PHARMACEUTICAL MICROBIOLOGY - V. A. Galynkin - 2015

PART I. BIOLOGY OF MICROORGANISMS

CHAPTER 5. VIRUSES

Viruses differ significantly from other life forms in their size, genome Structure, and reproduction mechanisms.

The size of Viral Particles (virions) ranges from 28 to 250 nm, making them visible only under an Electron microscope. A virion contains only one type of nucleic acid — either DNA or RNA. Viruses are incapable of synthesizing their structural elements (Proteins, Nucleic Acids, etc.) from nutrient media components, nor can they grow on nutrient media. Instead, they rely on the metabolic systems of a host Cell (human, animal, plant, or bacterium) for reproduction, meaning they are obligate parasites.

5.1 Virus Structure

A viral particle (Fig. 35) consists of genetic material (DNA or RNA) enclosed in a protein coat (capsid) [24]. DNA may form circular or linear structures. RNA is represented by single- or double-stranded molecules and, in some viruses, may be segmented. The advantage of a segmented genome is that discrete fragments carry information that a single molecule cannot provide. Depending on their Functions, single-stranded RNAs are divided into two groups:

(1) RNA capable of directly translating Genetic information onto the host cell's Ribosomes, i.e., performing mRNA functions, designated as +RNA (positive-sense single-stranded RNA, positive genome).

(2) Viral RNA that cannot function as mRNA, but serves as a template for +RNA Synthesis, designated as -RNA (negative-sense single-stranded RNA, negative genome).

Retroviruses contain +RNA, which serves as a template for the enzyme Reverse Transcriptase (RNA-dependent DNA polymerase) to synthesize a DNA provirus that integrates into the cellular genome.

Class="center">Fig. 35. Cytology/cytology/92.html">SCHEMATIC STRUCTURE OF an HIV virion.

The capsid protects The Genome from external influences, such as the action of host cell Nucleases. Its surface features systems for recognizing host cell receptors and adsorbing onto The Cell surface. These are typically Glycoproteins whose molecules surround the virion like villi. Bacterial viruses — Bacteriophages — often possess specialized structures that facilitate their penetration into the cell. Some viruses carry Enzymes within the virion that participate in destroying the host Cell wall (phage Lysozyme) or replicating its genome (for example, the HUMAN IMMUNODEFICIENCY VIRUS contains reverse transcriptase). The capsid is built from identical protein subunits called capsomeres. This subunit structure ensures efficient use of genetic material and enables self-assembly of the virion through non-covalent intermolecular interactions, akin to crystallization. Furthermore, this structure facilitates the release of genetic material inside the host cell via the dissociation of non-covalently bound subunits. The shape of the virion is determined by the pattern of capsomere self-assembly and can be cubic, helical, or combine multiple structural components (Fig. 36). On the surface of the protein capsid, many mammalian viruses feature a lipoprotein envelope, which is typically derived from the host cell membrane [12].

Fig. 36. Sizes and Morphology of major pathogens causing human viral infections.

5.2 Interaction of the Virus with the Host Cell

A virus enters a host cell and utilizes its metabolic systems for reproduction and survival [4]. The stages of these processes vary among different viral groups, and The ultimate outcome may be one of the following:

1. Virus Replication and death of the host cell.

2. Virus replication and release from the cell without significant damage to the latter.

3. Persistence of the virus within the cell in a latent state, typically as viral nucleic acid.

4. Integration of viral nucleic acid into the host genome, resulting in a mutation, such as The formation of Cancer Cells.

Bacteriophages (phages) are viruses that infect Bacteria (prokaryotes). Their genetic material is housed in a protein-coated HEAD (Fig. 37). Tail fibers and spikes are designed to recognize receptors on the bacterial cell surface and adsorb to it. This recognition is specific not only to the bacterial species but also to the strain, which serves as the basis for phage typing of bacteria (see below). In some phages, the tail features a sheath covering a central core. Following phage adsorption to the cell, the sheath contracts, driving the core inward. The phage nucleic acid penetrates the cell through this core. The process is facilitated by localized damage to The cell wall caused by phage lysozyme. Such a complex infection apparatus is found only in certain phages of Gram-negative bacteria. The cell walls of Gram-positive bacteria contain receptor sites that facilitate The entry of large molecules and bacteriophages. Pili represent a sensitive target for attack, to which phages can attach. Some phages inject their nucleic acid into the cell, while others enter intact. Based on the type of interaction with the bacterial cell, phages are classified as virulent or temperate. Virulent phages multiply within the cell. Mature phage particles destroy the cell wall from within and escape, causing the cell to perish. Temperate phages are also capable of lysing bacteria; however, in the majority of cells within a population, they persist as a prophage — a phage nucleic acid that, much like Plasmids, can integrate into the chromosome.

Fig. 37. Bacteriophage before (A) and after (B) sheath contraction.

