GROWTH AND CULTIVATION OF BIOOBJECTS - V. M. Samygin - 2016

CHAPTER 10. VIRUSES AS BIOLOGICAL OBJECTS

Viruses (from Latin virus meaning "poison") are Obligate Intracellular Parasites that typically cause diseases in humans, animals, plants, and microorganisms. Scientists have also discovered viruses that infect other viruses (satellite viruses). Outside a host Cell, Viral Particles show no signs of life and behave merely as biopolymer particles. Until the late 19th century, the term "virus" was used in medicine to describe any infectious agent causing disease. This word was first applied in 1728. The term "virion," coined in 1959, is used to designate a single, stable viral particle that has left the host cell and is fully capable of infecting other Cells of the same type.

In 1892, the Russian botanist D. I. Ivanovsky demonstrated the "filterability" of the CAUSATIVE AGENT OF tobacco mosaic disease. Five years later (in 1897), another filterable agent—the pathogen responsible for FOOT-and-Mouth disease in cattle—was discovered by the German bacteriologist F. Löffler. In 1901, the American surgeon W. Reed established that the yellow fever agent is also a filterable virus. In 1917, Félix d'Hérelle, a Canadian scientist working at the Pasteur Institute in Paris, discovered a virus that infects Bacteria: the bacteriophage. These discoveries marked the birth of virology, a new science dedicated to studying non-Cellular forms of life. The tobacco mosaic virus was the first virus to be crystallized, which provided profound insights into its Structure.

10.1. Hypotheses on the Origin of Viruses

Viruses are found wherever there is life and have presumably existed since the appearance of the first living cells. THE ORIGIN OF viruses remains unclear,

because they leave no fossil record, meaning their evolutionary relationships can only be studied using the Methods of molecular phylogenetics.

There are three primary hypotheses regarding the origin of viruses:

the regressive hypothesis;

the cellular origin hypothesis;

the coevolution hypothesis.

Regressive hypothesis. According to this hypothesis, viruses were once small cells that parasitized larger cells. Over time, these cells shed genes that became "superfluous" to a parasitic lifestyle. This hypothesis is supported by the observation that certain bacteria, specifically rickettsiae and chlamydiae, are cellular organisms that, much like viruses, can reproduce only inside another cell. This is also known as the degeneration or reduction hypothesis.

Cellular origin hypothesis. Some viruses may have evolved from fragments of DNA or RNA that "escaped" from The Genome of a larger Organism. Such fragments could originate from Plasmids (DNA molecules capable of transferring from Cell to Cell) or Transposons (DNA molecules that replicate and move to different positions within the genome). Transposons, formerly known as "jumping genes," are Examples of Mobile Genetic Elements and may have given rise to certain viruses. Transposons were discovered by Barbara McClintock in 1950 in maize. This hypothesis is also referred to as the vagrant or escape hypothesis.

The coevolution hypothesis suggests that viruses evolved from complex systems of Proteins and Nucleic Acids at the same time as the first living cells on Earth, and have depended on cellular life for billions of years. Besides viruses, other non-cellular forms of life exist. For instance, Viroids are RNA molecules that are not classified as viruses because they lack a protein coat. Nevertheless, they share several characteristics with certain viruses and are therefore classified as subviral particles. Viroids are major plant pathogens. Although they do not encode their own proteins, they interact with the host cell and hijack its machinery to replicate their RNA.

Each of these hypotheses has its weaknesses: the regressive hypothesis fails to explain why even the smallest cellular parasites bear no resemblance to viruses. The escape hypothesis does not account for The formation of the capsid and other viral particle components. The coevolution hypothesis contradicts the definition of viruses as non-cellular entities dependent on host cells. Nevertheless, many experts currently view viruses as ancient organisms that presumably emerged before cellular life split into three domains. This is supported by the fact that certain viral proteins show no Homology with proteins from bacteria, archaea, and eukaryotes, indicating that this group diverged in the distant past. Otherwise, the origin of viruses cannot be definitively explained by the three established classical hypotheses, making a revision and refinement of these theories necessary.

