza virus antigens can be studied after separating these antigens from the viral particle (Webster and Darlington, 1969) or by “genetically separating these antigens (Kilbourne et al., 1967). Thus, now with the use of monospecific antisera “to these two antigens, “it is possible to conduct detailed serological studies of the antigenic drift of individual influenza virus antigens.

V. MECHANISM OF ANTIGENIC DRIFT

(MINOR ANTIGENIC

CHANGE)

A. INTRODUCTION

The two distinct manifestations of antigenic variation observed among influenza A viruses, namely the sudden emergence of new antigenic subtypes and gradual drift within one subtype, are probably unrelated to each other.

It is generally accepted that drift—the sequential replacement of influenza A viruses by antigenically new strains—is the result

tat interaction of mutational variability of the virus and immunological selection

The importance of this selection mechanism is confirmed by the experimental production of antigenic variants by propagation of influenza viruses in the presence of small amounts of antiseizure (Burnet, Lind, 1949; Archetti, Horsfall, 1950; Isaacs, Edney, 1950; Edney, 1957; Laver, Webster, 1968) or in partially immune animals (Gerber et al.

1955, 1956; Magill, 1955; Hamre et al., 1958). Epidemiological

The observations are also consistent with such a mechanism, which

which offers a reasonable explanation for the disappearance of the mouth

emerging strains from the human population.

Several hypotheses have been put forward to explain the mechanism of antigenic drift. One of them (Francis, 1952, 1955, 1960; Jensen et al., 1956; Jensen, 1957) suggests that the surface of the influenza virus consists of a mosaic of antigens belonging to all strains of a given type, but present in individual antigenic strains in different proportions or in different places. Antigenic variability should be a consequence of the displacement of these antigens on the viral envelope from the protruding to the “Hidden position.” According to another hypothesis (Hilleman, 1952; Magil, Jotz, 1952; Andrewes,

1956, 1957; Takatsy, Furesz, 1957), antigens gradually

are located in the course of variability. Both of these hypotheses require

the existence of a relatively large number of antigens

but different protein molecules on the surface of the vi

Jensen et al. (1956) found that in each of the many strains in the vast collection of influenza A viruses available for research in 1953, the number of antigens present in different quantities and/or locations reached up to 18. Extension of these data to many new variants discovered since then would seem to lead to "the assumption of an even greater number of antigens in each virus, especially if accepted, and apparently

Well, it makes sense that the strains isolated from humans, pigs, horses and birds are part of the same complex.

The existence of such a large number of individual protein molecules in influenza viruses cannot be linked to the coding capacity of viral RNA (Laver, 1964). In addition, electron microscopic (Lafferty, Oertelis, 1963), immunochemical (Fazekas de St. Groth, 1961, 1962; Fazekas de St. Groth, Webster, 1963, 1964) and “biochemical (Laver, 1964) data are more consistent with the presence on the viral envelope of a very limited number of antigenically distinguishable protein molecules.

Based on recent experiments, it is assumed that antigenic drift is the result of selection of an immune population of mutant viral particles with “altered antigenic determinants, and therefore with advantages in growth in the presence of antibodies (Table 26). Moreover, it was “turned out that There are changes in the sequence of amino acids in polypeptides of hemagglutinating units of antigenic mutants isolated by selection by antibodies in an in vitro system (Laver, Webster, 1968) (Fig. 38).

Peptide maps have revealed that during natural antigenic drift there are also changes in the amino acid sequence of both the light and heavy polypeptide chains (39).

These results suggest that antigenic variation among influenza viruses is associated with changes in the amino acid sequence of their antigenic proteins. Although some of the changes in the sequence may be random, having little or no effect on antigenic determinants, it is likely that some of these changes affect antigenic determinants

HA subunits, making them less able to strictly “fit” the corresponding antibody molecules. The experiment, however, does not show whether these changes exist specifically in the antigenic determinants of viral proteins or in some other regions of the molecule.

Influenza viruses exhibit asymmetric crossover reactions in the RTGA. Fazekas de St.-Groth (1970) named viruses

which behave in a similar way, “older” and “younger” strains. Moreover, he “suggested (Fazekas de St. Groth, 1970) that in the process of natural antigenic drift, “older” influenza viruses replace “younger” strains. The last assumption “is confirmed only by very” sparse data.

B. IS IT POSSIBLE TO FORESEE THE DIRECTION OF DRIFT"

The ability of the influenza virus to undergo antigenic changes remains a major concern. Each new variant must be isolated and identified before vaccine production can begin, so each new variant has the potential to infect large numbers of people before it can be controlled with vaccines.

In this regard, attempts have been made to predict antigenic drift in the laboratory, but not entirely successfully. Hannoun and Fazekas de St. Groth at the Pasteur Institute in Paris, strain A/Hong Kong/68 (H3N2) was passaged in the presence of small concentrations of antiserum. After several such growth cycles, a variant was obtained that was no longer subject to antigenic mutations under these experimental conditions. This variant , the authors suggested, represented the end point of evolution within the NZ serotype, and was thus a virus of emergence (which might have been expected after 1970). This assumption was supported by the discovery that the London variant of influenza virus, isolated for the first time , in 1972 (A/England/42/72), was antigenically very similar to the first mutant that Hannoun and Fazekas de St. Groth obtained in their laboratory a year earlier (Fazekas de St. Groth, Hannoun, 1973) .

It was hoped that vaccines derived from the final "older" variant would provide protection against all NZ variants that might arise in humans. However, influenza A viruses subsequently isolated in 1973 and 1974 (e.g. A/Port Chalmers/1/73), which were antigenically different from the A/England/42/72 strain, were also significantly different from the artificially produced variant, suggesting that under natural conditions the drift did not go in the predicted direction.

In any case, the variant obtained in the laboratory by passage in the presence of antiserum experienced drift only in NA, whereas natural variants exhibit drift in both NA and NA. Thus, this attempt to prepare the “future” vaccine, lotidimoma, was unsuccessful.

B. POSSIBILITY OF SIGNIFICANT CHANGES IN CERTAIN ANTIGENIC DETERMINANTS DURING ANTIGENIC DRIFT

In section IV, it was shown that the HA subunits of the Hong Kong influenza virus possess at least two types of antigenic determinants and that in the process of evolution, through the antigenic drift of the Hong Kong influenza virus, a virus was formed (A/Memphis/102/72), in which one of these antigenic determinants

the termiyaant experienced a significant antigenic change (comparable in magnitude to the antigenic shift), while the other “drifted” (om. 37). We called the first of these determinants “specific” and the second “common” for these two viruses<(Laver et al., 1974).

Antibodies to the “specific” determinant do not detect any cross-reactions between the two viruses in immunodiffusion, HRT or neutralization of infectivity tests. Another determinant(s) was common to both viruses (although some antigenic drift occurred in this determinant), and cross-reactions were found between Hong Kong/68 and Memphis/72 viruses due to the same antibodies to this “common” determinant(s).

Different IB animals react to different determinants to varying degrees when immunized with the same preparation of isolated HA subunits. These variations in the immunological response may explain the variability in crossover reactions sometimes observed between two viruses when tested with different sera.

Despite the significant antigenic change in IB ONE

from determinants, peptide maps of heavy and light poly

peptides (HA1 and HA2) of HA subunits of Hong Kong/68 viruses

■and Memphis/72 were largely similar (see

39), on the basis of which it is assumed that in the process

evolution of the Hong Kong virus and education. Meme variant

fis/72 in the amino acid sequence of these polypeptides

only relatively small changes occur. Izme

differences occur in the peptide maps as heavy (HA1),

and light (HA2) polypeptide chains; some of them

may be random changes, others are selected

under the pressure of antibodies.

D. ANTIGENIC CHANGES IN NEURAMINIDASE

Antigenic drift observed in neuraminidase antigen

not influenza viruses of both type A and type B (Paniker, 1968;

Schulman, Kilbourne, 1969; Schild et al., 1973; Curry et al.