The lytic cycle of a virulent DNA-genomic phage begins with the penetration of its DNA into the cell and the synthesis of early mRNA (molecules synthesized prior to viral Nucleic Acid Replication are termed early). Early proteins are synthesized on the template of the viral mRNA. These proteins shut down host cell Protein Synthesis, degrade bacterial DNA, and initiate the synthesis of viral DNA components. Subsequently, Viral METABOLISM/36.html">DNA replication takes place, followed by the synthesis of late mRNA and proteins required to construct phage particles (head capsomeres and tail elements) and lysozyme. The newly synthesized structural units self-assemble into mature phage particles that escape the cell after its wall is destroyed by lysozyme. Up to 100 phage particles can be synthesized within a single microbial cell just 25 minutes post-infection.

The lytic activity of a virulent phage can be demonstrated experimentally by plating a mixture of the phage suspension and a susceptible culture onto nutrient Agar in a Petri dish. Zones of lysis will appear on the bacterial lawn As a result of cell death caused by the phage. Because each zone of lysis (phage plaque) is initiated by a single phage particle, this method makes it possible to determine their number in the original suspension (the phage titration method).

Lysogenic cultures are those that harbor a temperate phage. To detect The phenomenon of Lysogeny—that is, the release of phage from cells—an indicator culture is required for which the given phage is virulent. The lysogenic culture is mixed with an excess of indicator bacteria and spread as a lawn on a plate. Zones of lysis of the indicator culture are observed under the action of the phage. In the center of such a zone lie the Cells of the lysogenic culture. The MAIN STAGES OF Development of temperate and virulent phages are shown in Fig. 38.

Fig. 38. Schematic of The Development of virulent (A) and temperate (B) bacteriophages.

Integration of the phage NA with the bacterial chromosome ensures its transmission to daughter cells. Lysogenic bacteria are immune to infection by the phages with which they are lysogenized, as well as by closely related phages. This Immunity is associated with The production of a specific repressor that prevents phage multiplication. This same repressor prevents the prophage from transitioning to an active state and blocks the synthesis of phage proteins.

Spontaneously, lysogenic bacteria lyse rarely (10-²-10-⁵ per generation). The frequency of lysis depends on environmental conditions, such as the COMPOSITION OF THE nutrient medium. Mutagens (ultraviolet rays, H₂ O₂, mitomycin C, etc.) can induce the mass development of mature phage particles in the cells of a lysogenic culture, which is associated with a disruption of the repression mechanism. Mutations can also trigger the transition of a temperate phage into a virulent state. Such mutants either prove resistant to the repressor or lose The ability to direct repressor synthesis within the cell.

Usually, lysogeny is a very stable state; however, some cells are capable of losing the phage and, along with it, their resistance to this type of phage.

Lysogeny is an extremely widespread phenomenon: the majority of bacterial strains carry the NA of one or more phages, which determines

the phenotypic traits of the culture (morphological, cultural, antigenic, toxigenic, etc.). This phenomenon is known as phage conversion.

Infectious phages produced by a lysogenic culture are capable of lysogenizing other strains of a given bacterial species (or closely related species). During the transition from the state integrated with the bacterial chromosome to an autonomous state, the phage genome may incorporate adjacent GENES OF THE donor cell's nucleoid into its structure and transfer them to another cell (the recipient). This phenomenon is termed Transduction. Through transduction, many important bacterial traits can be transferred: Antibiotic Resistance, virulence, toxigenicity, etc.

Practical Applications OF phages. Phages are widely used in Introduction/32.html">Genetic Engineering as vectors—Gene carriers in the creation of recombinant DNA molecules. In medicine, phages are prescribed for prophylactic and therapeutic purposes in dysentery, typhoid fever, and other enteric diseases, as well as in purulent-inflammatory processes and dysbiosis. Phages are also widely employed in the Diagnosis of infectious diseases and the identification of microorganisms. The phage titer escalation test indicates the presence of the corresponding microorganism species in environmental objects (Water, food products, etc.). The phagetyping method makes it possible to determine the bacterial biovar and thereby identify the source of infection. Because many substances that induce prophage and its transition to an active state are oncogenic, lysogenic bacterial cultures can be used to detect potential carcinogens.

Replication of mammalian viruses.

Compared to bacteriophages, whose lytic cycle is completed within 30 minutes, mammalian viruses replicate slowly; in tissue culture, the replication cycle takes from 4 to 24 hours and includes the stages of adsorption, penetration into the cell, and the formation of mature viral particles.

Adsorption is driven by two mechanisms: non-specific (electrostatic and Van der Waals forces) and specific, stronger ones, which represent the interaction of viral receptors with corresponding cellular receptors based on THE PRINCIPLE OF biological recognition.

Penetration of mammalian viruses into the cell depends on The Nature of the virus. On The surface of virions of many virus groups, such as Influenza, there are specific spikes containing neuraminidase and hemagglutinin, which participate in the virion's entry into the cell. Poxviruses and Herpesviruses are engulfed by the cell through a process akin to phagocytosis.

Uncoating (the release of viral NA) occurs with the participation of host cell enzymes.