10.2. Viruses as a Form of Life

According to one definition, viruses represent a form of life; according to another, they are simply complexes of organic molecules that interact with living organisms. Viruses are often described as "organisms at the edge of life." They resemble living organisms in that they possess their own set of genes, evolve through natural Selection, and can reproduce by creating copies of themselves via self-assembly. Viruses carry genetic material, yet they lack cellular structure. They have no METABOLISM of their own and require a host cell to synthesize their molecules. Consequently, they cannot reproduce outside a cell. While recognized forms of life reproduce by Cell Division, viral particles spontaneously assemble inside an infected cell. Unlike the growth of crystals, viral reproduction involves the inheritance of Mutations and is subject to natural selection. The self-assembly of viral particles within a cell provides further support for the hypothesis that life may have originated from self-assembling organic molecules. Some Bacteriophages possess their own adaptive immune systems, which serves as an additional argument in favor of defining viruses as a form of life.

Thus, the main properties distinguishing viruses from the rest of the living world can be summarized as follows:

- Ultramicroscopic dimensions: ranging from 10–20 to 300–350 nm. On average, they are 50 times smaller than bacteria and cannot be seen with a Light Microscope because viral particles are smaller than the wavelength of light.

- Viruses contain only one type of nucleic acid—either DNA (DNA viruses) or RNA (RNA viruses).

- Viruses lack any cellular structures, their own metabolism, a protein-synthesizing apparatus (Ribosomes), and energy-generating mechanisms (ATP). Because of this, viruses are obligate intracellular parasites. Unlike other organisms that parasitize at the organismal or cellular level, viruses are ultra-parasites operating at the genetic level.

- Their natural habitats are living cells—bacteria (in the case of bacterial viruses or bacteriophages), as well as plant, animal, and human cells.

- Viruses are incapable of growth and binary fission; they reproduce by Replication inside an infected host cell using their own genomic nucleic acid.

All viruses exist in two qualitatively distinct forms: extracellular (the virion) and intracellular (the virus). The Taxonomy of these micro-organisms is based on the characteristics of virions—the terminal phase of the viral life cycle.

Viruses spread through numerous mechanisms: plant viruses are often transmitted from plant to plant by insects, while animal viruses can be disseminated by Blood-sucking vectors. Influenza Viruses spread via airborne droplets during coughing and sneezing. Noroviruses and rotaviruses, which typically cause viral gastroenteritis, are transmitted via the fecal-oral route through contaminated food or Water. HIV is one of several viruses transmitted sexually and through transfused contaminated blood. Each virus exhibits a specific host tropism, determined by the types of cells it can infect. This host range may be narrow or, if a virus affects multiple species, broad.

10.3. Virus Classification

Virus classification is based on the structural and Functional Characteristics of virions: their nucleic acid type (DNA or RNA, single- or double-stranded); the size and number of nucleic acid molecules in the virion; the geometry and Structural Features of the capsid and outer envelope; the host type (plants, bacteria, insects, mammals); the pathology caused by the virus; the antigenic properties of viral proteins, etc.

In biological taxonomy, viruses are assigned to a distinct taxon. The fundamental principles of virus classification based on the Linnaean hierarchical system were first developed by André Lwoff, Robert Horne, and Paul Tournier (1962). The principal taxa in this system are Class, order, family, genus, and species. Viruses were grouped according to their shared properties and the type of nucleic acids in their genomes. Later, the International Committee on Taxonomy of Viruses (ICTV) was established. However, The concepts of "division" and "kingdom" are not used in virus taxonomy because their small Genome Size and high mutation rate make it difficult to determine evolutionary relationships for groups higher than an order. Essentially, the Baltimore classification serves as an extension of the traditional classification system.

The systematics and taxonomy of viruses are currently codified and maintained by the International Committee on Taxonomy of Viruses (ICTV). The most widely recognized frameworks are the ICTV classification and the Baltimore classification.

