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Influenza: the evolution of the virus and the universal vaccine. Why flu mutates Viral diseases leading to mutations

Influenza virus is the champion of mutation
Between three and five million people annually suffer from severe influenza, up to 500,000 of whom die from the influenza itself or its complications (according to WHO data). Flu shots, of course, significantly reduce the likelihood of getting sick. However

unlike diseases such as measles or tuberculosis, for which immunity is developed after the first illness or vaccination and remains effective throughout life, many people get sick with the flu almost every year.

The effectiveness of immunity is determined by how successfully the immune system recognizes and neutralizes the source of infection - a virus or a bacterium. When you first get infected or vaccinated, the immune system learns to produce antibodies, molecules that bind to viral particles or bacteria and render them harmless. Once having developed antibodies, the immune system leaves them “in service” for the rest of life.

Therefore, if a person becomes infected with the same infection again, the immune system works and the infection is quickly neutralized. It is on this principle that vaccinations against measles, tuberculosis and other diseases work. Why, then, does this mechanism fail with the influenza virus and do you have to get vaccinated against influenza every year anew?

This is due to two reasons. The first is a feature of the interaction between our immune system and the virus. The surface of influenza virus particles is coated with molecules of two proteins called hemagglutinin (HA) and neuraminidase (NA) (see figure). Various variants of human influenza are classified according to the type of these proteins, for example, H1N1 (hemagglutinin type 1, neuraminidase type 1). The human immune system is able to produce antibodies that successfully bind to these proteins. The problem is that these antibodies are quite "finicky". Even small changes in the structure of HA and NA lead to the fact that antibodies lose their ability to bind to them and neutralize the virus.

From the point of view of the immune system, such modified versions of an already known virus look like completely new infections.

Secondly, the virus comes to the aid of an extremely useful (and harmful to us) property - the ability to rapidly evolve. Like all organisms, the influenza virus is subject to random mutations. This means that the genetic information of descendant viruses is slightly different from the genetic information of parent viruses. Thus, mutations are constantly creating new variants of the HA and NA proteins. However, unlike higher living organisms and many other viruses, influenza mutates very quickly:

it takes an influenza virus only a few years or even months to accumulate as many mutations as mammalian proteins accumulate over millions of years.

Thus, we can observe the evolution of the influenza virus literally in real time.

Some of the flu mutations cause the immune system, "trained" on the old strain, to recognize the mutated virus worse than the non-mutated one. While the immune system effectively fights unmutated viruses, mutant viruses multiply and infect more and more people. This is the classic process of natural selection discovered by Charles Darwin.

The selection is carried out by the immune system, which, by protecting us, unwittingly does us a disservice.

After some time - usually two or three years - the old, non-mutated strain (virus variant) dies out completely, and the mutant virus becomes the new dominant strain. Most people's immune systems learn to deal with the new strain as well, and the cycle repeats. This "arms race" between the virus and the immune system has been going on for decades.

How to fight the flu

How do you deal with the flu then? There are several ways to help our immune system. First, there are antiviral drugs, such as oseltamivir (known by the brand name Tamiflu) or amantadine, that prevent the virus from reproducing inside cells. Unfortunately, viruses eventually develop resistance to such drugs through the same process of mutation and natural selection:

for example, almost all of the H1N1 subtype virus circulating in 2009 was resistant to oseltamivir (Tamiflu).

Secondly, scientists are trying to teach the immune system to recognize less volatile parts of the virus (I wrote about this).

Third, scientists are trying to predict which strain of the virus will be the most prevalent next year. If we learn to do this, we can "retrain" our immune system as needed, pre-vaccinating against the strain that will prevail next season, and our immunity will have a head start in the arms race with the virus. Actually,

Today, the World Health Organization updates the composition of the influenza vaccine every six months.

However, sometimes - once every few years - the strain on which the vaccine was developed is not the predominant one; in this case, the vaccine is less effective. Therefore, an accurate prediction of the strain that will be most common next year is one of the important tasks in the fight against influenza.

Our group (Jonathan Dushoff, Joshua Plotkin, Georgy Bazykin and Sergey Kryazhimsky) has been studying the evolution of the influenza virus and other organisms for several years now. Our collaboration began at Princeton University in the laboratory of Professor Simon Levin, whose graduate students we were in different years. From the very beginning, we were interested in both practical questions (how to most effectively predict the next dominant strain) and fundamental evolutionary questions, for example,

whether the evolution of influenza is directed or random.

The aim of our last collaborative project was to determine the relationship between mutations occurring in different parts of the HA and NA proteins. The point is that the same mutation in, say, the HA protein can have very different consequences for the virus depending on whether the mutations occurred in other parts of the same protein. For example, mutation A allows the virus to become "invisible" to the immune system only when paired with mutation B, while each of the mutations by itself is useless for the virus. It is possible to detect such pairs of mutations, called epistatic ones, by analyzing statistical patterns in the genetic sequences of the virus. This is what we did.

Such an analysis has become possible only in recent years, when the cost of "sequencing", that is, elucidating genetic sequences, has fallen sharply.

The number of influenza virus genetic sequences registered in the database has increased more than six-fold over the past five years, reaching 150,000. This amount of data is enough to detect epistatic pairs of mutations that have occurred in the influenza virus over the past 100 years.

It turns out that the number of epistatic mutations in influenza is quite large, that is, only very specific variants of the virus that acquire the necessary combinations of mutations can, apparently, avoid an attack by the immune system or gain immunity to an antiviral drug. For example, immunity to the drug oseltamivir appeared in 2009 only in viruses with at least three specific mutations in the NA protein.

From a practical point of view, the fact that mutations in the influenza virus are epistatic allows us to hope that in the near future we will be able to predict subsequent mutations from previous ones. As long as the virus "gathers" all the necessary mutations for a successful combination, we will be able to develop a new vaccine against a strain that has the whole combination, which will spread only after a few months or even years.

To determine the success of one or another mutation in combination with others, it is necessary to understand exactly how the interaction between mutations occurs.

and how they, together and individually, affect the structure of HA and NA proteins, as well as understand how the immune system reacts to modified versions of these proteins. These issues are now being actively explored, especially in the Joshua Plotkin group at the University of Pennsylvania, with which we are actively collaborating, as well as other groups.