Synthesis of viral NAs and proteins is determined by the Nature of the virus. In DNA-genomic viruses, the process begins with the synthesis of early mRNA involving host cell or virion RNA polymerase. Early proteins, necessary for subsequent DNA replication, are synthesized on the early mRNA template. Replication also proceeds through the action of cellular or viral enzymes. Late mRNAs are synthesized on the template of the replicating DNA, which then direct the synthesis of viral proteins.

In RNA-genomic viruses containing +RNA, the latter is translated on host cell ribosomes. The viral -RNA is used as a template to build a complementary +RNA copy with the help of an RNA-dependent RNA polymerase, which then functions as Messenger RNA.

A necessary step in the retrovirus life cycle is the integration of its genome, in the form of a DNA provirus, into the host chromosome. The Synthesis of the DNA provirus on the viral +RNA template occurs with the participation of RNA-dependent DNA polymerase (reverse transcriptase). The viral DNA integrated into one of the host Chromosomes is transcribed by cellular RNA polymerase. Retroviruses are frequently oncogenic because the incorporation of their DNA into the host cell genome causes cellular transformation. For the same reason, DNA-genomic viruses can also be oncogenic.

Virion self-assembly is a physicochemical process resulting in the formation of a capsid with an embedded NA molecule. In enveloped viruses, virion formation takes place at The cell membrane, whose components become part of the viral envelope (Fig. 39).

Fig. 39. Schematic of the assembly and release of progeny influenza virus populations from infected cells [24].

Release of virions in some viruses is accompanied by cell death, while in others it results only in partial membrane damage.

5.3 Virus Cultivation

Cultivating viruses under laboratory conditions is a necessary step in the diagnosis of many viral diseases; moreover, it is essential for producing vaccine preparations. Because viruses are obligate parasites, they are capable of multiplying only within living cells, such as in tissue culture (human or animal tissue cells growing on a nutrient substrate, typically as a monolayer on the flat surface of a vessel). The presence of viruses can be detected by the cytopathic effect (CPE), i.e., the destruction of the cell monolayer. This method allows for the identification of a virus, for instance, in clinical material, using immune serum. Specific serum neutralizes the virus, and the CPE will not manifest in its presence.

Viruses are also propagated by infecting laboratory animals or avian embryos.

5.4 Effect of Chemical and Physical Factors on Viruses

Heat is the most effective method for destroying viruses. Most human pathogenic viruses are inactivated at 60°C for 30 minutes; however, the hepatitis B virus can withstand this Temperature for up to 4 hours. Viruses tolerate deep freezing and can be stored at temperatures ranging from -40°C to -70°C. Desiccation is lethal to some viruses while having no effect on others. Ultraviolet irradiation inactivates viruses by damaging their NA, a property utilized in the production of viral Vaccines.

Enveloped viruses are inactivated by organic Solvents (such as chloroform and ether); this phenomenon is widely used in Virus Classification. Many chemical disinfectants effective against bacteria (phenols, alcohols, QUATs) show little efficacy against viruses. The most active virucidal agents include chlorine, hypochlorites, iodine, aldehydes, and Ethylene oxide.

5.5 Principles of Antiviral Drug Development

For the prophylaxis and Treatment of viral infections, immunobiologicals and chemotherapeutic agents are employed [25]. Based on their spectrum of activity and clinical significance, medications used to treat viral diseases are categorized into the following groups: etiotropic, immunomodulating, pathogenetic (aimed at combating intoxication, dehydration, organ damage, and allergic reactions, as well as preventing bacterial complications), and symptomatic (alleviating specific symptoms such as headache and cough). Symptomatic and pathogenetic therapies are administered in nearly 100% of cases, whereas etiotropic chemotherapeutic agents have more limited applications. This limitation stems from the difficulty of developing drugs that selectively inhibit pathogen replication without disrupting the vital processes of the host Organism. Most inhibitors of virus-specific processes closely linked to cellular metabolism prove to be toxic.

Nevertheless, certain stages of The life cycle of specific viruses have been identified where inhibition has minimal impact on host cells. Primarily, these include viral adsorption and cell entry, NA deproteinization, and certain processes associated with NA synthesis, translation, and virion assembly.

Antiviral chemotherapeutic agents exhibit a narrow spectrum of activity (restricted to a single species or family), and their number is limited (Table 15).

Table 15. Spectrum of activity of antiviral drugs registered in the Russian Federation

Amantadine and rimantadine, which are tricyclic symmetrical adamantamines, are active against influenza A and rubella viruses. These substances interact

with the viral M2 protein, thereby blocking the fusion of the cellular and viral membranes as well as the entry of the nucleocapsid into the Cytoplasm. In addition, they inhibit primary Transcription and hemagglutinin activation. These drugs exert a prophylactic effect when administered prior to infection and during the Early stages of the disease.

Vidarabine (adenine arabinoside), the least toxic and most effective purine analog, blocks DNA assembly; its intermediate inhibits viral DNA polymerase. It is used in the treatment of herpetic infections.