1974). It probably occurs through selection (under pressure

antibodies) mutants that have an altered sequence

amino acid content in NA subunit polypeptides

(Kendal, Kiley, 1973). So far it has not been possible to achieve anti

genetic drift in the laboratory. Antibodies to NA are not neutral

the infectivity of the virus is known; therefore it is likely that

variability of this antigen is less important for survival

virus than the variability of HA (Seto, Rott, 1966; Dowdle et al.,

E. ANTIGENIC VARIABILITY OF INFLUENZA VIRUSES TYPE B

Antigenic drift occurs among influenza B viruses to approximately the same extent as among influenza A viruses, but the significant antigenic shifts seen in them were not found among influenza B strains. Antigenic drift (includes changes in both antigens - HA nd NA (Chakraverty, 1972a, b; Curry et al., 1974).The mechanism of antigenic variation of B strains is probably similar to that inherent in influenza A viruses, but “no biochemical studies have been carried out.

E. ANTIGENIC CHANGES IN BIRD AND ANIMALS INFLUENZA VIRUSES

Antigenic changes among influenza viruses infecting lower mammals and birds have not been well studied and little information is available about them. Based on some results, however, it can be assumed that antigenic drift also occurs in strains (influenza of mammals and birds, but to a lesser extent than in influenza viruses that infect humans.

Antigenic drift has been observed in swine and equine influenza viruses (erotype 2) (Meier-Ewert et al., 1970; Pereira et al., 1972), but there is no data on antigenic drift in avian influenza viruses. Perhaps the reason for this is that birds, especially domestic birds, live shorter lives than humans or horses. In humans, each subsequent variant of the influenza A virus quickly completely replaces the previous one, but viruses that differ from each other often circulate simultaneously among animals and birds.

VI. MECHANISM OF ANTIGENIC SHIFT (SIGNIFICANT ANTIGENIC CHANGES)

During antigenic changes of another kind, the surface subunits of the virus experience significant antigenic shifts. With these major shifts, there is a sudden and complete change in one or both surface antigens, so that “new” viruses arise to which there is no immunity in the population. These are the very viruses that cause influenza pandemics.

Human H2N2 influenza viruses provide a natural system for studying the molecular aspects of significant antigenic shifts. The viruses that emerged in humans in 1957 had HA and NA subunits that were completely antigenically different from those of the H1N1 strains. H2N2 viruses

experienced antigenic drift until 1968, when a “new” pandemic strain emerged; Hong Kong. .A2 viruses (H2N2) and the Hong Kong strain (H3N2) originated in China. The Hong Kong virus had the same NA as the previous A2 viruses, but an antigenically different NA (Coleman et al., 1968; Schulman and Kilbourne, 1969). This was clearly demonstrated using specific antisera to isolated HA subunits of representatives of type A2 influenza viruses (grown in chicken embryos. These monospecific sera were used in RTGA with viruses grown in duck embryos (Webster, Laver, 1972), which eliminated the problems of steric suppression of hemagglutinating antibodies to NA and host cell antigen, which can occur when using sera to whole viruses.

The results of these tests (Table 27) showed that the serological correspondence between the hemagglutinin antigens of the “old” A2/Asia strains isolated between 1957 and

1968, and there was no Hong Kong virus (1968). Among the three Hong Kong strains isolated during the first 3 years of the influenza pandemic, there was little or no variation (Webster and Laver, 1972). Where then did the “new” HA subunits of the Hong Kong influenza virus come from? There appear to be two possible reasons for the formation of “new” hemagglutinating subunits: either they arose as a result of mutation from an existing human influenza virus or came from some other source, such as animal or avian influenza viruses.

A single mutation of the “old” influenza A2/Asia virus could cause the polypeptide chains of the HA subunits to fold so that completely new ones are formed

antigenic determinants. If the HA subunits of the Hong Kong influenza virus were obtained by such a mutation from earlier A2 type viruses, then the sequence of amino acids in the polypeptides of the “old” and “new” subunits should be close. A complete shift in one of the antigenic determinants of the HA subunits, which occurred during the process of antigenic drift, was previously described, and this “shift” in one of the determinants is not accompanied, apparently, by any significant general changes in the follower of H"Osti amino acids in HA polypeptides. However, if the “new” subunits do not arise through mutation and selection, but come from the animal influenza virus, then their polypeptide chains may differ significantly in amino acid sequence from the lolipaptide chains of the “old” A2/Asia viruses.

HA subunits were isolated from three strains of influenza A2/Asia obtained in 1968 before the onset of the Hong Kong influenza pandemic, and from three strains of Hong Kong influenza virus isolated in different parts of the world in 1968, 1970 and 1971. Due to antigenic drift, the three viruses isolated at the end of the A2/Asia period exhibit significant antigenic differences. On the other hand, the three Hong Kong strains that were isolated during the first 3 years of the new pandemic show almost no antigenic variation.

HA subunits isolated from each of these six viral strains were dissociated by treatment with guanidine hydrochloride and dithiothreitol and their light and heavy targets were separated by centrifugation (Laver, 1971). Each of the isolated polypeptide targets was trypsinized and triltic peptides were mapped. The maps showed that the polypeptide chains from the hemagglutinating subunits of the “old” A2 viruses, isolated in 1968, differed significantly in amino acid composition from the lolileptid chains of the “new” Hong Kong strains! (40 and 41). At the same time, it was assumed that the “new” polypeltides were not obtained by mutation from the “old” ones (Laver, Webster, 1972).

One explanation for this result is that a frameshift mutation results in polypeptides with completely different amino acid sequences. However, it seems unlikely that such a mutation, if it occurs, would result in polypeltides capable of forming a functional hemagglutinating unit. Second, mutations may occur affecting mainly the basic amino acids, so that the maps of tristic peptides could differ significantly without any significant change in the overall amino acid sequence of the lolyletides.

Data have now been obtained indicating that some animal influenza viruses are possible precursors of the Hong Kong strain of human influenza virus. Two strains of influenza virus, A/horse/Miami/1/63 (Heq2Neq2) ■and A/duck/Ukraine /1/63 (Hav7Neq2), isolated from horses and ducks in 1963, i.e., 5 years before the emergence of Hong Kong influenza in humans, was shown to be antigenically similar to the Hong Kong strain (Coleman et al., 1968; Masurel, 1968; Kaplan, 1969; Zakstelskaja et al., 1969; Tumova, Easterday, 1969; Kasel et al., 1969).

The HA subunits of horse and duck viruses gave cross-reactions in the RTGA and in the immunodiffusion test with the subunits of the Hong Kong strain of human influenza virus A/Hong Kong/1/68 (H3N2). Moreover, the peptide maps of the light chains of the equine, duck, and human viruses were almost identical, leading to the assumption that the light chains from these three strains have almost identical amino acid sequences (Laver and Webster, 1973). This is clearly visible from 42, where the peptide maps of lolipeptide light chains from the HA subunits of the Hong Kong influenza virus and from the duck//Ukraine and horse/Miami strains (2nd serotype) are almost identical and significantly different from the map of lolipeptide light chains from the “old” virus Asia/68.

These results suggest that equine and avian viruses and the human Hong Kong strain virus may have arisen by genetic recombination from a common progenitor, and suggest an alternative mechanism to mutation to explain the origin of Hong Kong influenza virus.

Recent studies have shown that wild bird sera contain antibodies directed against antigens present in influenza viruses that infect humans (World Health Organization, 1972). In addition, influenza viruses have recently been isolated from wild birds distant from human populations, suggesting influenza has been a natural infection of birds for many thousands of years (Downie and Laver, 1973).

Rasmussen (1964) was the first to suggest that pandemic influenza viruses arise from such animal viruses as a result of the process of recombination. Subsequently, Tumova and Pereira (1965), Kilbourne (1968) and Easterday et al. (1969) obtained antigen-hybrid viruses by genetic recombination in vitro between human influenza viruses and strains of animal and avian influenza viruses.Recently, Webster et al (1971, 1973) simulated the emergence of a new pandemic strain of influenza virus in in vivo experiments (these will be described below).

VII. ADDITIONAL EVIDENCE,

CONFIRMING THE ROLE OF THE PROCESS

RECOMBINATIONS IN THE ORIGIN OF NEW

PANDEMIC FLU VIRUSES

The biochemical data presented do not support the theory that the HA antigen of the Hong Kong virus was due to a single mutation from previous Asian strains. Therefore, one may ask whether there is any evidence obtained from in vitro or in vivo laboratory studies or especially from observational

in natural conditions, which would support a theory suggesting that new viruses arise through recombination.