People are dying because of evolution. About 30% of the deaths that occur around the globe can be attributed to the evolution of simple microorganisms, from infectious agents that attack us all the time - viruses, fungi and bacteria - to our own cells, changes in which sometimes lead to cancer.

One of the most terrible infections is the most common flu. Every year it claims about 250 thousand lives, and in some years much more. The largest known influenza epidemic is the famous Spaniard of 1918, which killed several percent of the world's population.

Influenza virus strain

Like any biological object, each virus is constantly changing as a result of mutations occurring in its genome. The influenza virus is one of the fairly rapidly changing viruses. One reason is that its genetic information is encoded by RNA molecules, not DNA, as, for example, our genome; RNA is an easily mutable molecule. Another reason is that the virus is continuously affected by selection: many of the mutations that occur in its genome turn out to be “useful” for it, allowing it to be transmitted more efficiently, for example, between people.

Due to the accumulation of mutations, the properties of the influenza virus gradually change. The most noticeable result of mutations for us is changes in the antigenic properties of the virus, that is, the ability of the cells of our immune system to recognize this strain. Such gradual changes are called antigenic drift. It is now believed that most of the antigenic drift occurs in tropical latitudes, where influenza does not have pronounced seasonal epidemics and it stays at the same level in the human population all year round. But in the Northern and Southern hemispheres - respectively, in December-March and June-October - every year there are new epidemics. The WHO usually recommends a new vaccine formulation six months before the vaccine actually goes into use, because it has a long production cycle.

The evolution of the influenza virus

In addition to the gradual antigenic drift, the evolution of the influenza virus is also characterized by antigenic shifts - radical changes in the properties of the virus, which are usually associated with reassortment. The influenza virus has a genome written in eight separate segments, a bit like human chromosomes. When a host cell is simultaneously infected with two virus particles from two different strains, these segments can mix and a new virus particle with new properties can be created, consisting partly of segments from one parental strain and partly from the other. Such reassortant strains often differ in properties from the parental strains and sometimes lead to large epidemics. All the major pandemics of the 20th century that we know about - the pandemics of the 1950s and 1970s, and, most likely, the Spanish flu of 1918 - were apparently caused by such reassortments, when strains coming from different types of organisms, for example, from birds, pigs, horses, mixed up and gave something new that the human immune system had not encountered before.

Virus mutation prediction

Is the evolution of influenza predictable? In the short term, yes. Recent scientific work show that you can partly predict the future evolution of a virus if you know about its previous evolution. You can, as evolutionists love, build an evolutionary tree. Moreover, in the usual influenza A virus, it has a very characteristic shape: it is a separate trunk, from which short branches extend. When you see a tree of this shape, you can almost always be sure that you are dealing with a pathogen. There is only one lineage that is evolutionarily successful, and it is characterized by rapid changes, so that the collective immune system of mankind has to shoot at a moving target all the time. Other lines branch off from it, which eventually die out. However, there is always some variety.

In order to at least approximately understand, looking at the diversity of the current year, which of the strains observed in the current year will give rise to an epidemic next year, one must look at what mutations the strains differ from each other. If a virus has accumulated a large number of mutations in its epitopes, that is, in those places of its surface (protruding outward) proteins that are "visible" to the immune system, then most likely it will be invisible to the immune system, and therefore most likely effective. On the contrary, if it had any mutations in internal genes, then these mutations were very likely harmful - they make the virus less adapted, and such lines will die out. Can be built mathematical model, based on the number of mutations at epitopes and elsewhere, which predicts the future evolutionary success of the virus. In addition, it is possible to study how evolutionarily successful a given virus strain has been so far and extrapolate this into the future. Such approaches have limitations; for example, they do not yet take into account interactions between genes. The flu virus has 11 genes, and they all interact with each other in a rather complex way. For the time being, such considerations are generally omitted in forecasting, although various groups, including our own, have shown that they are indeed important. However, they are important in the short term.

Predicting the long-term evolution of the virus, including antigenic shifts, is much more difficult. At a minimum, this requires learning to understand which of the currently observed strains will produce a reassortant that could lead to the next serious epidemic. We do not know how to make such predictions at all, because there are a lot of incidental factors. Here it is important to look at who a person interacts with more, it is important to try to predict which strains are more likely to “learn” to be transmitted from person to person more easily.

How do epidemics occur?

Epidemics can be caused by strains that were present in the population before. For example, the current 2016 epidemic is caused by an influenza virus first seen in humans in 2009. However, the most serious epidemics are usually caused by strains new to humans. For such an epidemic to occur, several events must occur. In some kind of animal with which a person interacts, a pathogen variant capable of infecting humans must arise; this variant must be transmitted to a person; finally, as a general rule, it must acquire additional mutations to allow it to infect humans effectively. It is very difficult to estimate the probability of each of these events, so we cannot predict epidemics in advance.

Swine flu H1N1

This year, about two-thirds of all influenza cases are caused by the 2009 pandemic H1N1 strain, known as swine. This virus did indeed appear to have been acquired by humans from pigs, although the same is true for many other viruses: transmission from pigs is a fairly common mechanism for the emergence of new strains in humans. The distinguishing feature of H1N1/09 is its very interesting origin: some of its segments come from avian flu, some from swine flu, some from normal human H3N2, which has caused all infections so far. The melting pot where all these segments met each other was the pigs. It is now clear that the death rate from H1N1/09 is about the same as from the regular flu that we had every year before (although there are nuances). In fact, this year H1N1/09 has become a seasonal flu and it is possible that it will stay with us for many more years.

Universal flu vaccine

There is a fairly effective vaccine for the flu. But the problem is that it becomes outdated all the time, because every year the virus evolves, changing its antigenic properties and becoming again unfamiliar to our immune system. As a result, the vaccine has to be constantly updated. Every year, specialists from the World Health Organization (WHO) recommend to all manufacturers a new composition of the so-called trivalent vaccine, listing the three strains that should be included in it. Best of all, the trivalent vaccine protects against them. Although, of course, there is cross-immunity, and against strains that are similar in antigenic properties to these three strains, it will also protect well. Nevertheless, we are recommended to get vaccinated against influenza every year, and rightly so. This year's trivalent vaccine includes H1N1/09, so those who got vaccinated in the fall are likely to benefit now. The vaccine does not guarantee that you will not get sick, but it reduces the chance of it.