Cytosine arabinoside is a more toxic analog of vidarabine with a lower selectivity of action, primarily utilized in tumor Chemotherapy.

Halogenated deoxyuridine derivatives—iodoxuridine, trifluridine (trifluorothymidine)—are phosphorylated by viral thymidine kinase and incorporated into viral DNA, leading to the formation of defective viral proteins. They are applied topically for herpetic keratitis.

Nucleoside analogs selectively activated by virus-specific thymidine kinase—acyclovir, famciclovir, ganciclovir—exert a selective effect on virus-infected cells. Their activation requires conversion into high-energy triphosphate, which inhibits viral DNA polymerase. The initial phosphorylation step is induced by virus-specific thymidine kinase. The native form of these drugs is inactive, meaning they do not affect DNA Synthesis in uninfected cells. They are used for herpetic infections and administered orally, intravenously, or as an ophthalmic ointment.

Reverse transcriptase inhibitors are active against retroviruses, including HIV.

Zidovudine (azidothymidine), zalcitabine (dideoxycytidine), didanosine (dideoxyinosine), and stavudine (didehydrodeoxythymidine) act as competitive Enzyme Inhibitors and also terminate elongation during Protein Synthesis on Ribosomes. They exhibit significant toxicity.

Protease inhibitors—non-hydrolyzable synthetic Peptides such as saquinavir, ritonavir, and indinavir—competitively interact with HIV proteases. As a result, uncleaved gag polyprotein precursors accumulate in HIV-infected cells, exerting a cytotoxic effect. They are used in combination with reverse transcriptase inhibitors in HIV-infected patients.

Broad-spectrum nucleoside analogs

Ribavirin is a guanosine analog active against RNA and DNA genomic viruses. It is approved for the treatment of severe respiratory infections in children and other conditions. However, it causes side effects, including immunosuppression.

Foscarnet (trisodium phosphonoformate) inhibits The activity of reverse transcriptase and all herpesvirus DNA polymerases, as well as that of the hepatitis B virus. It is utilized in the treatment of herpetic infections.

N₁-methylisatin-β-thiosemicarbazone (methisazone, marboran) suppresses the synthesis of late mRNAs and late polysomes in poxviruses. It is used in the treatment of smallpox.

5.6 Viral Resistance to Chemotherapeutic Drugs

Like All living organisms, viruses possess the ability to adapt to changing environmental conditions, including exposure to biocides (Fig. 40). This adaptation occurs both through the Selection of resistant strains shaped during prior evolution and through the selective pressures favoring newly emergent strains. Overcoming drug resistance in clinical practice is achievable through the combined use of drugs with distinct Mechanisms of action and by employing agents that target the early stages of viral replication. Currently, instances of resistance are being observed against the following medications.

Fig. 40. Stages of viral production as targets for major antiviral drugs.

Acyclovir. Resistant strains of herpesviruses have been isolated, posing a risk to immunocompromised patients. Resistance is caused by the absence of thymidine kinase or modification of its structure, as well as mutations in the genes encoding DNA polymerase, which renders it resistant to inhibitors.

Ganciclovir. Resistance is associated with altered structures of viral phosphotransferase and DNA polymerase, leading to a reduced level of drug phosphorylation.

Zidovudine and nevirapine. HIV resistance is caused by mutations in genes encoding reverse transcriptase, which results in a decreased affinity of the enzyme for the inhibitor.

Resistant strains to cytarabine and ribavirin have not been identified, which is presumably due to the ability of these agents to target specific stages of cellular metabolism, explaining their broad spectrum of activity.

5.7 Interferons.

Interferons (IFNs, see Chapter 19) possess a universally broad spectrum of antiviral activity because they do not act on virions or their nucleic acids, but rather induce an antiviral state in the cell by stimulating the formation of a protein complex that blocks viral mRNA transcription. IFNs do not penetrate cells; instead, they interact with Membrane Receptors, inducing the production of cAMP, which transmits the signal to the corresponding DNA Operon. Additionally, IFNs activate genes encoding products with direct antiviral effects—protein Kinases, which disrupt protein molecule assembly, and adenylate synthetases, whose product activates an endonuclease that degrades viral mRNAs. Gamma-IFN activates cytotoxic lymphocytes, natural killer cells, monocytes, macrophages, and granulocytes, which contribute to the destruction of infected cells.

Medical IFN preparations are classified into natural and recombinant; their efficacy in various diseases is listed in Table 16.

IFN Inducers — are a structurally diverse group of natural and synthetic compounds capable of stimulating the body to produce its own (endogenous) IFN. Similar to IFNs, they exhibit a universally broad spectrum of antiviral activity (Table 17) as well as immunomodulatory effects, which determine their efficacy in many non-viral diseases.