The ICTV classification is based on viral properties. However, only a small fraction of the total diversity of viruses has been studied to date; analyzing viral samples from The Human Body revealed that about 20% of viral nucleic acid sequences had not been observed previously, while samples from environmental sources, such as seawater and the ocean floor, demonstrated that the vast majority of sequences are entirely novel. The primary taxonomic ranks are:

Order (virales)

Family (viridae)

Subfamily (virinae)

Genus (virus)

Species (virus)

The modern ICTV classification (2012) includes seven viral orders: Caudovirales, Herpesvirales, Ligamenvirales, Mononegavirales, Nidovirales, Picornavirales, and Tymovirales. The existence of an eighth order (Megavirales) has been tentatively proposed. This classification does not distinguish subspecies, strains, or isolates. In total, there are six orders, 87 families, 19 subfamilies, 349 genera, approximately 2,284 species, and over 3,000 unclassified viruses.

The ICTV classification is currently integrated with the Baltimore classification to form the modern system of viral taxonomy. The Baltimore classification of viruses is based on The Mechanism of mRNA synthesis. Viral genomes can be single-stranded or double-stranded, DNA- or RNA-based, and may or may not utilize reverse transcriptase. Furthermore, single-stranded RNA viruses may have a positive sense (+) or negative sense (-) RNA strand within their genome. This system comprises seven main groups:

(I) Viruses containing double-stranded DNA and lacking an RNA stage (e.g., Herpesviruses, Poxviruses, Papovaviruses, mimivirus).

(II) Viruses containing a single-stranded DNA molecule (e.g., Parvoviruses). In this case, the DNA is always of positive polarity.

(III) Viruses containing double-stranded RNA (e.g., rotaviruses).

(IV) Viruses containing a single-stranded RNA molecule of positive polarity (e.g., Picornaviruses, Flaviviruses).

(V) Viruses containing a single-stranded RNA molecule of negative or ambisense polarity (e.g., orthomyxoviruses, filoviruses).

(VI) Viruses containing a single-stranded positive-sense RNA molecule and having a DNA Synthesis stage from an RNA template in their life cycle — Retroviruses (e.g., HIV).

(VII) Viruses containing double-stranded DNA and having a DNA synthesis stage from an RNA template in their life cycle — retro-transcribing viruses (e.g., hepatitis B virus).

Further subdivision is based on features such as genome structure (presence of segments, circular or linear molecule), genetic similarity to other viruses, presence of a lipid envelope, host organism taxonomy, etc.

10.4. Structural Organization of Viruses

Virions of the simplest viruses consist of a nucleic acid and a protein coat called a capsid. The virions of certain more complex viruses possess an additional outer envelope, known as the supercapsid (peplos), On the surface of their protein capsid (Fig. 10).

Fig. 10. Schematic diagram of virion structure

Capsids are formed by protein subunits arranged in a highly specific manner. Moreover, the capsids of various animal, plant, and bacterial species are generally built according to a common architectural blueprint based on a relatively simple geometric principle of helical or isometric Symmetry. The capsid is constructed from identical repeating subunits called capsomeres. An additional lipoprotein envelope (supercapsid) is found in relatively large viruses (such as influenza and herpes). The protein capsid performs a protective function, shielding the viral genome from various Physical and Chemical factors, primarily Enzymes (Nucleases). The second function of the capsid is determined by the presence of a receptor within its structure that is complementary to the receptor of the target cell, thereby mediating the chemisorption of the virus onto The surface of the host cell.

The core of the virus consists of nucleic acids; unlike all other living organisms, viruses contain only a single type of nucleic acid (either DNA or RNA). Depending on the nucleic acid type, viruses are classified as DNA-genomic or RNA-genomic. Viral nucleic acids combine the Functions of both acids—DNA and RNA. Thus, in the realm of viruses, The Genetic Code can be stored and executed by RNA as well as DNA.

Viral nucleic acids are highly diverse. DNA genomic viruses contain both conventional double-stranded DNA and single-stranded DNA, and they may have linear or circular structures. RNA genomic viruses can contain either single-stranded (conventional) or double-stranded RNA. These nucleic acids most commonly exhibit a linear structure. Among RNA viruses, there are so-called "multichromosomal" viruses with a fragmented genome. In some of these viruses, genome fragments are distributed among multiple virions; viruses of this type are called coviruses.