Predictions about exactly how the flu will evolve would be less relevant if we learned how to make a universal vaccine that protects against all strains. There is no such vaccine yet, although several candidates are in clinical trials. The difficulty is that the immune system “sees” just those surface proteins of the virus (hemagglutinin and neuraminidase), which the virus can easily and painlessly change for itself. Therefore, vaccination is difficult to explain to the immune system what, in fact, it needs to aim at.

Artificial synthesis of influenza strain

There was some high-profile work by one group from Holland and a group from Japan where researchers were trying to manually synthesize a strain of avian flu that would be able to be transmitted between mammals. They succeeded. Their work was considered ethically questionable, because everyone was afraid that the synthesized strain might "escape" from the laboratory, that its genes should not be made publicly available, because someone maliciously could synthesize it. However, we now know what properties a strain of bird flu can have that can be transmitted to humans.

Heredity- this is the property of organisms to ensure material and functional continuity between generations, as well as to determine the specific nature of individual development.

Variability- a property opposite to heredity. The variability of viruses is due to mutation of genes, their combination during recombination, and various manifestations of traits that depend on external conditions (modification variability).

Genetic traits (markers) of viruses. All signs of viruses, information about which is encoded in genes, are called genetic. However, many traits are usually determined by several genes.

Genetic traits (markers) in strains are determined after their preliminary cloning. All of them are conditionally divided into main groups:

group and species: type and morphology of nucleic acid; capsid type and number of capsomeres; antigenic specificity; resistance to organic solvents and detergents; the presence of the enzyme neuraminidase and host antigens; hemagglutinating properties; pathogenicity for a certain type of living sensitive systems;
intrastrain: hemagglutinating activity; thermal resistance; attitude to UV rays, inhibitors; the nature of the plaques, etc.

For genetic research, intraspecific traits are of the greatest importance, making it possible to differentiate variants (mutants, recombinants) from each other.

In most studies, genetic traits are denoted by the initial letters of the Latin alphabet with the addition of a “+” or “-” sign, i.e. the presence or absence of a particular property. Individual traits, such as neurovirulence (N), are denoted by two letters depending on the type of animal in which this property is manifested: neurovirulence for a monkey - monN, neurovirulence for mice - mN, etc.

It is still difficult to unify the nomenclature of genetic characters, therefore, for each group of viruses, their specific symbolism is justified. However, a number of features may already have common designations for all types of viruses.

Types of virus mutation. Viruses change their properties both in natural conditions during reproduction and in experiment. Two processes underlie hereditary changes in the properties of viruses:

mutation - a change in the sequence of nucleotides in a certain section of the virus genome, leading to a change in phenotypic properties;
recombination - the exchange of genetic material between two viruses that are close, but differ in hereditary properties.

Mutation classification.

By mechanism:

division - loss of one or more nucleotides;
insertion of one or more nucleotides;
substitution of one nucleotide for another.

By the length of the changed nucleotide sequence:

1) point replacement of one nucleotide (RNA-containing viruses) or one pair of complementary nucleotides (DNA-containing viruses). Such mutations can sometimes be restored (reverted) to the original structure of the genome. Not always point mutations lead to a change in the phenotype. One of the main reasons why such mutations may not appear is the degeneracy of the genetic code, i.e., the coding of one amino acid by several triplets. Therefore, the structure of the protein and its biological properties will not be violated. If an amino acid is encoded by only one triplet, then some other amino acid is included in the protein, which can lead to the appearance of a mutant trait;

2) aberration - replacement of a significant part of the genome. Aberrations in viruses are caused by deletions of various numbers of nucleotides: from one pair to a sequence that determines one or more functions of the virus.

By reversibility:

1) irreversible (direct) mutations, in which the phenotype changes. The frequency of such mutations in viruses varies widely. It depends on the cellular system in which the development of the viral population occurs. At the same time, the cell system can also be a selection factor. Mutants that arise in a population do not remain genetically pure lines, and sometimes they can multiply rapidly and almost completely displace the rest of the population;

2) reversible mutations:

true reversions, in which the reverse mutation occurs at the site of the primary lesion; pseudo-reversions, in which the mutation occurs in another part of the defective gene (intragenous mutation suppression) or in another gene (extragenic mutation suppression). Reversions are not uncommon events, as altered organisms are usually more adapted to a given cell system.

By nature:

1) spontaneous mutations that occur extremely rarely in living nature and under the influence of causes that are difficult to establish in each individual case. Such changes in the virus population occur without external influence. There are no homogeneous populations, therefore, in the course of its development, spontaneous mutants appear with a certain probability in a viral population. The frequency of mutations of the same trait may be different depending on the strain.

Spontaneous mutations can occur as a result (according to Watson and Crick, 1953):

a) tautomeric transformation (rearrangements) of the bases that make up the nucleic acid. For example, a tautomeric shift in the position of the hydrogen atom in adenine leads to the fact that during replication adenine pairs not with thymine, but with guanine. When pairing bases, the error leads in subsequent replications to the replacement of the pair of AT and GC.

Spontaneous mutations that have arisen in the same gene are distributed unevenly along its length. Some parts of the gene change frequently ("hot" spots), others rarely. Therefore, the probability of errors in base pairing in different regions of the gene is different. This may be due to the specific conformation of the nucleic acid: individual nucleotides may be more likely to undergo tautomeric transformation than others;

b) errors in the work of enzymes: DNA or RNA polymerases.

Examples of spontaneous mutations.

1. Natural variability antigenic structure(hemagglutinin and neuraminidase) of human and animal influenza viruses (including birds), on the example of type A2 strains. The original strains of type A2 did not differ from each other in antigenic structure, agglutinating activity, thermal stability, reproductive ability at 40°C and transition and indicator state. (Indicator virus is a virus that makes it possible to detect another non-cytopathogenic virus in a cell culture.) Later, strains belonging to the antigenic subgroups A2 / 1, A2 / 2 and A2 / 3 (isolated from 1968 to 1970) showed a change in the antigenic structure and other biological properties of this pathogen. Inhibitor-resistant strains first disappeared, followed by inhibitor-resistant variants from a mixed population of inhibitor-sensitive strains.