Table 16. Antiviral activity of IFN preparations

Table 17. Spectrum of antiviral activity of IFN inducers

5.8 Causative Agents of Human Viral Diseases

5.8.1 DNA-containing viruses

Herpesviruses — icosahedral capsid, double-layered outer envelope, glycoprotein spikes, double-stranded linear DNA. They cause acute and latent infections (recurrent herpes, chickenpox, shingles, infectious mononucleosis, keratitis, encephalitis, etc.); they possess oncogenic potential.

Papovaviruses — naked icosahedral capsid, double-stranded cyclic DNA. They cause Papillomas and polyomas (warts, condylomas, local epithelial hyperplasias, laryngeal and cervical carcinoma).

Adenoviruses — naked icosahedral capsid, double-stranded linear DNA. They cause pharyngoconjunctivitis, epidemic keratoconjunctivitis, and gastroenteritis.

Poxviruses — brick-shaped virion, double-stranded DNA. They cause smallpox, cowpox, monkeypox, etc.

Parvoviruses — naked icosahedral capsid, single-stranded DNA. They cause aplastic crisis in children.

Hepadnaviruses — enveloped icosahedral capsid, incomplete (with a single-strand gap) circular double-stranded DNA; the virion contains a primer protein and DNA polymerase. Replication requires the synthesis of virus-induced reverse transcriptase, since viral DNA is formed on an RNA template; during the process, viral DNA integrates into the host chromosome. They cause hepatitis B.

5.8.2 RNA-containing Viruses

Orthomyxoviruses — spherical virion containing single-stranded segmented RNA. They cause influenza. In influenza A and B viruses, the genome contains 13,588 NUCLEOTIDES and consists of 8 segments, each encoding its own protein. The main virion protein, M-protein, is localized on the inner surface of The Lipid Bilayer, which is associated with surface glycoproteins—hemagglutinin and neuraminidase—forming spikes. These glycoproteins possess antigenic Specificity that can vary frequently during a single epidemic outbreak, complicating influenza vaccine prophylaxis.

Paramyxoviruses — spherical virion; the lipoprotein envelope contains an M-protein forming its inner layer, as well as surface hemagglutinin, neuraminidase, and glycoprotein F, which exhibits hemolytic and cytotoxic activity and is responsible for Cell Fusion; the genome

represented by a linear, unsegmented RNA molecule associated with a major protein. The virion contains an RNA-dependent RNA polymerase. They cause parainfluenza, measles, mumps, and subacute sclerosing panencephalitis.

Picornaviruses — naked icosahedral virions with an unsegmented +RNA molecule. They cause asymptomatic infections, meningitis, paralysis (poliomyelitis), myopericarditis, and acute respiratory infections.

Rhabdoviruses — bullet-shaped virions with helical Symmetry, featuring a double-layered lipid envelope containing external glycoprotein structures; the nucleocapsid contains single-stranded RNA, core proteins, and a transcriptase. They cause rabies, vesicular stomatitis, etc.

Togaviruses — icosahedral capsid, lipid envelope with glycoprotein spikes containing hemagglutinin, and a single-stranded +RNA molecule. They cause encephalitis, yellow fever, Dengue fever, Karelian fever, hepatitis C, and rubella.

Bunyaviruses — spherical virions with three nucleocapsids, each containing three separate linear -RNA segments and an RNA-dependent RNA polymerase. The lipoprotein envelope features glycoprotein spikes containing hemagglutinin. They cause fevers and encephalitis.

Arenaviruses — rounded or polymorphic virions, single-stranded segmented -RNA molecule; virions contain several types of both viral and host-derived ribonucleoproteins functioning as ribosomes. They cause severe hemorrhagic fevers, influenza-like illnesses, and meningitis.

Filoviruses — rod-shaped, branching virions with a helical nucleocapsid and -RNA. They cause severe hemorrhagic fevers.

Coronaviruses — rounded or oval virions, a glycolipoprotein envelope with characteristic projections (a crown), helical nucleocapsid, and unsegmented +RNA. They cause acute gastroenteritis and respiratory infections.

Caliciviruses — naked icosahedral capsid, unsegmented +RNA. They cause gastroenteritis and hepatitis E.

Reoviruses — naked virions, quasi-spherical capsids with icosahedral symmetry; the genome is fragmented, consisting of a double-stranded RNA composed of 10–11 segments. Orthoreoviruses cause fevers (Colorado tick fever, Kemerovo fever, etc.); rotaviruses cause acute enteritis in children.

Retroviruses — spherical virions enclosed in a lipid envelope with glycoprotein spikes; the genome consists of +RNA composed of two identical subunits. A characteristic feature is the presence of an RNA-dependent DNA polymerase within the virion. They cause malignancies; HTLV-1 causes T-cell lymphomas and myelopathies; HIV-1 and HIV-2 cause Acquired Immunodeficiency Syndrome (AIDS).

5.8.3 Unclassified Viruses

Hepatitis D virus — a defective virus (incapable of independent replication, requiring helper viruses) isolated only from patients infected with hepatitis B virus (HBV); its single-stranded RNA genome shares no Homology with HBV DNA, but its envelope includes HBV Antigens.

