Introduction in virology. Ultrastructure and chemical nature of viruses. Reproduction of viruses. (Prepared by Inzhevatkina S.M., Department of Microbiology and Virology of Russian National Research Medical University NI Pirogov) History of virology Famous great epidemics of measles and smallpox are documented in Roman Empire b.c. Clinical manifestations of rabies had been described from ancient time. Herman Cortez and his spanish warriors conquested the Aztec Empire due smallpox epidemy to which the native population of Mexico had lacked any immunity. English physician Jenner Edward conducted an experiment by scraping pus from a cowpox sore on the arm of a milkmaid and inserting it into two cuts on the arm of a young boy in 1796. Then he did exactly the same with pus from a smallpox sore. The boy caught cowpox, but did not catch smallpox. He had the courage even to vaccinate his own child. In 1798, he published his method of vaccination. His method was adopted for the British Army. After conducting this experiment on 23 different cases he concluded that those who had suffered cowpox were indeed immune to smallpox. Jenner called this new method 'vaccination' which mean 'from a cow' as a way of distinguishing it from the process of 'inoculation'. But now it’s known that he used the knowledge of the wife of English ambassador to Turkey Lady Worthey Montagu who observed that Turkish woman inoculated their children against smallpox the same way early in XVIII century. She tried to educate the English public, but without great success. She hadn’t been a physician! History of virology

Counting the history of virology should be conducted on February 12, 1892, when D.Y.Yvanovsky told the Academy of Sciences of the opening of the pathogen "tobacco mosaic." Interest in viruses growing with the opening of oncogenic viruses (1911) and bacteriophages (1915), when foundations culturing viruses. General properties of viruses Viruses are obligate intracellular parasites that are totally dependent on host cells. Viruses are simple, acellular, ultramicroscopic particles containing either RNA or DNA, which reproduce inside living cells, pass through filters that retain bacteria and are covered by a protein coat. The general characteristics of viruses are: 1. Do not possess cellular organization. 2. Contain one type of nucleic acid, either RNA or DNA but never both. 3. Lack enzymes necessary for protein and nucleic acid synthesis (exception for ss “-” RNA viruses: they possess RNA-dependent RNA-polymerase as a part of nucleocapsid) and so dependent upon synthetic machinery of host cells 4. They have no means to produce energy and contain a few enzymes at most. 5. They multiply by complex process and not by binary fission. 6. They are unaffected by antibiotics. 7. They are sensitive to interferon. Structure and chemical nature of viruses Viruses can exist in two phases: extracellular and intracellular. The basic infectious particle of virus (extracellular phase) is known as virion and contains a core of either DNA or RNA but never both enclosed in a coat of protein (capsid). Some viruses have additional layers that can be very complex and contain lipids, carbohydrates and additional proteins. Viruses can’t reproduce independently of living cells. Virions lack own enzymes or possess very few enzymes. Viruses change host cell metabolism to synthesize virion components, assembly and release of virus particles during intracellular phase. Structure of viruses The central core of nucleic acid is covered by protein coat called capsid . The capsid itself is composed of number of subunits called capsomers . The capsomers may be arranged as under: 1. Around coiled nucleic acid, which is known as helical arrangement (rabies virus, filoviruses, ortho- and paramyxoviruses, etc.). 2. As cubes around spheroidal nucleic acid known as icosahedral arrangement (all naked viruses: picorno-, adeno-, parvoviruses, etc.). 3. Complex arrangement means that such viruses possess a capsid that is neither purely helical nor purely icosahedral, and that may possess extra structures such as protein tails or a complex outer wall (several bacteriophages and smallpox virus). Structure of viruses Together, the nucleic acid and the capsid are referred as the nucleocapsid. Virions may be enveloped or nonenveloped (naked) . The envelope derived from host cell membrane when virus is released by budding. Envelope is lipoprotein in nature. Protein subunits may be seen as projecting spikes on the surface of the envelope. There are called peplomers . A virus may have more than one type of peplomer (influenza virus has two peplomers). The size of virions varies considerably, from 20 to 300 nm, in different families. Some filoviruses have a total length of up to 1400 nm; their diameters are only about 80 nm. Structure of viruses Kingdom Vira Sizes of representative viruses, bacteria, mycoplasmas, yeasts, protozoa and eukaryotic cells ( in µm) Principles of Virus Structure

• Virion is the complete extracellular virus particle General Structure of an Nonenveloped (Naked) Viruses

Nucleic acid + capsid → nucleocapsid

Structure of a nonenveloped virus Nucleocapsid of a helical virus showing icosahedral symmetry Symmetry of capsid