A. DATA OBTAINED FROM IN VITRO STUDIES

Antigenic hybrids (recombinants) of many influenza A viruses of mammals and birds were isolated after mixed infection of chicken embryos or cell cultures with various influenza A viruses (Tumova, Pereira, 1965; Kilbourne, Schulman, 1965; Kilbourne et al., 1967; Kilbourne, 1968; Easterday et al., 1969). These studies are summarized in reviews by Kilbourne et al. (1967), and Webster and La-ver (1971). It is now obvious that recombinant influenza A viruses with mixed surface antigens (Webster, 1970b) or growth potential (Kilbourne, Murphy, 1960; Kilbourne et al., 1971) or other biological characteristics (McCahon, Schild, 1971) can be made to order.

Thus, “new” influenza viruses can be created in the laboratory, but only recently has evidence been obtained that recombination and selection of “new” viruses can also occur in vivo under natural conditions (Webster et al., 1971).

B. DATA OBTAINED FROM STUDY IN THE IN VIVO SYSTEM

1. Demonstration of recombination in the in system

Kilbourne (1970) noted that recombination between two different strains of influenza A viruses has not yet been demonstrated in intact animals, even under experimental conditions. In order to find out whether recombination can occur in vivo, two systems were used. In the first, only one of the parental viruses multiplied in the host animal, and in the second, both parental viruses multiplied. The animals were injected with large doses of the parental viruses and on the 3rd day When at least one of the viruses was multiplied, the animals were killed. Lung suspensions were examined directly in the allantois membranes for the presence of recombinant (antigen-hybrid) viruses; parental viruses were suppressed with specific antisera (Webster, 1970b).

In the first system, pigs were injected with a mixture of swine influenza virus - HH"C (A/pig/Wisconsin/1/67) and fowl fever virus type A - HPV (Denmark/27) (43). The latter does not release infectious virus after administration to pigs . Lung suspensions collected through

In the second system, where both viruses replicated, turkeys were infected with VChV and the turkey influenza virus - VGI (A/I "ndyuk/Massachusetts/3740/65). As (it was indicated, in the allantoion membrane system antigenic hybrids with VGI (G) were isolated -VChP (N) (Hav6Neql) and VChP (N)-VGI(1Ch) (Havl-N2).

There are two possible objections to the idea that the described recombination occurs in vivo. First, recombination may occur in the cell culture system used for virus selection; second, it is unknown whether these antigenic hybrids were genetically stable and were not simply phenotypically mixed particles.

The first objection can be ignored, since the selection of antigenically hybrid viruses was carried out directly at very high concentrations of antibodies, which should neutralize the parent viruses. To obtain more rigorous evidence that anti-(HHH) hybrid viruses do not arise by isolation from outside an infected host, it was necessary to obtain mixed harvest virus plaques from a plaster suspension, to isolate individual plaques, and to characterize virus samples obtained from individual plaques. 25% plaques isolated from a suspension of lungs of turkeys mixed with HPV + HIV were recombinant viruses. Hybrid viruses were not isolated from control cultures infected with an artificial mixture of both parental viruses.

The genetic stability of recombinant viruses was established by “introducing cloned antigen hybrid viruses into animal hosts (Webster et al., 1971). For example, chickens infected with an antigen-hybrid virus carrying HPV(H)-CVI(N), (HavliN2), died from a transient infection, and the virus, again isolated from the lungs of these birds after 3 days, was a pure culture of the virus, possessing B4n(H)-(Havl-N2). Other antigen-tibrid viruses were also newly isolated from animals and turned out to be genetically stable.

2. Natural transmission of the virus and selection

The studies described have shown that two different strains of influenza A virus can recombine in vivo if they are simultaneously injected into the same animal.

The simultaneous administration of large doses of two different influenza A viruses to animals is, however, an artificial system that probably does not exist in nature. To investigate whether recombination could occur under more natural conditions, two different influenza A viruses were allowed to spread simultaneously in a flock of susceptible birds as follows: two turkeys infected with HIV (A/i-ndkj/Vieconsin/66 (Hav6N2]) , were placed in a flock of 30 sensitive protected turkeys. 2 days later, two more turkeys infected with HPV were introduced into the same flock. Two turkeys from the flock were slaughtered daily and lung samples were examined for the presence of each of the parental and antigen-hybrid viruses in the allantois membranes, and by plaque isolation and virus identification (Webster et al., 1971).IPV spread rapidly among protected birds and was detected 3 days after introduction; AIV was not detected until 9 days after introduction into a flock of infected birds (Webster et al. . Experiments of this kind were carried out three times, and in each experiment, antigenic hybrids were isolated on the 9-10th day; these hybrids possessed VChP (N)-VGI (N), but no reverse -hybrids were isolated. The isolated recombinant virus probably had a growth advantage over the parent viruses; in each experiment, this virus was isolated as dominant from one or more birds. In order for a “new” strain of influenza virus to appear in nature through this kind of recombination and become an epidemic strain, the “new” virus must have some selective advantage. This selective advantage may be the possession of antigens to which the population is generally not immune, but the virus must also have the ability to transfer to susceptible hosts. Both possibilities were studied in the experiments presented. For example, by the time the recombinant virus was already present, normal birds were introduced into the flock, but the recombinants failed to become the dominant strain, and all normal contacting birds died from infection caused by the parental HPV.

3. Selection and transmission of a “new” influenza virus in an in vivo system

If we hypothesize that new strains of influenza A viruses may arise naturally through recombination, it is important to show how these viruses can be selected to become dominant or new epidemic strains. A possible mechanism of selection may be that recombination and selection take place<в иммунных животных. Опыты Webster и Campbell (1974) показали, что рекомбинация и селекция «нового» штамма -вируса гриппа может происходить у индеек с низкими уровнями антител к НА одного родительского вируса и к NA другого родительского вируса (45).

Turkeys with low levels of antibodies to NA HIV (A/indkj/Wisconsin/bb) and to NA HPV were subjected to mixed infection with HPV and HIV. 1-2 days after mixed infection, both parental viruses and a recombinant influenza virus carrying HPV (H)-HIV (N) were present in the tracheas of turkeys. On day 6 after mixed infection, only the recombinant B4n(H)iBrH(N) virus was present. On the 7th day “after a mixed infection, the turkeys died, and only recombinant influenza viruses with HPV (H)-HIV (N) were isolated. All viruses were isolated at extreme dilutions from allantois membranes or from embryos, and no antibodies were used for selection of recombinant viruses. All non-immune birds introduced into the flock on the 5th day died from a transient infection and from “they were isolated only (recombinant influenza viruses.

After mixed infection of nonimmune or hyperimmune turkeys, there was no sequestration of the recombinant influenza virus. Thus, a mixed infection of birds that have low levels of antibodies to the NA of one virus and to the NA of another provides ideal conditions for the selection of recombinants. Following infection, both parent viruses replicate to a limited extent, thereby stimulating the immune system itself, which eliminates the parent viruses. In this way, recombinants can be selected and, provided that they have the necessary virulence properties and the ability to be transmitted to other individuals, these recombinants can cause an epidemic disease.

These experiments show that, under relatively natural conditions, recombination occurs between different influenza A viruses and that new viruses may have a selective advantage over both parental strains. These experiments do not prove that all new influenza viruses of lower mammals, birds and humans arise by this mechanism, but they establish that this mechanism is one of the ways “through which new” viruses appear.

B. DATA ON THE RECOMBINATION OF INFLUENZA VIRUSES IN NATURE

The above experiments leave no doubt that new strains of influenza virus can be “obtained in vitro and in vivo, and suggest that similar processes may also occur in nature. Is there, however, any evidence that recombination in occurs in nature? This evidence is indirect and includes: 1) antigenic correspondences between influenza viruses isolated from humans and from lower mammals and birds; 2) the absence of a strict host range for influenza viruses.