Astroviruses — spherical virions with a single-stranded RNA genome. They cause diarrheal infections in children.

5.9 Plant-Pathogenic Viruses

Viruses are the smallest (submicroscopic) infectious agents of plants, animals, and humans, lacking cellular structure and capable of replicating only within living host plant cells [20]. Approximately 600 phytopathogenic viruses have been registered; the exact number is difficult to determine because some viruses comprise numerous strains, sometimes described as independent species.

All phytopathogenic viruses are grouped into 20 categories.

Currently, viruses are believed to be the simplest forms of life, lacking a cellular structure and becoming active upon entering the cells of susceptible organisms. Plant viruses are characterized by the following features:

1. They replicate exclusively within the host or vector organism and do not grow on artificial nutrient media. They possess a unique mechanism of replication.

2. They lack Cellular Organization: they consist of RNA (ribonucleic acid, single- or double-stranded) or DNA (deoxyribonucleic acid) surrounded usually (though not always) by a protein coat.

3. The viral genome is represented solely by nucleic acid, which reproduces utilizing the host's enzymatic system.

4. The nucleic acid is responsible for infectivity, while the protein primarily serves to protect the RNA.

Viruses exhibit rod-shaped (TMV), filamentous (Potato virus X, Citrus tristeza), spherical (Tobacco necrosis), and bacilliform (Wheat striate mosaic) morphologies. Viral sizes range from 25 nanometers (nm) for tobacco necrosis virus up to 2500 nm for citrus tristeza virus (1 nm equals 10^-9 m = 0.001 µm). Based on their effect on the infected organism, viruses are divided into two major groups: mosaic-type viruses (mosaic) and yellows-type viruses (yellows).

Infection with mosaic viruses alters leaf coloration, resulting in alternating light-green, dark-green, and yellow areas on the leaves, as well as the appearance of necrotic spots, streaks, rings, etc.

Sources of infection for mosaic viruses can include dry plant debris, seeds, tubers, weeds, soil, etc.

The pathogenicity of viral diseases manifests primarily in reduced crop yields and deteriorated product quality. Viruses cause particularly severe damage when growing seed and planting material. Viral infections negatively affect the nutritional and fodder value of products, as well as their suitability for industrial Processing. Viruses induce sterility and incompatibility in plants, which adversely affects breeding programs. Ornamental crops lose their aesthetic appeal, causing significant economic losses. Viral action leads to a loss of varietal purity, frost hardiness, and winter hardiness, while also reducing seed germination. On average, losses from viral diseases account for approximately 20% of the total economic Damage caused by all groups of crop pathogens and pests.

5.9.1 Structure and Replication of Viruses.

Traditional microbiological Research Methods are not applicable to viruses; therefore, for a long time, nothing was known about their structure, methods of reproduction, or persistence. It was not until 1935 that the American virologist W. Stanley isolated the protein component from tobacco leaves infected with tobacco mosaic virus (TMV), obtaining a pure crystalline viral protein. In 1937, the British researchers F. Bawden and N. Pirie established that, In addition to protein, the virus contains nucleic acid. Tobacco mosaic virus consists of protein (95%) and nucleic acid (5%).

When discussing the Size and Structure of viruses, researchers refer to virions, or viral particles. In most cases, virion sizes range from 100 to 200 nm.

The shape of a virion is determined by the spatial orientation of the nucleic acid and The structure of the protein coat. The protein plays a protective role and also facilitates the penetration of the virus into the host plant Tissues. The nucleic acid serves as the bearer of infectivity and hereditary traits.

Most plant viruses contain single-stranded linear RNA; viruses with double-stranded RNA molecules twisted into a helix are less common. Only a few plant viruses (such as cauliflower mosaic virus) contain DNA.

The Mechanism of viral reproduction differs from the reproduction methods of other microorganisms. Phytopathogenic viruses enter a plant cell, for instance, through punctures made by the mouthparts of vector insects or via minor wounds (without severe cell damage) during mechanical transmission. Inside the cells of an infected plant, the virus replicates through the synthesis of individual nucleic acid and protein molecules followed by their assembly into virions. Upon entering The plant cell, the viral nucleic acid sheds its protein coat and, acting as a template, begins to direct the synthesis of plant cell enzymes in the direction required by the virus. It accumulates through the replication of nucleic acid chains using nucleotides already present in the cell and subsequently synthesized by it. Viral protein is synthesized on the ribosomes of the host cell. Subsequently, the nucleic acid and structural protein combine to form virions.

Virions frequently aggregate with each other, forming viral inclusions—crystals of various shapes (Ivanovskiy crystals)—or, if virions combine with cytoplasmic densities, inclusions resembling amorphous bodies are formed.

Viral infections involve an obligate type of parasitism, specifically in its absolute form. The pathogen integrates into the plant's genetic apparatus, altering it to suit the synthesis of its own enzymatic energy system and, subsequently, the corresponding viral structures.