The viral supercapsid consists of a lipid bilayer in which specific protein molecules are embedded. The lipid content in the supercapsid of large viruses reaches 20–40% of the dry mass of the virion. In addition to the lipid fraction, CARBOHYDRATES have been detected in the supercapsids of certain viruses.

10.5. Life Cycle

Viruses, unlike all pro- and eukaryotes, are incapable of reproducing by binary fission. Viral reproduction occurs through replication within a host cell. The replication of viral Nucleic Acids and Proteins takes place in different PARTS OF THE cell at different times. The reproduction cycle is a process wherein cellular mechanisms are hijacked by foreign Genetic information. Approximately 10 enzymes have been discovered in viruses, which are divided into two groups: 1) enzymes that facilitate the penetration of viral nucleic acid into The Cell and the release of newly formed virions into the environment; and 2) enzymes involved in the Transcription and Replication of viral nucleic acid.

The MAIN STAGES OF virus-host cell interaction can be outlined as follows:

Stage 1 — Chemisorption (attachment) of the virus to the surface of the host cell. This occurs only if the cell bears sensitive receptors on its surface that are complementary to the receptors of the given virus, that is, it involves the formation of a specific bond between viral capsid proteins and receptors on the host cell surface. This specific binding determines the host range of the virus. For example, HIV infects only a specific type of human cells (lymphocytes).

Stage 2 — Penetration of the virus into the host cell. Various viruses employ different strategies for cellular entry: viral penetration into the host cell via supercapsid-membrane fusion or via endocytosis (pinocytosis).

Stage 3 — Release of nucleic acids — "uncoating" of the nucleocapsid and activation of the nucleic acid, i.e., deproteinization of the virus (shedding of coats), which is The process of losing the capsid. This is achieved with the aid of viral or host cell enzymes, or it may be the result of simple dissociation. Ultimately, the viral genomic nucleic acid is released.

Stage 4 — Synthesis of viral components. This primarily involves the synthesis of nucleic acids and viral proteins, thereby commandeering the host cell systems and redirecting them toward viral reproduction.

Stage 5 — Virion assembly, or viral morphogenesis, which is the association of replicated copies of viral nucleic acid with the capsid protein. This process begins after a threshold amount of viral nucleic acid and proteins accumulates within the cell. In viruses such as HIV, this modification (maturation) occurs after the virus exits the host cell.

Stage 6 — Exit of viral particles from the cell, and acquisition of the supercapsid by enveloped viruses. This process varies among different viruses. The release of DNA genomic phages occurs through complete cell lysis by the phage enzyme Lysozyme, during which the cell dies due to the rupture of the membrane and Cell wall. Some viruses undergo a lysogenic cycle, where the viral genome is integrated into a specific site of the host cell chromosome via genetic recombination. In this state, the viral genome is called a provirus, or a prophage in the case of bacteriophages. Large Human and Animal viruses exit the cell with a portion of the Cytoplasm by budding through The Plasma Membrane and envelope, simultaneously acquiring a supercapsid.

10.6. Features of the Life Cycle in Various Groups

The genetic material within viral particles and its mode of replication differ significantly among various viruses.

Introduction/6.html">DNA-containing viruses. Genome replication in most DNA-containing viruses occurs in the Cell Nucleus. These viruses enter the cell either by direct fusion with The cell membrane or via endocytosis. The eukaryotic viral genome must cross the nuclear membrane to gain access to DNA- and RNA-synthesizing enzymes, whereas in bacteriophages, simply entering the cell is sufficient.

RNA-containing Viruses. Replication of such viruses typically takes place in the cytoplasm. The replication mechanism is determined by whether the viral genome is single-stranded or double-stranded. A second important factor for single-stranded genomes is their polarity (whether it can directly serve as a template for ribosomal Protein Synthesis).