1. Antigenic relationships between influenza viruses of humans, lower mammals and birds

Evidence suggesting that recombination between human and animal influenza viruses is possible in nature comes from the finding that some influenza viruses from humans, lower mammals, and birds have similar, if not identical, surface antigens.

a) Antigenic relationships due to NA. The NA of some avian influenza viruses is antigenically very similar to the NA of early human influenza viruses. For example, the duck virus (A/uzha/Germany/1868/68) has an NA similar to the NA of the human viruses HOS and H1N1 (Schild and Newman, 1969). Influenza viruses isolated from pigs also carry an NA antigen, which is related to the NA antigen of human viruses

H0N1 (Meier-Ewert et al., 1970). Similarly, HIV (A/indkj/MA/65) has an NA similar, if not identical, to that of human influenza viruses H2N2 (Pereira et al., 1967; Webster and Pereira, 1968; Schild and Newman, 1969). Other avian influenza viruses have NA antigens, ■ closely related to the NA of equine influenza viruses types 1 and 2 (Webster and Pereira, 1968; World Health Organization, 1971). Thus, the NA of VChP (A/ VChP/ Holland/27) is similar to the NA of equine influenza virus type 1 (A/ losha, d/ Prague/1/57). These interspecies relationships are used in the revised nomenclature of influenza viruses (World Health Organization, 1971). There are eight different subtypes of avian influenza viruses and four of them have NA antigens related to the NA antigens of human and equine influenza viruses.

b) Antigenic matches caused by the HA antigen. Fewer similar examples were found with influenza viruses isolated from lower mammals and birds, which would have HA antigens related to the HA antigens of human viruses. The correspondence between the HAs of the Hong Kong, duck/Ukraine/63 and horse/type 2 viruses was discussed above. Recently, it was found that a virus isolated from ducks in Germany (A/ut-ka/Germany/1225/74) has an HA similar to the HA viruses influenza family Asia. Thus, as more viruses are isolated, the number of detected matches increases.

2. Circle of hosts

Influenza A viruses are not always strictly defined

high specificity to the host (see Easterday, Tumova, 1971;

Webster, 1972). For example, the Hong Kong influenza virus was

isolated from pigs, dogs, cats, baboons and gibbons. Viru

Influenza A/Hong Kong (H3N2) viruses have also recently been isolated

from chickens and calves (Zhezmer, 1973). These viruses are experimental

but were transferred to calves and chickens; in all cases

the virus replicated in the host from which it was isolated

linen. Thus, the calf influenza virus caused a respiratory infection

tion in calves, and the chicken influenza virus replicated, but not

showed signs of disease in chickens (Schild, Campbell, Web

russ of Hong Kong influenza could not replicate in chickens.

In the case of the Hong Kong influenza virus, it is clear that this virus

has adapted to cause natural infection

tion from other owners, and thus conditions were created

when double infection and genetic

interaction

D. SUMMARY OF DATA SUPPORTING A POINT

VIEWS ABOUT THE EMERGENCE OF NEW STRAINS

FLU VIRUS BY RECOMBINATION

1. Influenza pandemics in humans are caused only by viruses

mi influenza type A, and only influenza viruses of this type were

isolated from lower mammals and birds. Influenza viruses

type B constantly recombine in vitro, but in nature they can

Such a combination of genetic information may not occur

mation [which would allow "the emergence of a pandemic

strain of influenza virus type B. Recombinations between viruses

Influenza types A and B were not shown.

2. Biochemical data presented earlier, as follows:

indicate the unlikely possibility of occurrence

“new” pandemic strains of influenza virus by

means mutation from previous influenza viruses

person.

3. New influenza viruses that can cause a pandemic

may arise through recombination and selection under conditions

in vivo experiment.

4. Based on antigenic and biochemical correspondences

vii between hemalglutinating and neuraminidase an

tigens of human influenza viruses, lower mammals

and birds suggest that genetic exchanges exist

and in nature.

The evidence presented is circumstantial; More direct evidence may be obtained if future pandemic strains are found to have antigens identical to those already isolated from domestic or wild animal influenza viruses (see also Chapter 15).

VIII. FUTURE ANTIGENE CHANGES

FLU VIRUSES AND OPPORTUNITIES

VARIABILITY PREDICTIONS

AND DISEASE CONTROL

A. POSSIBLE EXPLANATIONS FOR THE CYCLICAL NATURE OF THE PANDEMIC

Based on the study of antibodies in the sera of elderly people, it can be assumed that an influenza virus similar to the Hong Kong thrip virus existed among people in earlier times and may have been the cause of the influenza pandemic of the late 19th century (see section II). elderly people - antibodies to NA of equine influenza viruses type 2 and Asia were also detected in low titers. Antibodies to NA of influenza viruses. Hong Kong or Asia were not detected in the same ayatis-vortok, while antibodies to NA of equine influenza virus

type 2 have been identified. This suggests that viruses with similar HA subunits but different NA subunits are responsible for the previous and current epidemics. Epidemiological data have led to the belief that pandemic human influenza viruses appear cyclically. The lack of data on NA homology makes it unlikely that the same Hong Kong influenza virus exists at the end of the 19th century and again in 1968. It seems more likely that the influenza virus that existed at the end of the 19th century had an HA subunit that showed some antigenic similarity to the Hong Kong influenza virus, but carried a completely different NA antigen. Based on serological data, this NA is antigenically related to equine influenza NA type 2. A new cycle of influenza viruses may occur as a result of the emergence of viruses from some animal reservoir, with or without the participation of recombination, when herd immunity “no longer protects the human population from it.

Another phenomenon associated with the emergence of new influenza strains is the apparent disappearance of previous strains. It could simply be due to a lack of interest in collecting samples of influenza viruses that are no longer dangerous to the majority of society (Fenner, 1968), but this explanation is unlikely, since experience has shown that human influenza viruses do not coexist in nature for any long period of time. time. The disappearance of strains that appeared as a result of antigenic drift can be explained by self-eradication; serologically, the new virus increases the levels of older antibodies, thereby preventing the spread of the older virus. The disappearance of older strains (Fazekas de St. Groth, 1970) of each subtype after a significant antigenic shift is less clear and does not yet have a satisfactory explanation.

B. POSSIBILITIES FOR CONTROL OF ANTIGENIC CHANGES IN THE INFLUENZA VIRUS IN THE FUTURE

The biological, biochemical and immunological data presented above provide only indirect evidence that significant antigenic shifts in human influenza viruses occur through recombination. More definitive data will be obtained if reassortment between different influenza viruses can be detected in nature to produce a new pandemic strain. The rarity of such an event effectively rules out this possibility. An alternative approach to this problem is to isolate influenza viruses from animal populations before the next one emerges. pandemic strain for humans, i.e.

creating a “bank” of influenza viruses. After the emergence of the next strain that causes a pandemic among people, this virus can be compared with the viruses in the “bike”, and it will be possible to obtain data about its occurrence. Wildlife populations as sources of new influenza viruses have been largely ignored. Bird populations around the world live in high-density colonies for longer periods than mammals or humans. Interestingly, eight different subtypes of avian influenza viruses have already been identified, six of them - from domestic birds. It is therefore logical to begin the search for influenza viruses in nature in large bird colonies, especially at the end of the nesting season. Such ecological studies will help establish the number of different subtypes of influenza virus that exist in nature and may eventually reveal how new strains are emerging.If there are only a limited number of influenza A viruses, then in the future it will be possible to think about controlling these viruses, which represent a huge disaster for humans.

LITERATURE

Ada G. L., Lind P. E., Laver W. G. J. gen. Microbiol., 1963, v. 32, p. 225.

Andrewes S. N. Calif. Med., 1956, v. 84, p. 375.

Andrewes S H. N. engl. J. Med., 1957, y. 242, p. 197.

Andrewes S. N . In: Perspectives in Virology (M. Pollard, ed.); New York,

Wiley, 1959, p. 184-196.

Archetti I. , Horsfall F. L. J. exp. Med., 1950, v. 92, p. 441. Becht H., Hammerling U., Rott R. Virology, 1971, v. 46, p. 337. Brand S M., Skehel J. J. Nature (London ). New Biol., 1972, v. 238, p. 145. Bucher D. J., Kilbourne E. D. J. Virol., 1972, v. 10, p. 60. Burnet F. M. “Principles of Animal Virology”, 1st ed. New York , 1955, p. 380. Burnet F. M., Clarke E. Influenza, Melbourne , Walter and Eliza Hall Inst., 1942.