5.9.2 Symptoms of Plant Viral Diseases

Based on their manifestation, symptoms of viral diseases can be divided into 5 main types: 1. Growth retardation; 2. Changes in leaf coloration resulting in a mosaic pattern; 3. Organ deformation; 4. Local necrosis; 5. Impairment of plant reproductive functions.

A single viral disease usually manifests multiple types of symptoms on a plant. The symptoms of viral diseases may change as the pathological process progresses.

5.9.3 Modes of Transmission of Phytopathogenic Viruses

Viruses that cause plant diseases can be spread in various ways. Many viruses are disseminated by vectors that feed or parasitize on the plant. These are primarily insects, mites, nematodes, Fungi, and parasitic flowering plants (dodder). Only a relatively small number of phytopathogenic viruses are transmitted by insects with chewing mouthparts; such transmission has low specificity and is significant only for viruses capable of surviving in the sap of a diseased plant.

Depending on the characteristics of insect transmission, viruses are classified as persistent or non-persistent. Persistent viruses retain their infectivity in the vector's body for several days and sometimes for the entire lifespan of the vector. Non-persistent viruses can be transmitted by vectors within a limited timeframe, often no more than an hour.

Viruses can be transmitted through contact and mechanical means, i.e., via mutually damaging contact between parts of healthy and diseased plants. This occurs when aboveground or underground plant parts Touch. Some viruses (about 20%) can be transmitted via seeds. Certain viruses affecting fruit and berry crops can be transmitted through pollen. In vegetatively propagated crops (such as potatoes, strawberries, and tulips), viruses are spread mainly through planting material. Viral diseases are also transmitted through various types of grafting (transplantation). All known phytopathogenic viruses can be transmitted using this method. A few isolated viruses (tobacco mosaic virus, tobacco necrosis virus) can be spread via plant debris, soil, or hydroponic solutions. The spread of viruses through dodder stems is of minor significance (specifically for viruses affecting forage legumes).

5.9.4 Plant Protection Against Viral Diseases

1. Plant quarantine is a system of government measures aimed at preventing the introduction of quarantine plant disease pathogens from other countries (external quarantine), and in the event of their entry, localizing their outbreaks (internal quarantine).

2. The use and production of virus-free seed and planting material.

3. The breeding method, which focuses on developing new varieties resistant to both the virus and its vector.

4. Organizational and economic measures, including the disinfection of tools in a solution of formalin, potassium permanganate, or alcohol, as well as heat treatment. Regular visual inspection of plants.

5.9.5 Viroids as Plant Pathogens

Viroids as a new class of pathogens were discovered by T. Diener in the 1970s. This group of phytopathogens comprises virus-like infectious agents that do not form the nucleoprotein particles characteristic of viruses. They consist solely of low-molecular-weight single-stranded RNA, which serves as the carrier of infectivity and utilizes the biosynthetic System of the host plant cell for its replication. They are characterized by having exclusively covalently closed circular RNA with an extremely low molecular weight (2.5 × 10⁴ – 15 × 10⁴). A protein coat is absent.

The most characteristic symptoms of viroid diseases include growth suppression, reduction in the size of the plant and its individual Organs (leaves, flowers, fruits), fading of color intensity, and leaf chlorosis.

Viroids are spread through planting material and seeds, and are transmitted from plant to plant mechanically. For example, the citrus exocortis viroid spreads rapidly through grafting. Viroids are characterized by high infectivity, thermostability, and resistance to various chemical compounds.

The main symptoms of viroid diseases include stunted plant growth or damage to specific plant organs, discoloration (chlorosis, anthocyanosis), and various structural deformations. Viroids are highly infectious and exhibit strong resistance to chemical and thermal treatments. They are typically spread through propagation material, seeds, and mechanical contact. Principal diagnostic methods for viroids include visual inspection, indicator plant assays, Electron Microscopy, gel Electrophoresis, and DNA Hybridization probes. Protecting plants from viroid diseases is largely similar to managing viral pathogens.

Due to the exceptionally close mutual organization between fungi and plants, their interactions are remarkably diverse. Estimates suggest there are between 120,000 and 250,000 fungal species, of which over 8,000 are phytopathogenic, whereas only about 200 phytopathogenic species are known among bacteria.

Agrobacteria contribute to the development of various plant tumors. Tumor formation is triggered by an oncogenic plasmid transferred by agrobacteria into plant cells, which induces crown gall tumors. Once the tumor develops, agrobacteria are typically no longer present in the tissues. Phytopathogenic bacteria spread via infected seeds, crop residues, soil, water, air, and vectors such as insects, Mollusks, and nematodes. Bacteria invade plants through Stomata, nectaries, and other natural openings, as well as through minor physical injuries.

5.10 Prions

Prions as Infectious Agents

Prions are glycoproteins capable of inducing a conformational transition in normal cellular proteins, converting them into an infectious conformer isoform (Fig. 41) [26]. The source of the normal protein is the host cell itself, where the constitutive expression of the PRNP gene maintains a pool of PrPc—a normal component of cell membranes. Contact with the infectious prion PrPsc (sc standing for scrapie) triggers the refolding of the normal protein into the PrPsc conformation. This conversion takes place during the post-translational Processing of the pre-existing normal cellular protein.