Viruses utilizing reverse transcription. These viruses contain single-stranded RNA (Retroviridae, Metaviridae, Pseudoviridae) or double-stranded DNA (Caulimoviridae and Hepadnaviridae). RNA viruses capable of reverse transcription (retroviruses, e.g., HIV) use a DNA copy of the genome as an intermediate molecule during replication, whereas DNA-containing viruses (pararetroviruses, e.g., hepatitis B virus) use RNA. In both cases, Reverse Transcriptase or RNA-dependent DNA polymerase is utilized.

An actively multiplying virus does not necessarily kill the host cell. The relationship between a virus and a host cell can develop in various ways. These relationships can be broadly categorized into three types.

Abortive infection — when cells clear the virus while maintaining viability. This occurs in several scenarios:

- upon infection with a defective virus whose replication requires a helper virus, meaning independent replication of these viruses is impossible (so-called viroids);

- upon infection of genetically insensitive cells;

- upon infection of susceptible cells under inappropriate conditions.

Productive infection — replication (production) of viruses is observed:

- if the viral reproduction cycle in the cell concludes with the generation of a large progeny of viruses, typically accompanied by cell death;

- during stable interaction that does not lead to cell death (persistent and latent infections) — the so-called viral transformation of the cell.

Integrative infection — integration (insertion) of the viral nucleic acid into the host cell genome, which subsequently leads to the synchronous replication of both the cellular DNA and the viral nucleic acid. The cell continues to survive and transmits the viral genome along with its own DNA to subsequent generations. This is a special variant of the productive process characterized by stable interaction. Only DNA viruses can integrate into the host DNA genome ("DNA-to-DNA" principle). The only RNA viruses capable of integrating into the host cell genome are retroviruses, which possess a specialized mechanism for this purpose. A key feature of their reproduction is the synthesis of proviral DNA based on genomic RNA using the enzyme reverse transcriptase, followed by the insertion of the DNA into the host genome.

Most frequently, the viral reproduction cycle culminates in a productive infection yielding 100–200 mature virions, usually accompanied by cell death.

10.7. HUMAN IMMUNODEFICIENCY VIRUS

10.7.1. Origin and Spread of HIV

One of the most dangerous human viruses is the human immunodeficiency virus (HIV), which causes AIDS (Acquired Immunodeficiency Syndrome). AIDS, dubbed the "plague of the 20th century," is currently a fatal disease. The first cases of AIDS in humans were identified in 1981, and HIV was discovered by Montagnier in 1984. During the first decade, the spread of the pathogen occurred primarily among populations known as "risk groups" (drug users, sex workers, homosexuals, hemophiliacs). However, since 1992, AIDS has spread into the general population. Unfortunately, exact figures for the number of infected individuals and patients worldwide remain unknown. This is due to imperfections in statistical reporting, as the WHO registers only patients with clear clinical manifestations and does not account for asymptomatic virus carriers. Furthermore, some countries (mainly in Asia and Africa) provide incomplete data because, due to a lack of resources, only a small fraction of the population undergoes testing. Finally, the governments of certain nations withhold publication of such data to avoid harming international tourism, which serves as a major source of national income.

Russia is among the countries with the highest rates of AIDS spread. Specialists project that by the mid-21st century, Russia could lose up to half of its population—primarily young people—to the AIDS epidemic. The Volgograd Region has emerged as one of the leaders in these grim statistics. The average population infection rate in our region is 2.5 times higher than the national average.

AIDS is caused by an infectious pathogen belonging to the retrovirus group. HIV is characterized by extreme Variability—it is 30 to 100 times, and by some estimates up to a million times, higher than that of the influenza virus. This property severely hinders The Development of effective HIV Vaccines. The process of genome transcription in HIV proceeds a thousand times faster than in cellular genes, which explains its astonishing rate of reproduction.

Several hypotheses exist regarding the origin of HIV:

- The impact of adverse environmental factors on a pre-existing virus;

- biological warfare;

- viral mutation resulting from radiation exposure from uranium deposits in the pathogen's putative homeland of Zambia and Zaire.