Burnet F. M., Lind P. E. Aust. J. Sci., 1949, v. 22, p. 109.

Burnet F. M., Lind P. E. J. gen. Microbiol., 1951, v. 5, p. 67.

Burnet F. M., White D. O. Natural History of Infectious Disease, 4th ed. London - New York, Cambridge Univ. Press, 1972, p. 202-212.

Chakraverty P. Bull. Wld Hlth Org., 1972a, v. 45, p. 755.

Chakraverty P. Bull. Wld Hlth Org., 1972b, v. 46, p. 473.

Chu C.-M. J. Hyg., Epidemiol., Microbiol., Immunol., 1958, v. 2, p. 1.

Coleman M. T ., Dowdle W. R., Pereira H. G., Schitd G. C, Chang W. K-Lancet, 1968, v. 2, p. 1384.

Compans R. W., Klenk H. D., Caliguiri L. A., Choppin P. W. Virology, 1970

The first data on the influenza virus were obtained when the virus was isolated from a patient in 1933 (Smith W. et al., 1933). The isolated virus isolate and similar ones (with similar properties) were called influenza virus type A. Subsequently, this type of virus was constantly identified during the seasonal influenza epidemics that it caused. In 1940, the influenza virus type B was identified, which is recognized as the second most important virus isolated during epidemics (Francis T., 1940; Frank A.L. et al., 1983). The virus isolated from an influenza patient in 1949 was neither type A nor type B, and was designated influenza virus type C (Francis T. et al., 1950). The course of the disease caused by influenza virus type C was not severe, and its activity did not obey the laws of seasonal epidemics (Shaw M.W. et al., 1992).

Influenza viruses types A and B represent the genus Orthomyxovirus within the family Orthomyxoviridae. The influenza virus genome is represented by a single-net segmented RNA, each segment has independent transcription (Table 1).

As shown in Fig. 1, on the surface of spherical or filamentous influenza virions there are “spikes”, which are surface glycoproteins: hemagglutinin (H) and neuraminidase (N). Influenza A viruses are described by nomenclature, including biological host, geographic origin, strain number, and year of isolation. The antigenic classification of H and N is given in parentheses, for example, A/Hong Kong/1/68(H 3 N 2). In total, there are 14 antigenic subtypes of hemagglutinin (H 1 - H 1 4) and 9 subtypes of neuraminidase (N 1 - N9).

Table 1
Implementation of genetic information of the influenza virus

All subtypes are found in waterfowl, and only some of them are found in humans (H 1 N 1, H 2 N 2, H 3 N 2), pigs and horses (Hinshaw V.S., Webster R.G., 1992). Influenza B virus does not have such subtypes.

1.1. Surface proteins

1.1.1. Hemagglutinin

Glycoprotein (H), which accounts for up to 25% of the total viral protein, performs at least three functions: a) attachment of the virus to specific receptors on the cell membrane; b) fusion of the virion and the cell when the virus enters the latter; c) providing a “platform” to which antibodies are predominantly attached that neutralize the infectivity of the influenza virus (Ada G.L., Jones P.D., 1986). The H molecule is present in the virion as a trimer. Each monomer is represented by two polypeptides, HA1 and HA2, connected by a single disulfide bond. The distal segment of the HA1 polypeptide contains active sites for binding to receptors and antibodies. Variations in the active sites designated for antibody binding are largely responsible for frequent outbreaks of influenza and failure to consistently control the virus by active immunization (Webster R.G. et al., 1982). In contrast to the variability of HA1, the HA2 polypeptide is quite unchanged (Krystal M., 1982).

1.1.2. Neuraminidase

The N virion spikes are tetramers anchored in the lipid bilayer of the virus. Neuraminidase has activity aimed at destroying receptors and prevents the aggregation of immature virions, which significantly reduces their infectivity (Palese P., 1974).

Antineuraminidase antibodies inhibit the release of immature viral particles from an infected cell by forming cross-links between budding virions. The presence of antineuraminidase antibodies is inversely correlated with the incidence and severity of clinical manifestations of infection (Schulman I.L., 1975; Ada G.L., Jones P.D., 1986). Based on these data, the concept of a “neuraminidase-specific vaccine” appeared (Couch R.B. et al., 1974).

1.2. Internal proteins

Nucleoprotein (NP) is one of the type-specific antigens by which viruses of types A, B and C differ. It belongs to the main antigens to which the activity of cytotoxic T-lymphocytes - CTLs - is directed (Tite I.P. et al., 1988). The M1 matrix protein membrane is also a type-specific antigen of the virion. Its role in the induction of immunity is not clear (Webster R.G. et al., 1982). The second polypeptide, M2, encoded by genome 7, is specifically associated with resistance to the antiviral drug amantadine (Hay A.I. et al., 1985). Polymerase proteins (PA, PB1 and PB2) transcribe viral RNA and are probably not essential in the development of immunity.

Nonstructural proteins (NS1, NS2) are encoded by the smallest segment of RNA and are probably not involved in the formation of the immune response.

2. Molecular genetics of the influenza virus

2.1. Antigenic shift

The segmented nature of the influenza virus genome is responsible for significant variations that are possible in both genotype and phenotype (Tables 2, 3) (Palese P., Kingsbury D.W., 1983). When a cell is infected with more than one strain, it can produce progeny viruses with new combinations of genes. This process, which can occur both in natural conditions and in the laboratory, is called recombination, or reassortment (Webster R.G. et al., 1971). With such an antigenic shift (literally from English “shift”), an exchange of genome sections encoding H and N occurs.

table 2
Variability of the influenza virus

Pandemic strains of influenza A virus arise when the genes of human and animal strains are reassorted while simultaneously infecting an intermediate host, possibly a pig, which serves as a “mixing vessel” (Hinshaw V.S. et al., 1978; Scholtissek C. et al., 1985).

2.2. Antigenic drift

Less radical changes - antigenic drift of components H and N occur with point mutations in genes, as a result of which changes in amino acid sequences accumulate, which in turn leads to changes in the active centers of the antigen, at which they are no longer recognized by the host's immune system.

The re-emergence of the influenza A (H 1 N 1) subtype in 1977 illustrated the phenomenon of recirculation, because the strains isolated in Northern China were highly similar to the strains circulating in 1950 (Scholtissek C. et al., 1978) .

H variability can also manifest itself during the period of adaptation of the influenza virus to the chick embryo (Schild G.C. et al., 1983; Robertson J.S. et al., 1987). Because the influenza virus used as a vaccine is cultured in chicken embryos, the question has arisen whether changes in antigenicity would be sufficient to reduce the vaccine's protective properties against circulating human strains. However, experimental data indicated that strains of virus A (H 3 N 2), grown both in chicken embryos and in mammalian cells, induce the same protective properties in humans upon contact with the virus (Katz J.C. et al., 1987).

The nature of the immune response to vaccination

Humoral (antibody) response

There is evidence that serum antibodies, measured by hemagglutination inhibition, infectivity neutralization, and N inhibition, are good predictors of an individual's resistance to infection. Serum antibodies, immunoglobulins G (IgG) to H play a major role in protection against influenza (Potter C.W., Oxford J.S., 1979; Ada G.L., Jones P.D., 1986). In high concentrations they provide complete protection. At lower concentrations, they prevent the development or weaken the manifestations of the disease after infection in a significant number of patients. Antihemagglutinin titer (1:40) or (1:32) is defined by many authors (Hobson D. et al., 1972; Longini I.M. et al., 1988; Davis J.R., Grillis E.A., 1989) as a protective threshold. Field studies suggest that preexposure titers above this value provide at least partial protection.

Thus, determination of serum antihemagglutinin levels can be considered as an indicator of the level of immunity against influenza.

Significant antibody production was sometimes recorded as early as 4 days after vaccination in adults who had previously been exposed to the virus (Zuckerman M.A. et al., 1991). Despite evidence of an extremely rapid response and high levels of protection, high protective levels of antibodies occur within 14 days after vaccination (Pyhala R. et al., 1993). The duration of protective levels of immunity after vaccination with an inactivated vaccine rarely exceeds 1 year, which is important in practical terms (Clark A. et al., 1983).

