Practice general virology RNRMU


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Practice general virology RNRMU

  1. 1. GENERAL VIROLOGY Department of microbiology and virology of PIROGOV’s RSMU
  2. 2. Viruses and virions • VIRUSES are obligate intracellular genetic parasites that use the host cell’s machinery for replication • VIRUSES lack any cellular organization • VIRUSES have one type of nucleic acid (DNA or RNA), which, following the adsorption of the virus into the host cell, is then translated inside the host cell to make viral proteins • Viruses assemble new virus particles within the host cell • Virion is complete virus particle
  3. 3. Structure of viruses • Virus contains a core of either DNA or RNA surrounded by a capsid. Together, the nucleic acid and the capsid is referred to as nucleocapsid • Capsids are composed of smaller repetitive subunits (capsomers). Capsomers are arranged in two fundamental patterns of capsid structural symmetry, icosahedral and helical • Viruses with icosahedral symmetry contain a defined number of structural subunits (20 triangular faces and 12 vertices). HIV have a mixed symmetry: icosahedral in the capsid, and helical in the nucleic acid core • Many viruses possess an envelope - enveloped viruses; viruses without an envelope are called nonenveloped viruses. The viral envelope is composed of virus-specific proteins plus lipids and carbohydrates derived from host cell membrane.
  4. 4. Enzymes • Large number of viruses carry out virion- associated enzymatic activities: RNA- dependent RNA polymerase (RNA- transcriptases) or a DNA-dependent RNA polymerase • Retroviruses contain an RNA-dependent DNA polymerase known as reverse transcriptase.
  5. 5. Classification of viruses Basic principles • The type and structure of the viral nucleic acid (single- or double-stranded RNA or DNA) • The presence or absence of an envelope • The size • The type of capsid symmetry • The strategy for genome replication • The viral antigens • The route of transmission • Epidemiologic features (susceptible hosts)
  6. 6. Classification of viruses enveloped viruses nonenveloped viruses Double-stranded DNA Double-stranded DNA Single-stranded RNA Single-stranded RNA Single-stranded RNA Double-stranded RNA enveloped viruses nonenveloped viruses
  7. 7. Differences in size of typical biological objects
  8. 8. DNA viruses, double-stranded • Adenoviruses (Adenoviridae), nonenveloped, icosahedral symmetry, • Herpes simplex virus (Herpesviridae), enveloped, icosahedral symmetry • Poxvirus, vaccinia (Poxviridae), enveloped, • Hepatitis B (Hepadnaviridae), enveloped (mixed strandedness: 1 strand = party DNA)
  9. 9. Adenovirus
  10. 10. Herpesvirus
  11. 11. Poxvirus
  12. 12. Hepatitis B virus
  13. 13. RNA viruses RNA viruses, single-stranded Poliovirus (family Picornaviridae), nonenveloped, icosahedral capsid symmetry Yellow fever virus (Flaviviridae), enveloped, icosahedral symmetry Influenza viruses (Orthomyxoviridae), enveloped, helical symmetry Rabies virus (Rhabdoviridae), enveloped, helical symmetry HIV (Retroviridae), enveloped, icosahedral capsid and helical nucleocapsid RNA viruses, double-stranded Rotaviruses
  14. 14. Measles virus
  15. 15. Rubella virus
  16. 16. Influenza virus (RNA)
  17. 17. Influenza virus
  18. 18. Rabies virus (RNA)
  19. 19. HIV
  20. 20. HIV
  21. 21. Virus-cell interaction • Once a virus reaches its target organs, it must then infect and successfully replicate in host cells. Three possible outcomes follow infection of a host cell by virus: lytic infection, latent infection, or chronic infection • In a lytic infection (polio, influenza viruses), the virus undergoes multiple rounds of replication that results in the death of the host cell • A latent infection (Herpes simplex, retroviruses) does not result in the immediate production of progenity virus. During cell growth, the genome of the virus is replicated along with the chromosomes of the host cell. Upon reactivation of the herpes simplex virus type 1, fever blisters or cold sores result. • A chronic (persistent) infection: virus particles continue to be shed after the period of acute illness has passed. This kind of infection is usually associated with RNA viruses. Chronic infections are associated with a defective host immunity • Transformation of normal cells to tumor cells
  22. 22. The stages of viral-cell interaction • I stage. Adsorption. The attachment of viruses to host cells and specific binding of viral proteins to receptors on the host cell surface • II stage. Penetration. The viruses use different strategies for penetration. Receptor-mediated endocytos, for example. Adherence of the virus to clathrin-coated pits and the gradual invagination of the membrane carrying the virus into endosome • III stage. Fusion of membranes. The viral envelope fuses with the endosomal membrane • IV stage. Uncoating: entry of the viral nucleic acid into the cytoplasm • V stage. Viral genome replication and macromolecular synthesis. The synthesis requires the translation of viral messenger RNA (mRNA)