1. Helical Repeated units of a single polypeptide (protomer) Protein subunits are in association with the viral nucleic acid Self-assemble into a helical cylinder (rod-shaped, filamentous) 2. Icosahedral (cubic) More complex, with several different polypeptides Polypeptides grouped into identical capsomers and form an icosahedron (20 faces, 12 vertices). Nucleic acid genome is located within the empty space. 3. Complex capsid that is neither purely helical nor purely icosahedral, and that may possess extra structures such as protein tails or a complex outer wall (poxviruses, bacteriophages) Virus Structure

Capsid is a protein shell Functions:  Protection of the viral genome  Site of receptors necessary for naked viruses to initiate infection  Induces antibody production  Site of antigenic determinants, important in some serologic tests. Virus Structure Genome: types of viral nucleic acid RNA or DNA (either may be single-stranded (ss), mixed strandedness (partially double stranded), or double-stranded (ds)):  Positive-sense RNA genome  Negative-sense RNA genome  Non-segmented RNA genome (linear)  Segmented RNA genome  Linear configuration DNA  Circular configuration DNA General Structure of an Enveloped Viruses [Nucleic acid + capsid] + envelope with glycoproteins (peplomers) Virus Structure

Envelope  Lipid-containing membrane surrounding the nucleocapsid  Derived from host cell membranes  Contain virus-specific glycoproteins (peplomers) Resistance of viruses

Naked viruses are resistant and survive well in the outside world. They may also be bile resistant , thus, causing infection in gastrointestinal tract. Enveloped viruses are more susceptible to environmental factors such as drying, gastric acidity and bile. Virus Structure Diagrammatic view of influenza A virus Enveloped virus Structure of influenza virus Enveloped virus Structure of HIV particle Enveloped virus. Rabies virus (helical symmetry) Enveloped filamentous virus Smallpox virus (complex symmetry) Transmission electron microscopy

• A - Tobacco mosaic virus • B - Bacteriophage фX174 • C – Bacteriophage T4Viruses Electron microscopy of polioviruses

Electron microscopy of rotaviruses Electron microscopy of herpesviruses Transmission electron micrograph showing mature forms of HIV Transmission electron micrograph of different viruses

• A - Poliovirus • B - Simian virus • C - Vesicular stomatitis virus • D - Influenza virus • E - Adenovirus Classification of viruses The International Committee on Taxonomy of Viruses (ICTV) developed the current classification system and wrote guidelines that put a greater weight on certain virus properties to maintain family uniformity. All viruses are classified into families on the basis of: 1. Presence or absence of envelope. 2. Characteristics of nucleic acid (type of nucleic acid (DNA or RNA), nucleic acid strandedness, the sense (positive or negative) RNA. 3. Strategy of genome replication. Classification of viruses Further classification of viruses is based on the symmetry of nucleocapsid, size and shape of envelope and number of capsomers; immunological, cytopathological, pathogenic, and epidemiological (natural host, mode of transmission, tissue tropisms) features; physicochemical properties including molecular mass, pH, thermal stability, susceptibility to chemicals and physical extremes and to ether and detergents; etc. Classification of viruses

• The general taxonomic structure is as follows: • Phylum Vira (divided into 2 subphyla) Subphylum Deoxyvira (DNA viruses) Subphylum Ribovira (RNA viruses) • Class ( -ica, -ala) • Order (-virales) • Family (-viridae) • Subfamily (-virinae) • Genus ( -virus ) • Species ( -virus ) Examples of medically important families of viruses Nonenveloped viruses are: 1. single-stranded RNA viruses - Picornaviridae, Caliciviridae 2. single-stranded DNA viruses - Parvoviridae 3. double-stranded RNA viruses - Reoviridae 4. double-stranded DNA viruses - Polyomaviridae, Papillomaviridae, Adenoviridae Enveloped viruses are: 1. single-stranded RNA viruses – “+“Togaviridae, Flaviviridae, Coronaviridae, “-” Paramyxoviridae, Orthomyxoviridae, Rhabdoviridae, Bunyaviridae, Arenaviridae, Filoviridae 2. double-stranded RNA viruses (two identical molecules ss”+”) – Retroviridae 3. double-stranded DNA viruses - Poxviridae, Herpesviridae 4. mixed strandedness (DNA-viruses) - Hepadnaviridae Classification of Viruses