2. Hemagglutinin and neuraminidase of the influenza virus undergo changes independent of each other. For example, an avian influenza virus isolated from turkeys in Wisconsin (USA) contained a neuraminidase antigenically related to neuraminidase of human influenza viruses A2/Hong Kong/68 and not similar to hemagglutinin, while an influenza virus isolated from a pig on about. Taiwan, contained hemagglutinin and neuraminidase antigens related to human influenza A2/Hong Kong/68 antigens.

3. There are 11 known serovariants of foot-and-mouth disease virus (family picornaviruses) in type O, 32 serovariants in type A, 5 in type C, 7 in type Sat-1, and 3 each in types Sat-2 and Sat-3. FMD virus strains of type Asia 1 are also heterogeneous in antigenic structure.

4. Increasing or weakening the virulence of the pathogen Changes can occur in a short time, since the life cycle of viruses is incomparably shorter than the life cycle of the hosts. The presence of a large number of pathogenic variants of Newcastle disease virus strains (paramyxoviruses) is known. When the disease first appeared in Europe, it was acute, with a high percentage of bird deaths. Currently, foci with low mortality and a mild course of the disease are increasingly being recorded. There is not only a decrease in the number of foci, but also an attenuation of epizootic strains, which made it possible to isolate naturally weakened vaccine strains B1, La Sota, F Bor / 74 / VGNKI.

5. There are reports of the isolation of naturally attenuated strains of swine fever viruses (Miyagi strain), Aujeszky's disease (Russ strain), rabbit myxoma and tick-borne encephalitis:

2) induced mutations that occur when a virus (virion or vegetative form) is exposed to various chemical and physical mutagens, as well as when the virus adapts to unusual biological systems (adaptive variability).

After determining the hereditary units of the virus, the prospect of obtaining live vaccine strains by direct exposure to physical or chemical mutagens that cause structural and functional changes in the viral acid molecule arose. The use of artificial mutagens has two advantages:

they cause mutations tens and hundreds of times more effective than natural factors;
the action of artificial mutagens has a certain direction; it is possible to foresee in advance which elements of the structure of nucleic acids and how this or that mutagen acts and what changes it will cause in them.

Chemical mutagens. According to the Freese classification (1960), chemical mutagens are divided into two main groups:

mutagens that react with nucleic acid only during its replication (analogues of purine and pyrimidine bases);
mutagens that react with a resting nucleic acid molecule. However, subsequent replications of the molecule (nitrous acid, hydroxylamine, alkylating compounds) are necessary for the formation of mutations.

Molecular mechanisms of the mutagenic action of chemical compounds are divided into two main groups (according to E. Fries):

1) base substitution, which is of two types: a) simple (transition), when one purine base is replaced by another (for example, instead of adenine - guanine) or one pyrimidine base is replaced by another (cytosine - uracil). Such substitutions occur upon induction of mutations by nitrous acid, a number of alkylating compounds, and hydroxylamine;

b) complex (transversion), in which instead of one purine base, a pyrimidine or pyrimidine base appears is replaced by a purine base. Transversions occur when mutations are induced with ethyl ethane sulfonate;

2) loss (deletion) or insertion of a base, which leads to deeper changes in the genetic code than a simple base substitution. Mutational damage in one region of the genome often leads to changes in several genetic traits that have different phenotypic manifestations (pleiotropy). At the same time, the basis for a change in a genetic trait that has the same phenotypic expression (for example, the ability to reproduce at elevated temperatures) may be mutational damage to various genes.

Mutagenic activity of nitrogenous base analogs(5-bromouracil, 5-fluorouracil, 5-ioduracil, 2-aminopurine, 2,6-diaminopurine) lies in the fact that they induce mutations only when exposed to replicating DNA and RNA molecules. The most well studied are 5-bromouracil and 2-aminopurine. Thymine is uracil, in which the hydrogen atom (H) in one of the CH groups is replaced by a methyl group (CH 3) - methyluracil. However, in uracil, this hydrogen atom can be replaced by another atom, such as bromine (Br). As a result of such a replacement, a new compound is obtained - bromuracil, which is an analogue of thymine. The structure of the main ring in both compounds is exactly the same, and the difference is only in one group (Br instead of CH 3).

Mutations induced by alkylating compounds, are manifested by simple (transition) and complex (transversion) substitutions in the nucleic acid molecule. Purines (mainly guanine) are removed from DNA, and depending on which nucleotide is found opposite the gap during replication, either a substitution type mutation occurs or it does not occur at all. This group of substances includes alkylating compounds - mustard gas and its analogues, ethyleneimine and its analogues - ethylmethanesulfonate and ethylethanesulfonate, etc. Ethylmethanesulfonate and ethylethanesulfonate cause alkylation of guanine and, to a lesser extent, adenine. This results in the hydrolysis of the bond between the purine base and the sugar and the loss of the purine base. These compounds have been shown to be mutagenic with Newcastle disease and tick-borne encephalitis viruses.

Mutagenic effect of hydroxylamine consists in the formation of simple base substitutions in the nucleic acid molecule (the direction depends on the type of nucleic acid of the virus). In DNA-containing viruses, this mutagen reacts exclusively with cytosine. In RNA-containing viruses, it reacts with both cytosine and uracil, which causes the replacement of cytosine with uracil and vice versa.

With the help of hydroxylamine, mutations were induced in herpes viruses, Newcastle disease, and poliomyelitis. Spontaneous mutants of the herpes simplex virus (on the basis of sensitivity to agar inhibitors) are not able to revert under the action of hydroxylamine and, therefore, are due to the replacement of HC by AT.

Synthesized and studied one of the analogs of hydroxylamine - hydroxymethylhydroxylamine, which reacts only with cytosine, but not with RNA uracil, i.e., has a higher specificity and one direction of mutagenic action.

Hydrazine, which acts only on pyrimidine bases, also belongs to substances that chemically change bases in a resting nucleic acid molecule.