The composition of antibodies produced in response to vaccination depends mainly on the patient's previous contacts with the antigen. Patients primed with certain viral subtypes in childhood respond differently to homologous or heterologous infection or vaccination subsequently compared with those exposed to other viral subtypes (Davenport F.M. et al., 1953; Francis T. et al. , 1953; Webster R.G., 1966). This phenomenon may affect some of the vaccinated, but in no case can it be a reason to refuse annual vaccination, since new antibodies provide protection against epidemic strains of influenza A virus arise when the genes of human and animal strains are re-sorted while simultaneously infecting an intermediate host, possibly pig, which serves as a “mixing vessel” (Hinshaw V.S. et al., 1978; Scholtissek C. et al., 1985).

Since the isolation of the first influenza virus, two major antigenic shifts have occurred in the human influenza A virus, not taking into account the re-emergence of strains of this A (H 1 N 1) virus in 1977. Retrospective seroepidemiological studies have identified subtypes of epidemic strains that circulated before 1933. (Masurel N. et al., 1973). It has also been established that influenza viruses types B and C do not undergo antigenic shifts, possibly because they do not have an animal reservoir, unlike influenza A virus type. Antigenic shifts occur at irregular intervals of 10-40 years.

Local antibody response

The use of inactivated vaccines does not usually lead to the production of IgA in the upper respiratory tract, but leads to the formation of IgG antibodies in the upper and lower respiratory tract (Clements M.L., Murphy B.R., 1986). A local immune response, consisting of the production of IgG, was noted in 94% of primed patients after administration of an inactivated vaccine; IgA class antibodies were observed in only 38% (Zahradnik J.M. et al., 1983; Clements M.L. et al., 1986).

Effect of age on the immune response

Numerous observations suggest that the immune response following influenza vaccination may decline with age. It is believed that the immunodeficiency associated with aging depends on the gradual involution of the thymus, which leads to insufficient production of T cells, while B cells remain intact (Thomas M.L., Weigle W.O., 1989). It is also believed that T-cell function declines in older people, so both the quantity and quality of Tx may change with age. Thus, a decrease in the production of interleukin-2 (IL-2) with age in response to the administration of the influenza vaccine was detected (Huang J.P. et al., 1992).

Data have been published on a decrease in the effectiveness of the hepatitis B vaccine in old age, which also confirms the assumption of an age-related decrease in the immune response (Denis F. et al., 1984). However, most vaccinated hepatitis B patients living in the Northern Hemisphere have no recent exposure to the virus, and older adults have had one or more exposures to one or more types or subtypes of influenza virus circulating during their lifetime. Thus, vaccination acts as a booster and activates clones of pre-existing memory B cells.

With age, changes occur in the subclasses of antibodies produced. The decline in IgG1 antibody production with age may be responsible for the lower vaccine efficacy reported in at least several studies in older patients compared to younger patients (Hocart M.J. et al., 1990; Remarque E.J. et al., 1993; Powers D.C., 1994). IgG1 antibodies are the most effective subclass in promoting complement activation and virus neutralization. Hemagglutinin inhibitory activity correlates more with IgG1 titers than with any other IgG isotype (Burton D. et al., 1986). Activated lymphocytes obtained from elderly individuals secrete less IL-2 than IL-4, IL-6 and gamma interferon compared to cells obtained from young individuals (control) (Daynes R.A. et al., 1993). Changes in the cytokine profile may be responsible for age-related changes in the relative amounts of specific IgG subclass antibodies. Several studies have examined the CTL response to vaccine administration to prevent influenza in old age (Gorse G.J., Belshe R.B., 1990; Powers D.C., Belshe R.B., 1993).

In a comparative study of the cellular immune response in elderly individuals, significantly lower initial and peak post-vaccination levels of specific lysis of autologous target cells infected with influenza A virus were noted, but the CTL-mediated response was comparable to that in young adults. Influenza-specific CTL activity decreased in elderly individuals 3 months after vaccination (Powers D.C., Belshe R.B., 1993). The observed limited persistence of CTL memory in vaccinated elderly individuals may have some significance due to the interval between vaccine administration in the late fall and the winter influenza season.

Epidemiology of influenza

Influenza infection in humans is determined by complex interactions of several factors, including the virulence and genetic specificity of the virus, host immunity, and possibly both genetic and environmental factors that influence transmission of the virus in the human population (Hemmes J.H. et al., 1960) . Influenza is characterized by a certain seasonality with the highest activity in winter and early spring. Environmental factors during this period may be important for the transmission of influenza virus (Ghendon Y., 1991). In the tropics, influenza epidemics occur during the rainy season. The main activity of the epidemic process is observed during random peaks, sporadic activity throughout the year; endemic persistence between epidemics is a well-documented fact (Ghendon Y., 1991).

The increased incidence of influenza in closed groups (nursing homes) is due to overcrowding. The influenza virus is transmitted by airborne droplets (by coughing or sneezing), so frequent contact between people in closed groups, sharing meals and living together contributes to cross-infection (Longini I.M. et al., 1982).

Most studies have found that the incidence rate in preschool and school-age children is much higher than in adults (Monto A.S., Kiomehr F., 1975). Consequently, families with children suffer significantly more from the flu. Therefore, vaccination of children who live in direct contact with high-risk individuals, such as the elderly, is recommended to reduce the likelihood of transmission of the virus to these individuals (Immunization Practices Advisory Committee (ACIP), 1992).

According to studies of epidemic processes, the incidence of influenza is from 10 to 20% of all respiratory diseases in an epidemic year. The incidence of influenza type A is slightly higher than influenza type B (Ghendon Y., 1991). However, the incidence of type A influenza in closed groups can reach 60% (Glezen W.P., 1982).

Catastrophic pandemic 1918-1920 (“Spanish flu”), during which more than 20 million people died worldwide, the pandemics of 1957 (“Asian flu”) and 1968 (“Hong Kong flu”) indicate the danger of suddenly emerging strains of the influenza virus, against which the population lacks immunity (Collins S.D. et al., 1930). Annual winter epidemics caused by drifting variants of influenza A and B are less dramatic and therefore their danger is often underestimated. Nevertheless, they are responsible for increased morbidity and mortality in risk groups (Barker W.H., Mullooly J.P., 1980; Choi K., Thacker S.B., 1981; Blackwelder W.C. et al., 1982; Cameron A.S. et al., 1985; Lui K. , Kendal A.P., 1987; Baron R.C. et al., 1988; Glathe H., Rasch G., 1992; McBean A.M. et al., 1993; Sprenger M.C. et al., 1993).

Frequently occurring clinical complications such as pneumonia, secondary bacterial infections or exacerbations of existing pathology are dangerous for the elderly and patients with chronic diseases. In the USA, annual epidemics cause the death of 20,000-40,000 patients and hospitalizations of 150,000 - 200,000, mainly elderly people with chronic diseases (Choi K., Thacker S.B., 1981; Blackwelder W.C. et al., 1982; Lui K. , Kendal A.P., 1987; Baron R.C. et al., 1988). During the epidemic of 1989-1990. 26,000 people died from complications of influenza in the UK, 55,000 in the USA and 4100 in the Netherlands (Curwen M. et al., 1990; Sprenger M.I.W. et al., 1990). Hospitalization rates for adults with high-risk comorbidities increase 2- to 5-fold during large epidemics, reaching a peak above-ground hospitalization rate of 800 per 100,000 high-risk subjects, resulting in 1,600 “additional” hospitalizations per 1 million population, if taken as a point the estimate that 20% of the total population is at high risk (Barker W.H., Mullooly J.P., 1980; Sprenger M.C. et al., 1993).

Flu as a medical problem

Among the medical community today there is already a general understanding of the problem of influenza and its serious health consequences. The disease is associated with a high incidence of serious complications and absolute mortality rates sometimes reach millions, although deaths directly attributable to influenza may sometimes be underestimated or undetected.