+ -or + - - +

- - - - + +

+ + + - Viruses

ATYPICAL AGENTS (Agents, Simpler Than Viruses) Defective viruses are viruses, which are genetically deficient and so incapable of producing infectious daughter virion (delta hepatitis virus). Viroids are infective agents with protein free, low molecular weight RNA resistant to heat and organic solvents but sensitive to nucleases. Prions are small particles of molecular weight of 50,000 Da and 4 to 6 nm in diameter. Term “ Prion” is derived from the words protein and infection. Prions lack detectable nucleic acid resistance to heat (90°C for 3 minutes), ultraviolet rays and nucleases. They are sensitive to proteases. Examples are causative agents of scrapie and Creutzfeldt-Jacob disease. DDefective Viruses Defective virus is a virus particle that is functionally deficient in some aspect of replication. 1. Defective interfering particles (DIP). They are deletion mutants which lacks a portion of its genome but contain normal capsid proteins, they require infections homologous virus as helper for replication. 2. Satellite (virusoids for RNA agents) This category of defective virus requires an unrelated replication heterologous- competent virus as helper. - adenoassociated - delta agent (hepatitis D virus, co-infection hepatitis B virus). It uses the hepatitis B virus as its helper virus. 3. Pseudovirions Defective particle which contain only host cell DNA and capsid. Viroids • Small infection agents that cause diseases of plants, including potato spindle-tuber disease, exocortis disease of citrus trees, and chrysanthemum stunt disease. • They are nucleic acid molecules without a protein coat. • Plant viroids are single-stranded, covalently closed, circular RNA molecules, consisting of about 250 to 370 nucleotides. • Viroid RNA does not encode any gene products, so they cannot replicate themselves. • Viroid is replicated by one of the host cell`s DNA-dependent RNA polymerases. The host polymerase synthesizes a complementary RNA molecule, a negative-strand RNA. This then serves as template for the same host polymerase, and new viroid RNAs are synthesized. Both steps may occur by a rolling-circle-like mechanism. Prions The prion protein (PrP) is encoded by the host’s chromosomal DNA. The normal cellular form of the protein (PrP c) is expressed on the surface of neurons both infected and uninfected brains. An abnormal isoform of this protein (PrP sc )- is a post- translationally modified normal host form, which is insoluble in detergents and resistant to proteases and nucleases. Proteins are extremely resistant to inactivation by UV light and ionizing radiation and unusually resistant to alcohol, formalin, boiling. When a prion enters a healthy organism, it induces existing, properly folded proteins to convert into the disease- associated, prion form; the prion acts as a template to guide the misfolding of more proteins into prion form. These newly formed prions can then go on to convert more proteins themselves; this triggers a chain reaction that produces large amounts of the prion form (chain reaction). Prions

Prions are differ from both viruses and viroids in that they consist of protein and not nucleic acid Prions

Normal prion Pathogenic prion

Chain reaction Prions

• Prion’s diseases is a group of human and animal fatal neurodegenerative diseases. • Histologically, all prions diseases, are characterized by spongiform vacuolization in the central nervous system, the accumulation of a unique protein in cytoplasmic vesicles of neurons, the formation of extracellular amyloid plaques, composed of PrP sc . • The incubation period of ″slow″ prion diseases is long (many years). • Chronic progressive pathology without remission or recovery (lethal outcome). Prions Animal diseases: 1. Scrapie of sheep 2. Bovine spongiform encephalopathy of cattle ( ″mad cow disease ″). 3. Transmissible mink encephalopathy. • Human diseases: 1. Creutzfeldt- Jakob disease (sporadic, familial, acquired- iatrogenic) 2. Gerstmann-Straussler-Scheinker syndrom 3. Fatal familial insomnia. 4. Kuru (related to ritualistic cannibalism in Papua New Guinea). Prions

A unique characteristic of the prion diseases is that there is - no immune response - no inflammatory reaction in the diseased tissue - no interferon production or interference by conventional viruses - no alteration in pathogenesis by immunosuppression or immunopotentiation Mechanism of Reproduction The fundamental difference between viruses and all other infectious agents is in their mechanism of reproduction. Unlike cellular forms of life, viruses do not simply divide. Virus replication is carried out by the host cell machinery , which synthesizes multiple copies of the viral genome and viral proteins. Those viral components assemble spontaneously within the host cell to form progeny virus particles. Viruses have no means to produce energy and contain a few enzymes at most. Thus, totally dependent on host cells, viruses are obligate intracellular genetic parasites. Steps in the Replication Cycles of Viruses

1. Adsorption Attachment of a virion to a specific host cell receptor

Steps in the Replication Cycles of Viruses

2. Penetration (taken up inside the cell)

By receptor-mediated By membrane fusion (direct endocytosis penetration) Steps in the Replication Cycles of Viruses