Mutagenic effect of intercolour agents is the ability of compounds to fit between the turns of the DNA molecule - the phenomenon of intercolation. When acridine is introduced between DNA bases, the distance between them changes by 0.34-0.7 nm. The mutagenic effect of acridine has been studied mainly on bacteriophages.

In human and animal viruses, proflavin induced Various types mutations when exposed to the intracellular virus of poliomyelitis, tick-borne encephalitis and cowpox. In combination with visible light, acridine dyes have a pronounced lethal effect on DNA-containing viruses.

Nitroso compounds (N-nitrosomethylurea, N-nitrosoethylurea, 14-methyl-M-1-nitpo-N-nitrosoguanidine) and their derivatives have a mutagenic effect. At alkaline pH, the mutagenic effect of nitroso compounds is due to the resulting diazomethane, and at acidic pH, nitrous acid.

For human and animal viruses, formaldehyde is also a mutagen; Mutants were induced in poliomyelitis virus and western equine encephalomyelitis virus when exposed to purified RNA and intracellular virus, respectively. The mechanism of the mutagenic action of formaldehyde is not well understood.

Mutagenic effect of nitrous acid is to change the bases in a resting nucleic acid molecule. The most well studied are nitrous acid (HNO 2) and hydroxylamine. Nitrous acid as a mutagen deaminates organic bases [cleaves off molecules of the amino group (NH 2)].

As a result of the action of nitrous acid, adenine (A) is converted into hypoxanthine (Gk), guanine (G) into xanthine (K), and cytosine (C) into uracil (U). Deaminated organic bases have new properties.

Physical mutagens. The mutagenic effect of elevated temperature (40-50 ° C) was discovered by E. Friz in an experiment with the bacteriophage T4 and Yu. 3. Gendon during the processing of RNA of the poliomyelitis virus. Temperature promotes the removal of purines (apurination, predominantly guanine) from DNA.

When such DNA is replicated against the gap caused by the loss of purine, any nucleotides can be included in the synthesized chain. If a base turns on, which was not previously at this place, then this means the appearance of a mutation (transition or transversion).

Mutagenic effect of ultraviolet radiation. Ultraviolet rays (UV) interact with nucleic acid molecules and are "absorbed" by organic bases, especially at a wavelength of 260-280 nm. Thymine (T), uracil (U) and cytosine (C) are more sensitive to UV than adenine (A) and guanine (G). Upon irradiation, the structure of these pyrimidines changes; two adjacent thymine molecules pair with each other, forming the so-called thymine dimers.

For the first time, the mutagenic effect of UV rays on bacteriophages was established by the American scientist Krieger (1958), then by Elmer and Kaplan (1959).

Examples: a mutation in the extracellular CD phage was obtained by A. S. Krivinsky, using the method of UV exposure to an intracellular virus, it was possible to obtain mutants of the Newcastle disease virus that differ from the original strain in reproduction in cell culture.

Under the influence of UV rays, a small-plaque mutant of the Western equine encephalomyelitis (WEE) virus was obtained, which has a stable S-phenotype in the cell culture of the FEC. The possibility of obtaining mutations under the influence of UV rays on a reproducing virus and its nucleic acid, in which RNA structural disorders occur: uracil forms limers and hydrates, has been established.

There are at least eight factors affecting the efficiency and direction of mutagenesis:

1) the nature of the mutagen;

2) specific features of the virus. Under the same experimental conditions, the same mutagen can induce mutations differently in different viruses and even strains of the same virus;

3) the period of interaction of the virus with the cell. Mutations can occur when mutagens act on the dormant and vegetative forms of the virus. In the second case, the mutagenic effect is associated not only with the possibility of penetration of the mutagen into the cell, but also with the possibility of an inextricable link with the replication of the viral genome.

At different stages of reproduction, the sensitivity of the genetic material of the virus to the action of mutagens is different, which is especially pronounced in RNA-containing viruses with a single-stranded genome. For example, ethyleneimine was first successfully used to induce mutations in vertebrate viruses when exposed to a reproducing population of tick-borne encephalitis virus.

The dependence of the lethal and mutagenic effects of the substance on the stage of virus reproduction was established: the greatest mutagenic and lethal effect was observed when exposed to this mutagen in the first 2 hours of the latent period, i.e., in the initial stage of the formation of replicative forms;

4) the number of replications occurring in the virus after exposure to the mutagen;

5) selectivity of the interaction of the mutagen with the virus genes. The effectiveness and specificity of the action of mutagens depend on the concentration of the mutagen, pH, salt composition of the medium, and a number of other factors. For many mutagens, a direct relationship has been found between the intensity of mutagenesis and the dose. However, with an increase in the dose and an increase in the mutagenic effect, the survival of the virus also decreases. There is a mathematical relationship between the value that determines the frequency of mutations, and the survival of the latter;

6) processing conditions (pH of the medium, its composition, temperature);

7) type of cell system;

8) cultivation conditions. Induced mutagenesis depends on the composition of the nutrient medium in which the "virus - cell" system is located. In the absence of thymine in the growth medium, a more intense incorporation of 5-bromouracil into the nucleic acid occurs.

The impact of the listed mutagenic factors on the native (original) virus is less effective, due to:

less accessibility of viral nucleic acid to mutagens that are unable to penetrate the protein coat of the virus;
a certain stabilizing effect of the viral protein, which has close internal bonds with the nucleic acid.

Mutant stability. Not all mutations formed under the influence of mutagens are equally stable. Differences in stability are associated with different molecular mechanisms of action of the mutagens used. Mutants obtained under the action of elevated temperature, acidic environment, UV rays and ultrasonic waves give about 20% reversions. This is due to the fact that they cause mainly local changes in the viral nucleic acid, leading to the replacement of individual bases. When exposed to proflavin, all mutants were completely stable. Mutations are caused by deletions or base insertions. When obtaining vaccine virus strains by exposing the virus to mutagens, it is advisable to use mutagens that cause deeper changes in the genetic code, such as dropouts or insertions, since such mutants have stable hereditary properties.

Mutations arising from passages (adaptation) of viruses. Mutant populations of viruses can also arise as a result of their adaptation to biological systems in vitro (cell cultures) and in vivo (animals, chicken embryos).