  23. 23. VI stage of viral-cell interaction • Assembly of virions and release from the host cell • Assembly of the nonenveloped and the nucleocapsid of enveloped viruses proceed by the self-assembly of viral capsomers into crystal-like arrays. Once the capsid is formed, it becomes filled with the viral nucleic acid to make a viable virion • Nonenveloped virions are usually released when the cell lyses. • Enveloped viruses are typically released from infect cells by budding. Virus-specified proteins inserted into host cell membranes displace some of its normal protein components, which results in the restructuring of the membrane
  24. 24. Viral genome replication and protein synthesis
  25. 25. Viral replication • The single-stranded positive polarity RNA viruses • The genomes of picornaviruses (Polio) and togaviruses are said to have positive (+) strand polarity: the nucleic acid of the virion to function directly as mRNA. • The cellular ribosomes bind to the mRNA to form large polyribosomes that produce a single polyprotein. This precursor molecule is then cleaved in a series of proteolytic steps to produce the proteins of the core and the capsid • RNA polymerase known as transcriptase synthesized a complementary (-) strand RNA using the genomic RNA as template
  26. 26. The single-stranded, RNA+
  27. 27. The single-stranded negative polarity RNA viruses (RNA-) • The RNA of negative-strand viruses (Measles virus, Rabies virus) does not carry coding sequences for protein: these viruses synthesize mRNA by transcription of genomic RNA. The genome is replicated via a (+) single- stranded RNA intermediate • RNA-containing, negative polarity Influenza viruses have segmented genomes consisting of more than one RNA molecule. RNA replication result in a unique mRNA for each viral protein. Replication in the nucleus.
  28. 28. HIV • These RNA viruses contain (+) single-stranded RNA but employ a unique replicative strategy using a DNA intermediate • Viral RNA serves as a template for a virion RNA- dependent DNA polymerase (reverse transcriptase). The DNA is then integrated into host chromosomal DNA
  29. 29. DNA viruses • In cells infected with adenoviruses and herpesviruses transcription of viral DNA in to mRNA occurs in the nucleus of the host cell • The first viral proteins produced after infection are called early proteins. mRNAs encoding the capsid polypeptides (late proteins) are transcribed
  30. 30. Hepatitis B virus • The structure of HBV DNA is unique: it is a partly double- stranded circular molecule • The ‘minus’ strand (noncoding) is nicked and a polymerase molecule is attached to its 5’ end • The ‘plus’ strand contains a short RNA oligonucleotide at its 5’ end and is shortened at its 3’ end • Thus, the circular DNA genome has a single-stranded gap • HBV travels to the liver. After uncoating viral genome is converted into a fully double-stranded partial ds DNA >ssRNA Viral RNA is used as a template for reverse transcription, resulting in the formation of viral DNA ss RNA > ssDNA> dsDNA
  31. 31. Cultivation of viruses Isolation of virus from clinical specimens is done in • cell cultures, • embryonated eggs, • animals (such as suckling mice, monkeys, rabbits). • Cell culture techniques involve the use of primary cultures of cells prepared from organs of freshly killed animals (e.g. monkey kidney cells); of human diploid cell lines; and continuous (heterodiploid) cell lines such as HeLa, Hep-2, BHK-21, and Vero
  32. 32. Structure of embryonated chicken egg
  33. 33. Cultivation of viruses Example • Inoculation into the amniotic cavity or the allantoic cavity of embryonated chicken eggs is useful for the isolation of influenza virus
  34. 34. Indication of viruses • Indication of viruses can be achieved by cytopathic effect (CPE), plaque assay, color probe, hemagglutination and hemabsorbtion • E.g., Orto- and paramyxoviruses (influenza, parainfluenza, measles, mumps) may be detected by the ability of infected cultures to adsorb erythrocytes of animals (hemadsorption)
  35. 35. Identification of viruses • Identification of viruses can be achieved by neutralization cytopathic effect (CPE), plaque assay, color probe, hemagglutination and hemadsorbtion • Examples • Once cell cultures have been inoculated, the specimens are examined for distinctive patterns of cytopathic effect (CPE) . Herpes simplex virus and many enteroviruses produce early CPE, whereas CPE due to CMV, rubella, and some adenoviruses may take weeks • Cultured cells are examined for cell lysis and vacuolization. • The presence of syncytium suggest HSV, respiratory syncytial virus, measles, or mumps virus. • Cytomegaly is seen with HSV, varicella-zoster virus, and CMV. • Immunocytochemical staining of cell cultures to detect viral antigens using fluorescein or enzyme-conjugated specific antiviral antibodies may aid in the detection and identification of many viruses.