3. Uncoating Separation of the viral genome from the outer structural components of the virion such that it can function. 4. Macromolecular synthesis of viral components  Genome replication  Transcription  Translation 5. Assembly and maturation of the virus 6. Release of virus  Lysis (disintegration of the cell)  Budding – process by which enveloped viruses obtain their envelope. Steps in the Replication Cycles of Viruses (budding of enveloped virus) Steps in the Replication Cycles of Viruses (budding) Mechanism of DNA Virus Genome Replication (usually occurs in host nucleus; only one exception is Poxviridae )) Replication of herpesviruses Mechanism of RNA Virus Genome Replication Type I: viruses with an ssRNA genome of (+) polarity that replicates via a complementary (-) strand intermediate Replication of rubella virus Mechanism of RNA Virus Genome Replication Type II: viruses with a ssRNA genome of (-) polarity that replicates via a complementary (+) strand intermediate Replication of Influenza A virus Mechanism of RNA Virus Genome Replication Type III: Viruses with a dsRNA genome Replication of Rotaviruses (Reoviridae) Mechanism of RNA Virus Genome Replication

Type IV: Viruses with a genome of ss-RNA of (+) polarity that is replicated via a DNA intermediate Mechanism of RNA virus Genome Replication For Retroviruses (HIV) Mechanism of Virus Genome Replication for Oncogenic Viruses Oncogenic viruses exhibit a unique replicative strategy. Each group of viruses ( Adenoviridae, Herpesviridae, Hepadnaviridae, Papillomaviridae, Retroviridae, Flaviviridae ) has its own unique mechanism of oncogenesis. Most of oncogenic viruses are DNA- containing, exceptions are Retroviridae and Flaviviridae. Mechanism of RNA Virus Genome Replication for Oncogenic Retroviruses

Example of replication for oncogenic Retroviridae . Their single stranded RNA genome is converted into an RNA:DNA hybrid by the viral reverse transcriptase (RNA directed DNA polymerase) enzyme. Double stranded DNA is then synthesized from the RNA: DNA hybrid. The double stranded DNA form of the virus (provirus) is integrated into the host cell chromosome. The integration may lead to the transformation of the cell and development of neoplasia. Pathways of mRNA synthesis Cultivation of viruses

All viruses are obligate intracellular parasites and cannot grow on inanimate culture medium. Three methods are used for their cultivation: 1.Animal inoculation. 2. Chick embryo. 3. Cell culture. Inoculation in animals Inoculation into Chick Embryo Inoculation into Chick Embryo Classification of Cell Cultures • 1) Primary cell cultures . These are normal cells freshly taken from the body and cultured. They are capable of only limited growth in culture and cannot be maintained in serial culture. Common examples of primary cell cultures are monkey kidney, human embryotic kidney, human embryo and chick embryo cell cultures. • 2) Diploid cell lines . These are cells of a single type that retain the original diploid chromosome number and karyotype during serial subcultivations for a limited number of times. After about 50 serial passages, they undergo death. Diploid cell lines developed from human fibroblasts are susceptible to a wide range of human viruses and are very useful for the isolation of some fastidious pathogens. They are also employed for the production of viral vaccines. • 3) Continuous cell lines (tumor cell lines, immortalized cell lines) . These are cells of a single type, usually derived from cancer cells that are capable of continuous serial cultivation without senecing. Standard cell lines derived from human cancers, such as HeLa, Hep-2 and KB cell lines have been used in laboratories throughout the world for many years. These cell lines may be maintained by serial subcultivation or stored in the cold (-70 0C) for use when necessary. Inoculation in Cell Cultures Types of Viral Infection in a Host Cell

In lytic, productive infection virus goes through a cycle of replication, producing many virus particles. These are released by cell lysis (cytocidal effect) –for polio or influenza viruses, etc. Persistent infection means that virus -infected cell remains alive and continue to release virus particle at slow rate – for hepatitis B virus. Thus, the person is symptomless carrier of virus and a continuing source of infection. In both lytic and persistant infection virus undergoes replication. Types of Viral Infection in a Host Cell

Abortive ( nonproductive) infection is an infection in which some or all viral components have been synthesized but no infective virus is produced. The situation may result from an infection with defective viruses or because the host cell is nonpermissive and prohibits replication of the particular virus. In latent infections the virus remains quiescent (dormant) , and the genetic material of virus may exist in the cytoplasm (herpesvirus) or be incorporated into the genome (retroviruses, hepatitis B virus). Replication does not take place until some signal triggers a release from latency. Effects of Viral Infection on a Host Cell