Mutations in animal passages. When adapting viruses to naturally susceptible animals or to heterogeneous tissues of experimentally susceptible animals, many factors are of decisive importance in the work:

properties of the virus and method of its introduction. The properties of the strain are also of considerable importance. For example, different strains of street rabies virus require different numbers of passages to convert to a fixed virus strain;
type and age of the animal. The weakening of host resistance (exposure to cortisone, temperature, γ-irradiation, etc.) is of importance.

There are many examples of the influence of the animal species used in passages on the change in the biological properties of the virus.

Examples that had a positive outcome in the form of obtaining genetically stable harmless live vaccines:

1) by serial passages of the rabies virus (Flury strain) through the brain of one-day-old chickens and further in chicken embryos (infection in the yolk sac), a vaccine strain (virus fixe) was obtained, which is apathogenic for rabbits, mice and dogs (options Hep and Lep);

2) multiple passages of the yellow fever virus through the brain of mice significantly increased the neurotropic properties of the virus for mice and lost the properties of pathogenicity for monkeys. After 258-260 passages, the adapted virus has been used since 1939 to immunize people against this infection;

3) long passages of rinderpest virus through the body of goats led to a weakening of its virulence for susceptible animals. In 1948, an attenuated version of the virus was obtained, which is used for vaccination of cattle (the Edwards strain);

4) similarly, Nakamura and Miyamato adapted the rinderpest virus (L strain) to the rabbit, which is used as a highly effective live vaccine;

5) by passages on mice and guinea pigs, it was possible to transform the viscerotropic nature of the horse distemper virus into a neurotropic one and obtain vaccine strains harmless to horses;

6) long-term passages of the FMD virus through the body of newborn rabbits or mice led over time to the attenuation of the virus in relation to cattle. At the same time, the type affiliation did not change;

7) as a result of a series of passages of the influenza virus (the most variable both under experimental and natural conditions) in mice (intranasal administration), it acquired a high pathogenicity for these animals and, at the same time, lost its pathogenicity for humans. In strains adapted to mice, a number of other properties also changed: inhibitory sensitivity, thermal resistance, etc. This indicates profound changes in the heredity of the virus.

Mutations during passages in chick embryos. Hereditary variability of viruses was also observed during their passages on chicken embryos. Vaccine strains have been obtained for the prevention of infectious bronchitis, infectious avian laryngotracheitis, dog distemper, bluetongue, rinderpest, Newcastle disease, etc.

Mutations at passages in cell cultures. Many viruses are successfully grown and attenuated in cell and tissue cultures:

after long passages in the culture of surviving tissue of chicken embryos, the yellow fever virus lost its neurotropic and viscerotropic properties, retaining its immunogenicity. The obtained strain 17D is successfully used as a live vaccine;
received a number of attenuated strains of poliovirus (three types) by passage in a culture of monkey kidney cells. The vaccine prepared from these strains is harmless to humans, causes intense and long-term immunity, providing immunity to wild strains of the polio virus circulating in nature;
by the passage method in combination with selection in cell culture of the rinderpest virus (strain LIII Nakamura), an attenuated areactogaein vaccine strain LT was obtained.

There are many reports of obtaining hereditarily attenuated immunogenic strains of foot-and-mouth disease virus, infectious rhinotracheitis, viral diarrhea, rabbit myxomatosis and parainfluenza-3 in cattle during adaptation to various types cell cultures.

Reasons for the occurrence of mutations in the process of adaptation. The change in the properties of the virus during the passages occurs in steps. In the first passages, mainly virions are found that have changed any one genetic trait. With an increase in the number of passages in the population, virions are detected that have changed two or more genetic traits; the number of such particles is constantly increasing, and in the future, in the vast majority of viral particles, a change in many genetic traits is observed.

The mechanism of hereditary variability of the viral population during passages is based on two processes: mutation and selection. In both processes, an important role is played by the external environment, which is both a mutation inducer and a selective factor.

If a heterogeneous viral population, which includes modified and original viral particles, is cultivated under normal conditions, then its reversion occurs. For example, studies with attenuated poliomyelitis virus type 3 have shown that the reversion of altered traits can be associated not only with the heterogeneity of the viral population, but also with the presence of virus particles with low stability of hereditary properties in a genetically homogeneous population.

A large number of facts have accumulated about the variability of the virus caused by the host (host-controlled variation). The changes lie in the fact that the cell affects the nature of the components of the virus synthesized in it. Such modifications do not affect the nucleotide sequence of the virus genome. They are described in DNA-containing bacteriophages, the Sendai virus, Newcastle disease, influenza, etc. The composition of proteins encoded in the virus genome can be modified by the host cell. This is due to the presence in the cell of special mutagenic forms of tRNA with an impaired correspondence between the anticodon and the codon of the corresponding amino acid.

Modifications are possible caused by the inclusion of host cell components (mainly proteins and lipids) into the viral particle: myxoviruses, paramyxoviruses, the shell of which is a modified cell membrane. Such a membrane contains both cellular lipids and cellular proteins of the host. Therefore, when the host cell (cell system or species of organisms) changes, cellular antigens also change in the structure of the virus envelope.

Antigenic variants of alphaviruses and flaviviruses are associated with culture conditions in different host systems. The protein components of the host cells are included in the supercapsid shell of the virion, causing changes in its antigenic characteristics.

Thus, the host cell can significantly influence the phenotype of the virus or block (partially or completely) its reproduction.

Consequences of mutations:

change in phenotypic manifestations under normal conditions. For example, the size of plaques under agar gel increases or decreases; increases or decreases neurovirulence for a particular animal species; the virus becomes more sensitive to the action of a chemotherapeutic agent, etc. Morphological or structural mutations may relate to the size of the virion, the primary structure of viral proteins, changes in genes that determine early and late virus-specific enzymes that ensure the reproduction of the virus;
a lethal mutation that disrupts the synthesis or function of a vital virus-specific protein, such as viral polymerase;
a conditionally lethal mutation in which a virus-specific protein retains its functions under certain optimal conditions for it and loses its functions under unfavorable conditions. An example of such mutations are temperature-sensitive (from English temperature-sensitive) - ts-mutations, in which the virus loses the ability to multiply at elevated temperatures (39-42 ° C), while maintaining this ability at normal growing temperatures (36-37 ° C ).