  36. 36. Development and progression of viral cytopathology Human embryo skin muscle cells were infected with human cytomegalovirus and stained at selected times to demonstrate (A) uninfected cells, (B) late virus cytopathic effects (nuclear inclusions, cell enlargement), (C) cell degeneration, and (D) a focus of infected cells in a cell monolayer (i.e., a plaque).
  37. 37. Inclusions caused by different viruses
  38. 38. Respiratory syncytial virus. Paramyxoviridae
  39. 39. Formation of multinucleated cells The figure represents the cytopathology of measles virus- induced syncytia.
  40. 40. Methods of diagnosis for viral infections • 1. Viroscopical method (microscopy) • 2. Virological method means isolation and identification of viruses in cell cultures and embryonated eggs • 3. Biological method uses laboratory animals for isolation and identification of viruses • 4. Serological method • 5. Genetic-engineering method (PCR, molecular hybridization)
  41. 41. Identification of viruses by specific antisera • In some cases, examination of specimens by immune electron microscopy is of diagnostic value. The use of specific antisera to aggregate virus in prepared stool specimens facilitates electron microscopy detection of rotaviruses, hepatitis A virus • A 4-fold or greater increase in the antibody titer to a specific viral agent in a patient’s acute and convalescent (3 to 4 weeks later) sera is usually considered diagnostic of acute infection • A number of different types of antibodies including neutralizing, complement-fixing, and hemagglutination- inhibiting antibodies are routinely assayed.
  42. 42. PCR • Hybridization and polymerase chain reaction technique may enable the detection of even single copies of virus genomes in tissue samples or cells from body fluids
  43. 43. Mechanism of PCR
  44. 44. Antiviral immunity (NK-cells) • Cytotoxity by natural killer (NK) cells provides one of the earliest host defenses against viral infection (peak activity at 2 to 3 day) and precedes the appearance of antibody (7 days). Natural killer cells are large granular lymphocytes that bind to infected cells and then secrete cytotoxic molecules –perforins and granzymes • These cells are activated by virus-induced interferons.
  45. 45. Apoptosis (action of NK-cells)
  46. 46. Antiviral immunity (CTLs) • Cytotoxic T lymphocytes (CTLs) constitute a specific virus-induced immune response • CTLs (CD8+) recognize protein fragments of viruses with major histocompatibility complex (MHC) antigens • CTLs product granzymes ( action via apoptosis)
  47. 47. The antibody response • The specific antibodies (production of СD4+ cells) do not usually play a primary role in terminating acute viral infections but are very important in preventing reinfection. • Antibodies that protect the host by destroying the infectivity of virus are called neutralizing antibodies • Neutralizing antibodies reduce viral infectivity by possibly inhibiting attachment, penetration, or uncoating of virus. In addition, such antibodies may produce aggregation of virions, accelerate viral degradation
  48. 48. Structure of IgG
  49. 49. Interferons • Interferons inhibit viral replication indirectly by inducing the synthesis of cellular proteins that minimize viral replication. • There are three main kinds of interferons, called α- , β-, γ-
  50. 50. IFNα
  51. 51. IFNβ
  52. 52. IFNγ