AIDS has now affected nearly all continents. It is the first human epidemic disease caused by retroviruses. The primary clinical sign of the disease is the destruction of The Immune System. However, accumulating evidence each year demonstrates that the AIDS pathogen also damages The Nervous system.

Currently, the existence of at least three pathogen genotypes can be recognized: HIV-1, HIV-2, and HTLV-4. Of these, HIV-1 is the most widespread, better known simply as HIV. HIV-2 has been detected in the blood of patients primarily in West and Central Africa (with a few cases in Europe). HTLV-4 is likely a variant of HIV-2.

10.7.2. Structure and Life Cycle of HIV

The viral particle comprises genetic material, internal proteins, and envelope proteins enclosed within a lipid membrane. The viral genome consists of single-stranded RNA. Each virion contains two identical RNA molecules, which harbor three primary genes:

- gag — encodes the synthesis of core structural proteins;

- pol — dictates the synthesis of three enzymes (reverse transcriptase, integrase, and protease);

- env — encodes the synthesis of envelope proteins.

Like all other viruses, HIV is capable of replicating only in specific cells possessing complementary receptors. The primary targets of HIV are T-lymphocytes (specifically helper-inducer T-lymphocytes, which mature in the Thymus). Their destruction manifests as a decline in antiviral, antimicrobial, and antitumor Immunity. In addition to these cells, the virus frequently infects macrophages (monocytes, histiocytes) and Central Nervous System cells.

Following attachment to the cell surface, viral penetration occurs, accompanied by virion uncoating and the release of RNA into the cytoplasm. Here, Stage 1 of the HIV life cycle takes place — reverse transcription, namely the synthesis of DNA on an RNA template facilitated by the enzyme reverse transcriptase (revertase). The resulting proviral DNA enters the cell nucleus, where it is integrated into one of the host cell Chromosomes with the aid of the enzyme integrase. Once integrated into the chromosomal DNA, the cell permanently becomes a carrier of HIV genetic information, and should it subsequently divide, the resulting daughter cells will also harbor the HIV genes.

The subsequent (2nd) stage of The life cycle occurs in The Nucleus of the cell: under the action of cellular enzymes, RNA is synthesized using the HIV proviral DNA template (forward transcription). Messenger RNA molecules migrate into the cytoplasm and serve as templates for the synthesis of viral proteins. The resulting components — HIV virion RNA and proteins — assemble to form new viral particles. During this process, structural viral proteins are synthesized as large precursor molecules that migrate to the cell membrane, bind RNA molecules, and bud off from the cell, acquiring a portion of the membrane along with cellular proteins and Lipids.

During the budding process, a portion of the precursor molecule spontaneously cleaves to form the protease enzyme molecule, which chops the remaining precursor into fragments of specific sizes corresponding to mature virion proteins, thereby completing virus formation (maturation). A single cell releases several hundred virions.

10.8. Artificial Viruses and Antiviral Drugs

Many viruses can be generated de novo, that is, from scratch, with the first artificial virus produced in 2002. In this process, it is not the virus itself that is synthesized, but rather its genomic DNA (in the case of DNA viruses) or a complementary DNA copy of its genome (in the case of RNA viruses). For viruses of many families, artificial DNA or RNA (the latter obtained via reverse transcription of synthetic complementary DNA), when introduced into a cell, exhibits infectious properties. In other words, they contain all the information necessary to generate new viruses. This technology is currently employed to develop new-type vaccines. The ability to create artificial viruses has far-reaching consequences, as a virus cannot become extinct as long as its genomic sequence is known and susceptible cells are available.

Antiviral drugs frequently function as nucleoside analogues. They incorporate into the viral genome during replication, halting the viral life cycle because the newly synthesized DNA is inactive. This occurs because these analogues lack hydroxyl groups which, together with phosphorus atoms, bond to form the rigid "backbone" of the DNA molecule. This phenomenon is termed DNA chain termination. Examples of nucleoside analogues include acyclovir, ribavirin, and lamivudine. A broad class of drugs known as protease inhibitors, which inactivate these enzymes, has also been developed.