Mortality due to influenza or its complications is especially high among people in closed institutions, in patients with chronic diseases, tuberculosis and bronchial asthma, arteriosclerosis, arterial hypertension, rheumatic heart disease, cerebrovascular diseases, generalized arteriosclerosis, diabetes mellitus, Parkinson's disease and multiple multiple sclerosis. For example, during the 1957 Asian influenza epidemic in the Netherlands, mortality among patients with endocrine diseases (mainly diabetes mellitus) increased by 25% (Polak M.F., 1959; Ashley J. et al., 1991; Foster D.A. et al., demic influenza A virus strains arise when the genes of human and animal strains are re-sorted while simultaneously infecting an intermediate host, possibly a pig, which serves as a “mixing vessel” (Hinshaw V.S. et al., 1978; Scholtissek C. et al., 1985).

Since the isolation of the first influenza virus, two major antigenic shifts have occurred in the human influenza A virus, not taking into account the re-emergence of strains of this A (H 1 N 1) virus in 1977. Retrospective seroepidemiological studies have identified subtypes of epidemic strains that circulated before 1933. (Masurel N. et al., 1973). It has also been established that influenza viruses types B and C do not undergo antigenic shifts, possibly because they do not have an animal reservoir, unlike influenza A virus type. Antigenic shifts occur at irregular intervals of 10-40 years.

Influenza as a socio-economic problem

The socioeconomic impact of influenza is even more difficult to assess, and the data we have are mostly published in the United States and Western Europe. Thus, in France during the influenza epidemic of 1989-1990. the total number of days of disability was 17 million (Sprenger M.J.W. et al., 1992). In 31% of cases, the reason for employee absence from work from September to March was influenza (Nicol K.L. et al., 1994) (Table 4). According to published data, coverage rates for risk groups in Europe range from 30 to 78%. In the United States, the primary goal of the health service for the year 2000 is to achieve 60% coverage of patients at risk. Low coverage rates are likely due to factors such as national health policies.

In most countries of the world, budget funds are primarily directed to solving problems of cardiovascular diseases, cancer, AIDS/HIV and sexual health, and disaster medicine. The press and scientific literature widely cover the problems of controlled randomized and uncontrolled studies, mainly devoted to the clinical effectiveness of vaccines. Unfortunately, these works cannot be used as evidence of the effectiveness of preventive medicine itself, much less to strengthen the position of prevention in the minds of officials and managers at all levels. Even experts sometimes cannot prove the effectiveness of preventive work with specific vaccines or predict the turnover of investments. However, it is estimated that the benefits of vaccination, and in particular in children, are greater than all other medical interventions, including the use of antibiotics. At the same time, it is significant that less than 10% of the global budget is spent on vaccination. The cost-effectiveness of preventing influenza is one of the most cost-effective strategies in the field of preventive medicine, second only to preventing hepatitis B. The market for all anti-infective vaccines is approximately equal to the market for only one drug for the treatment of stomach ulcers. Why is such a cost-effective strategy for the NHS not being used? Why is it still not possible to completely eradicate certain infectious diseases with the availability of modern highly effective vaccines? There are many reasons, but the main ones seem to be the following:

• Vaccination coverage must be adequate to achieve herd immunity. For example, it was hoped that measles would be completely eliminated by 1991. It is now clear that 99% of the population must be vaccinated to achieve this goal.
• Inaccurate or incorrect information about side effects, which is given too much attention. To avoid this, education and training of both health workers and the public is required.

additional literature

A feature of influenza viruses is their ability to be variable (variability of the influenza virus). Small changes in the antigenic structure of viral proteins are called antigenic drift (from the English word drift - slow flow). Such changes occur in influenza viruses every year, and for this reason influenza epidemics occur every year.

Changes in the influenza virus.

Sometimes more significant changes in the antigenic structure of the influenza virus occur, which are called shift (from the English word shift - jump). As a result of such variability, influenza viruses emerge with completely new properties, and therefore the entire population of the planet is susceptible to infection. Such viruses cause influenza pandemics. Drastic changes in the genome of the influenza virus occur once every 20-40 years and, as a rule, as a result of gene reassortment, that is, the exchange of genes between human and animal influenza viruses. It is believed that this is how a new influenza virus A/California/4/2009 (H1N1) appeared, which caused an influenza pandemic on the planet in 2009. Special studies have shown that the causative agent of this influenza pandemic is a complex reassortant, which includes genes from the classic swine flu virus, genes from the swine influenza virus of the Eurasian line, passed on to them from the avian virus in the late 70s, and genes from the virus swine influenza of the North American line, which in turn is a reassortant and includes a gene from the human influenza A (H3N2) virus.

Since the summer of 2011, a new variant of the influenza A(N3N2) virus has been isolated from some influenza patients in the United States, which in its genetic characteristics differs from the currently circulating seasonal influenza A(H3N2) virus. This variant of the influenza virus is designated influenza A(H3N2)v virus.

Experts are closely monitoring the circulation of influenza viruses in different regions of the world. This is necessary for recommendations on the composition of influenza vaccines and forecasting the epidemiological situation.

Is the swine flu pandemic over?

According to information from the World Health Organization published in August 2010, the new A(H1N1)pdm09 virus has largely completed its development cycle. The world has entered a post-pandemic period. But this does not mean that the new A(H1N1)pdm09 virus has disappeared. Experts believe it will behave like the seasonal flu virus. This virus will still cause illness. Therefore, precautions must be taken to reduce the risk of infection.

Type antigens influenza viruses type A - hemagglutinin and neuramidase; The classification of influenza viruses is based on the combination of these proteins.

In particular, 13 are isolated from the influenza A virus antigens various types of hemagglutinin and 10 types of neuraminidases. Antigenic differences among influenza viruses types A, B and C determine differences in the structures of NP and M proteins.

All strains of type A viruses have a group (S-) antigen, detected in RTGA. Type-specific influenza virus antigens- hemagglutinin and neuraminidase; variation in their structure leads to the emergence of new serological variants, often in the dynamics of one epidemic outbreak.

Changes antigenic structure of the influenza virus can happen in two ways:

Antigenic drift of the influenza virus.

Causes minor structural changes antigens caused by point mutations. To a greater extent, the structure of hemagglutinin changes. Drift develops in the dynamics of the epidemic process and reduces the specificity of immune reactions that have developed in the population as a result of previous circulation of the pathogen.

Antigenic shift of the influenza virus.

Causes the appearance new antigenic variant of the virus, unrelated or distantly antigenically related to previously circulating variants. Presumably, antigenic shift occurs as a result of genetic recombination between human and animal virus strains or latent circulation in a virus population that has exhausted its epidemic capabilities. Every 10-20 years, the human population is renewed, but the immune “layer” disappears, which leads to the formation of pandemics.

Influenza A/H1N1 as a typical emerging infection: General characteristics of influenza viruses, variability, emergence of new pandemic strains

Influenza viruses - RNA viruses - belong to the family. Orthomyxoviridae and are divided into viruses A, B and C (Table 1).

Table 1.

Comparative characteristics of influenza viruses

The influenza virus has a spherical shape and size of 80-120 nm. The core is a single-stranded negative strand of RNA, consisting of 8 fragments that encode 11 viral proteins.

Influenza A viruses are widespread in nature and infect both humans and a wide range of mammals and birds. Influenza viruses types B and C have been isolated only from humans.

Epidemially significant are 2 subtypes of influenza A virus - H3N2 and H1N1 and influenza virus type B (A.A. Sominova et al., 1997; O.M. Litvinova et al., 2001). The result of such co-circulation was the development of influenza epidemics of various etiologies in different countries during the same epidemic season. The heterogeneity of the population of epidemic viruses also increases due to the divergent nature of the variability of influenza viruses, which leads to the simultaneous circulation of viruses belonging to different evolutionary branches (O.M. Litvinova et al., 2001). Under these conditions, prerequisites are created for the simultaneous infection of humans by various pathogens, which leads to the formation of mixed populations and reassortment both between viruses of co-circulating subtypes and among strains within the same subtype (O.I. Kiselev et al., 2000).