Selection of mutants. When working with modified organisms, it is important to study the mutational variability of a particular virus and to select mutants of interest. For selection, the physicochemical and biological properties of mutants are determined:

the relationship between a change in a certain trait (marker) and virulence (reactogenicity, immunogenicity and other properties of the virus) - covariance of genetic traits in mutants;
the nature of the mutant phenotype: the ability to reproduce in a particular system, thermal resistance, hemagglutinating, hemolyzing and other properties.

Since the properties of the population as a whole are studied in virology, a selection process is necessary for the manifestation of a change in the heredity of a virus, i.e., the creation of such conditions under which the predominant reproduction of viral particles with altered heredity occurs. As a result, the entire viral population will consist of homogeneous genetic mutant virions.

When obtaining a genetically homogeneous population in experimental studies, the following selection methods are used:

isolation of clones from single pustules on the chorioallantoic membrane of the chick embryo;
selection of clones from plaques in cell culture;
selection by the method of limiting dilutions;
selection by selective adsorption and elution;
passage selection under modified cultivation conditions.

For genetic analysis, only those traits are suitable that are easily detected, sufficiently stable, and controlled by a single mutation. In animal viruses, such complex traits as pathogenicity, virulence, and antigenic structure are controlled not by one, but by many genes.

Currently, animal virus mutants are selected based on the analysis of conditionally lethal mutations. All conditionally lethal mutants of this class (for example, all ts-mutants) have one common trait and are phenotypically indistinguishable in most cases. One of the reasons for the emergence of ts-sensitivity as a result of a mutation is a change in the primary structure of a protein, such as an enzyme. Moreover, this change is such that the enzyme works at a permissive temperature, and an increase (decrease) in temperature changes its conformation more strongly than the conformation of the wild-type protein, and leads to the absence of enzymatic activity in the mutant protein.

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Introduction

Improving the safety and productivity of farm animals is impossible without further improvement of veterinary services for animal husbandry. Among the veterinary disciplines, an important place belongs to virology. A modern veterinarian should know not only the clinical and pathological side of the disease, but also have a clear understanding of viruses, their properties, laboratory diagnostic methods and features of post-infection and post-vaccination immunity.

Viruses change their properties both in natural conditions of reproduction and in experiment. Two processes can underlie the hereditary change in the properties of viruses: 1) mutation, i.e., a change in the nucleotide sequence in a certain part of the virus genome, leading to a phenotypically pronounced change in the property; 2) recombination, i.e., the exchange of genetic material between two viruses that are close, but differ in hereditary properties.

Mutation in viruses

Mutation - variability associated with a change in the genes themselves. It can be intermittent, spasmodic and lead to persistent changes in the hereditary properties of viruses. All mutations of viruses are divided into two groups:

· spontaneous;

· induced;

According to their length, they are divided into point and aberration (changes affecting a significant part of the genome). Point mutations are caused by the replacement of one nucleotide (for RNA-containing viruses). Such mutations can sometimes reverse, restoring the original genome structure.

However, mutational changes are also capable of capturing larger regions of nucleic acid molecules, i.e., several nucleotides. In this case, dropouts, insertions and displacements (translocation) of entire sections and even turns of sections by 180 ° (the so-called inversions), reading frame shifts can also occur - larger rearrangements in the structure of nucleic acids, and consequently, violations of genetic information.

But not always point mutations lead to a change in the phenotype. There are a number of reasons why such mutations may not show up. One of them is the degeneracy of the genetic code. The protein synthesis code is degenerate, i.e., some amino acids can be encoded by several triplets (codons). For example, the amino acid leucine can be coded for by six triplets. That is why, if in the RNA molecule, due to some influences, the triplet of CUU was replaced by CUC, CUA by CUG, then the amino acid leucine will still be included in the synthesized protein molecule. Therefore, neither the structure of the protein nor its biological properties will be violated.

Nature uses a peculiar language of synonyms and, replacing one codon with another, puts the same concept (amino acid) into them, thus preserving its natural structure and function in the synthesized protein.

Another thing is when some amino acid is encoded by only one triplet, for example, the synthesis of tryptophan is encoded by only one UGG triplet and there is no substitution, i.e., a synonym. In this case, some other amino acid is included in the protein, which can lead to the appearance of a mutant trait.

Aberration in phages is caused by deletions (dropouts) of various numbers of nucleotides, from one pair to a sequence that determines one or more functions of the virus. Both spontaneous and induced mutations are also divided into forward and reverse.

Mutations can have different consequences. In some cases, they lead to a change in phenotypic manifestations under normal conditions. For example, the size of the plaques under the agar coating increases or decreases; increases or decreases neurovirulence for a particular animal species; the virus becomes more sensitive to the action of a chemotherapeutic agent, etc.

In other cases, the mutation is lethal because it disrupts the synthesis or function of a vital virus-specific protein, such as viral polymerase.

In some cases, mutations are conditionally lethal, since the virus-specific protein retains its functions under certain conditions and loses this ability under nonpermissive (nonpermissive) conditions. A typical example of such mutations are temperature-sensitive - ts-mutations, in which the virus loses the ability to multiply at elevated temperatures (39 - 42°C), while retaining this ability at normal growing temperatures (36 - 37°C).

Morphological or structural mutations may relate to the size of the virion, the primary structure of viral proteins, changes in genes that determine early and late virus-specific enzymes that ensure the reproduction of the virus.

According to their mechanism, mutations can also be different. In some cases, a deletion occurs, i.e., the loss of one or more nucleotides, in others, one or more nucleotides are inserted, and in some cases, one nucleotide is replaced by another.

Mutations can be direct and reverse. Direct mutations change the phenotype, and reverse (reversions) restore it. True reversions are possible when a back mutation occurs along with the primary damage, and pseudo-reversions if the mutation occurs in another part of the defective gene (intragenous mutation suppression) or in another gene (extragenic mutation suppression). Reversion is not an uncommon event, as revertants are usually more adapted to a given cellular system. Therefore, when obtaining mutants with desired properties, for example, vaccine strains, one has to take into account their possible reversion to the wild type.