Fig. 41. Schematic representation of prion infection, dissemination within the organism, and Stages of the infectious process in the Central Nervous system (B — B-lymphocytes; FDC — follicular dendritic cells [7].

Normal PrPc protein localizes to the cytoplasmic membrane and participates in cellular signaling pathways, particularly in Neurons, and is believed to play a role in the biogenesis and development of The Nervous System. Its conformational modification disrupts these physiological processes. Furthermore, conformers induce apoptosis in infected cells and generate neurotoxic Polypeptides that are thought to form pores in neurons, bind nucleic acids, and block mitochondrial replication, leading to cellular degeneration. This latter mechanism underlies many neurological disorders.

Prion-induced diseases are characterized by central nervous system degeneration and include Creutzfeldt-Jakob disease, Gerstmann-

Sträussler-Scheinker syndrome, fatal familial insomnia, kuru, sheep and goat scrapie, and bovine spongiform encephalopathy, among others. The source of infection is the tissue of an infected organism. Transmission to humans can occur via the alimentary route, as well as through medicinal products derived from infected animal tissues or improperly sterilized medical instruments. In cattle and sheep, infectious agents are transmitted through feed containing tissues of dead animals.

Prion diseases are characterized by an unusually long incubation period. The Development of the infection is closely tied to genome functions and cellular processes that drive the accumulation of the PrPsc protein and the gradual, progressive onset of symptoms, which may span months or even years.

Prion diseases typically feature a virtually complete absence of an Immune Response due to the high evolutionary conservation of the protein's Primary Structure; these infections do not respond to Immunomodulatory therapy, although research is underway to develop immunotherapeutic agents targeting specific Stages of Protein conformation conversion.

Prions in Pharmaceutical Practice

Prions are individual proteins with a molecular weight ranging from 20 to 30 kDa and a polypeptide chain length of approximately 254 amino acid residues. They can pass through filters with a pore diameter of 25–50 nm.

Prions remain stable at 90 °C for 30 minutes and are inactivated only by autoclaving at 135 °C for 30 minutes. Nevertheless, cases of transmission have been documented following The Use of autoclaved medical instruments (dental, otolaryngological, and neurosurgical). Prions exhibit strong resistance to chemical agents (glutaraldehyde, formaldehyde, β-propiolactone, ethanol, toluene, xylene), nucleases, UV radiation, and ionizing radiation. They show somewhat lower resistance to acetone, sodium hydroxide, ionic detergents such as sodium dodecyl sulfate, phenol, chloroform, strong oxidizing agents, ethylene oxide, and proteases.

Prions have a relatively narrow host range, yet they possess the ability to adapt to new hosts, thereby overcoming interspecies barriers.

The risk of prion disease transmission poses a serious challenge for pharmaceutical operations, particularly in monitoring animal-derived raw material suppliers (preventing the sourcing of Materials from regions reporting cases of bovine spongiform encephalopathy) and selecting appropriate sterilization protocols. For thermostable medical equipment contaminated with prion-containing material, the WHO recommends immersion in a sodium hydroxide solution (1 N) or sodium hypochlorite solution (20,000 ppm available chlorine) for 1 hour, followed by autoclaving, cleaning, and standard sterilization.

Methods for verifying the complete inactivation of prions are labor-intensive, time-consuming, and expensive, typically involving the inoculation of animals with biocide-treated infected tissue combined with mathematical modeling to calculate the biocide concentration and exposure time required for prion inactivation. Consequently, strict adherence to validated and standardized treatment protocols is essential in practice to guarantee sterilization efficacy.

Conclusion

Viruses differ from other life forms by the following characteristics:

— sizes ranging from 28 to 250 nm;

— presence of a single type of nucleic acid—either DNA or RNA;

— obligatory parasitism.

Viruses parasitize bacterial cells (bacteriophages), plants, animals, and humans, causing various diseases. Retroviruses contain single-stranded RNA (+RNA), which serves as a template for the enzyme reverse transcriptase to synthesize a DNA provirus that integrates into the host cell genome and induces cellular transformation.

The outer shell of a virus, the capsid, is composed of protein subunits known as capsomeres.

Virulent bacteriophages cause cell lysis, whereas temperate phages persist within The Cell as a prophage—phage nucleic acid integrated into the bacterial cell's genome.

Viruses are cultivated using tissue cultures or by inoculating animals or avian embryos.

The development of antiviral drugs focuses on inhibiting stages of the viral life cycle that do not affect the host cell: adsorption, viral entry into the cell, and the functioning of viral nucleic acids.

Interferons are proteins exhibiting a universally broad spectrum of antiviral activity.

Prions are infectious glycoproteins that cause central nervous system disorders and are transmitted via the alimentary route or through infected materials and instruments. Prions are exceptionally resistant to sterilizing agents.