The classification of influenza virus types is based on antigenic differences between two surface glycoproteins - hemagglutinin (HA) and neuraminidase (NA). According to this classification, influenza viruses are divided into 3 types - influenza viruses type A, type B and type C. There are 16 HA subtypes and 9 NA subtypes.

Rice. 1. Classification of influenza A viruses and types of animals and birds - intermediate and final hosts in the chain of transmission of infection to humans.
Subtype 16 (H16) of hemagglutinin was recently discovered
Note: ∗ NA 7 and NA 7-NA8 were also detected in horses

In Fig. 1 shows the subtypes of influenza A viruses and their intermediate hosts and natural reservoirs (migratory birds). The main hosts of influenza A viruses include those species that are associated with influenza.

In the human population, only three subtypes of influenza A viruses have been identified so far: HA1, HA2 and HA3. Moreover, viruses contain only two types of neuraminidase - NA1 and NA2 (Fig. 1). Their stable circulation has been proven over the past century, starting with the 1918 pandemic (R.G. Webster et al., 1978; K.G. Nicholson et al., 2003).

Influenza A viruses (to a lesser extent B) have the ability to change the structure of HA and NA. The influenza A virus is characterized by two types of variability:

• point mutations in the viral genome with a corresponding change in HA and NA (antigenic drift);
• complete replacement of one or both surface glycoproteins (NA and NA) of the virus through reassortment/recombination (antigenic shift), as a result of which a fundamentally new variant of the virus appears that can cause influenza pandemics.

For influenza B virus, antigenic variability is limited only by drift, because it apparently has no natural reservoir among birds and animals. The influenza C virus is characterized by greater stability of the antigenic structure and only local outbreaks and sporadic cases of the disease are associated with it.

Of some interest emergence of new strains of influenza virus in the human population and associated pandemics (Fig. 2). In Fig. Figure 2 presents the main antigenic shifts associated with pan-epidmias of the twentieth century caused by influenza A viruses:

• in 1918, the pandemic was caused by the H1N1 virus;
• in 1957 - H2N2 strain A/Singapore/1/57;
• in 1968 - H3N2 strain A/Hong Kong/1/68;
• in 1977 - H1N1 strain A/USSR/1/77 (many scientists did not consider this as a pandemic, but with the appearance of this strain, a situation arose with the simultaneous co-circulation of 2 strains of influenza A virus - H3N2 and H1N1).

In 1986, in China, the A/Taiwan/1/86 virus caused a widespread epidemic of influenza A/H1N1, which lasted until 1989. Drift variants of this virus survived until 1995, causing local outbreaks and sporadic cases of the disease. According to the results of molecular biological studies, multiple mutations arose in the genome of the A/H1N1 virus during these years. In 1996, two antigenic variants of the A/H1N1 influenza virus appeared: A/Bern and A/Beijing, their feature was not only antigenic, but also geographic disunity. Thus, in Russia, the influenza A/Bern virus took an active part in the influenza epidemic of 1997-98. During the same season, circulation of strains of the A/Beijing virus was registered in the east of the country. Subsequently, in 2000-2001. influenza A/H1N1 virus became the causative agent of the influenza epidemic in Russia. Modern influenza A/H1N1 viruses have low immunogenic activity; fresh isolated virus isolates interact only with the erythrocytes of mammals (human group 0 and guinea pigs).

Rice. 2. The emergence of new strains of influenza virus in the human population and associated pandemics

Influenza A viruses have undergone significant genetic changes over the past century, resulting in global pandemics with high mortality rates in humans. The largest influenza pandemic (H1N1) was in 1918-1919. ("Spaniard"). The virus, which appeared in 1918, has undergone a pronounced drift; its initial (Hsw1N1) and final (H1N1) variants are considered shift. The virus caused a devastating epidemic that claimed 20 million lives (half of the dead were young people aged 20 to 50 years (M.T. Osterholm, 2005).

Research by J.K. Tanbenberger et al., (2005) showed that the virus that caused the 1918 pandemic was not a reassortant between the avian influenza virus and the human influenza virus - all 8 genes of the H1N1 virus were more similar to variants of the avian virus than to the human one (Fig. .3). Therefore, according to R.B. Belshe (2005) avian influenza virus must infect (bypassing the intermediate host) humans, transmitted from person to person.

It is important to note that avian influenza viruses “participate” in the emergence of new “human” influenza viruses, which are characterized by high pathogenicity and the ability to cause pandemics (E.G. Deeva, 2008). These viruses (H1N1, H2N2 and H3N2) had a different set of internal genes, the origin of which indicates their phylogenetic relationship with avian and swine viruses.

What are the mechanisms of origin of pandemic strains and what biological characteristics are necessary for the emergence of a highly pathogenic virus with pandemic potential?

Influenza A viruses are characterized by a high frequency of occurrence of reassortants as a result of mixed infection, which is due to the segmentation of the viral genome. The predominance of a reassortant of a certain gene composition is considered the result of selection, in which from an extensive set of different reassortants the one that is most adapted to reproduction under given conditions is selected (N.L. Varich et al., 2009). Strain-specific properties of genomic segments can have a strong influence on the gene composition of reassortants under non-selective conditions. In other words, a distinctive feature of influenza viruses is that frequent and unpredictable mutations occur in eight of the gene segments, especially the HA gene. Reassortment plays an important role in the emergence of new viral variants, particularly in the origin of pandemic strains. And sometimes the possibility of a virus with higher virulence emerging during a pandemic cannot be ruled out.

Modern research has shown that the gene structure of the new A/H1N1 virus is complex and, as we noted in the introduction, its composition includes the genes of swine flu that affects pigs in North America; genes for swine flu, which affects pigs in Europe and Asia; avian influenza genes; human influenza genes. Essentially, the genes for the new virus come from four different sources. A micrograph of the influenza A/H1N1 virus is shown in Fig. 4.

Rice. 4. Microphotograph of influenza A/H1N1 virus

WHO published “Guidelines for influenza laboratories” and presented new data on the viral gene sequence and their length of the reassortant new influenza A/H1N1 virus (isolate A/California/04/2009): HA, NA, M, PB1, PB2, RA, NP, NS. These data indicate the formation of a new pandemic variant of the virus, creating universal vulnerability to infection due to the lack of immunity. It is becoming clear that pandemic variants of the influenza virus arise through at least two mechanisms:

• reassortment between animal/avian and human influenza viruses;
• direct adaptation of the animal/avian virus to humans.

To understand the origin of pandemic influenza viruses, it is important to study the properties of the natural reservoir of infection and the evolutionary paths of this family of viruses when changing hosts. It is already well known and can be argued that waterfowl are a natural reservoir of influenza A viruses (adapted to these intermediate hosts for many centuries), as evidenced by the carriage of all 16 HA subtypes of this virus. Through bird feces, which can survive in water for more than 400 days (Bird flu..., 2005), viruses can be transmitted to other animal species when drinking water from a reservoir. (K. G. Nicholson et al., 2003). This is confirmed by phylogenetic analysis of nucleic acid sequences of different subtypes of influenza A viruses from different hosts and from different geographical regions.

Analysis of nucleoprotein gene sequences showed that avian influenza viruses evolved with the emergence of 5 specific host lineages: viruses of wild and domestic horses, gulls, pigs and humans. Moreover (!) the human and swine influenza viruses form a so-called sister group, which indicates their close relationship and, naturally, a common origin. The predecessor of the human influenza viruses and the classic swine virus appear to have been entirely of avian origin. In the countries of Central Asia, for known reasons, pork is not popular, and these animals are practically absent from livestock farming. This leads to the fact that (unlike China, for example), this region does not have the main intermediate host in the domestic animal population - pigs, therefore the probability of the “emergence” of pandemic viruses in the Central Asian region is lower than in China, which practically follows from data on the analysis of their origin (Avian influenza, 2005). A permanent source of genes for pandemic influenza viruses exists (in a phenotypically unchanged state) in the natural reservoir of viruses of waterfowl and migratory birds (R.G. Welster, 1998). It should be borne in mind that the predecessors of the viruses that caused the Spanish flu pandemic (1918), as well as the viruses that were the source of the genome of the Asia/57 and Hong Kong/68 pandemic strains, still circulate among the wild bird population with minor mutational changes (Influenza birds..., 2005).

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