Viruses differ from other representatives of the living world not only in their small size, selective ability to reproduce in living cells, structural features of the hereditary substance, but also in significant variability. Changes may relate to the size, shape, pathogenicity, antigenic structure, tissue tropism, resistance to physical and chemical influences and other properties of viruses. The significance of the causes, mechanisms and nature of the change is of great importance in obtaining the necessary vaccine strains of viruses, as well as in developing effective measures to combat viral epizootics, during which, as is known, the properties of viruses can significantly change one of the reasons for the relatively high ability of viruses to change their properties. is that the hereditary substance of these microorganisms is less protected from the effects of the external environment.

Mutation of viruses can occur as a result of chemical changes in cistrons or a violation of the sequence of their location in the structure of the viral nucleic acid molecule.

Depending on the conditions, the natural variability of viruses, observed under normal conditions of reproduction, and artificial, obtained in the process of numerous special passages or by exposing viruses to special physical or chemical factors (mutagens), are distinguished.

Under natural conditions, variability does not manifest itself in all viruses in the same way. This feature is most pronounced in the influenza virus. The pangolin virus is subject to significant variability. This is evidenced by the presence of a large number of variants in different types of these viruses, and significant changes in its antigenic properties at the end of almost every epizootic.

2) recombination, i.e., the exchange of genetic material between two viruses that are close, but differ in hereditary properties.

Mutation in viruses

Mutation is the variability associated with a change in the genes themselves. It can be intermittent, spasmodic and lead to persistent changes in the hereditary properties of viruses. All mutations of viruses are divided into two groups:

· spontaneous;

· induced;

However, mutational changes are also capable of capturing larger regions of nucleic acid molecules, i.e., several nucleotides. In this case, dropouts, insertions and displacements (translocation) of entire sections and even turns of sections by 180° (the so-called inversions), shifts of the reading frame can also occur - larger rearrangements in the structure of nucleic acids, and consequently, violations of genetic information.

In other cases, the mutation is lethal because it disrupts the synthesis or function of a vital virus-specific protein, such as viral polymerase.

Mutation of viruses can occur as a result of chemical changes in cistrons or a violation of the sequence of their location in the structure of the viral nucleic acid molecule.

Mutation frequency and mechanisms of their occurrence

Mutations in bacteriophages have been studied very intensively, not only for the purpose of genetic analysis, but also to obtain information about the properties of the phages themselves. The frequency of occurrence of certain mutants in phage progeny varies within a very wide range: for example, some mutants are formed with a frequency of no more than 10, while others arise with a frequency of 10 or more. The adverse effect of a high mutation rate is usually offset by the action of selection. For example, a phage mutant may be out-competed by a wild-type that yields a higher yield of phage.

A high frequency of spontaneous occurrence is usually characteristic of such mutations, which can occur at many sites of the same locus. In those cases where the normal trait corresponds to the functional form of the gene, and the mutant one appears as a result of some change at any point of the given locus, the frequency of direct mutations will be higher than the frequency of reverse mutations, since reverse mutations should lead to the restoration of the normal state. Sometimes revertants are actually pseudo-revertants, either as a result of changes in some other gene (suppressor mutations) or as a result of changes in the same gene that cause a different, but also active, form of the product.

In mature phage particles, the frequency of spontaneous mutations is very low, but they can be induced by exposure to any mutagenic factors, such as x-rays or ultraviolet rays, nitrous acid, hydroxylamine, or alkylating agents. Nitrous acid deaminates the bases of nucleotides, and ethyl methylsulfonate ethylates them. Hydroxylamine converts schitosin into uracil. When infected with modified phages, due to errors that occur during the replication of a chemically modified nucleic acid, mutations occur, and phage progeny released from one bacterium contain both normal and mutant particles. However, as expected, treatment of a phage containing single-stranded DNA with a mutagen produces a pure clone of the mutant.

The study of the mutation process that occurs during phage reproduction is of great direct relevance to the analysis of phage development. Let us first consider the spontaneous mutation process. In a bacterial cell in which phage mutation has occurred, both normal and mutant phage are formed. The number of mutant phage particles contained in a phage population emerging from a given single bacterial cell is obviously determined by the nature of phage reproduction, since new genes can be formed only by replication of pre-existing ones. If the probability of a given mutation is the same for each replication, then the number of mutants that have arisen depends on the mechanism of replication. For example, if each new copy gene is formed independently of the others, then the distribution of mutant copies in the progeny of the phage from various infected bacteria will be random. If, on the other hand, each of the resulting copies is reproduced in turn, then the mutant copies will occur in groups, or clones, consisting of mutant "siblings".

Host Modifications

In addition to mutations, bacteriophages undergo non-genetic changes in which the host cell plays the main role. This phenomenon is called host-induced modifications. The importance of these modifications for molecular biology is that they have demonstrated the ability of the intracellular environment to induce such changes in the chemical structure of the genetic material, which can be used to identify cell lines that synthesize DNA. Similar phenomena were first discovered on phage DNA, but they are also true for any DNA of a bacterial cell. There are also observations according to which this phenomenon is also true for eukaryotic cells. In special cases, more complex situations may arise. Bilateral restriction of the phage by two hosts is sometimes observed, but it is not mandatory.

The phage that is rejected by the cells is able to adsorb on them and inject their DNA. However, part of the latter is rapidly destroyed and replication does not occur. DNA degradation is caused by specific endonucleases (restriction enzymes, or R-nucleases), which are able to recognize specific sections of DNA and cleave them if they have not been modified under the influence of M-enzymes. After that, DNA is cleaved by exonucleases to individual nucleotides. A bacterial strain may have one or more R-nucleases and at the same time M-enzymes that protect the cell's own DNA. A convenient nomenclature of these enzymes has been proposed. According to some data, the sites of recognition of R-nucleases do not always coincide with the sites of DNA cleavage; perhaps the enzyme is able to migrate along the strand before it finds a site where the DNA is to be cleaved.

The functional role of host-induced modifications is unclear. They are able to protect a given strain of bacteria from massive destruction by phages growing on various bacteria. More generally, the role of modifications can be defined as protection against the entry of unacceptable foreign DNA into a bacterial cell and its subsequent “engraftment”. Bacterium A, which rejects phage propagated on strain B, also rejects bacterium B's DNA if introduced by conjugation or transduction.

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