Bohomolets Microbiology Lecture #5


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By Ms. Kostiuk from Microbiology department

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  • Viruses can replicate in host cell only if: The host cell must be permissive and the virus must be compatible with the host cell The host cell must not degrade the virus The viral genome must possess the information for modifying the normal metabolism of the host cell The virus must be able to use the metabolic capabilities of the host cell to produce new virus particles Abortive infections occur because the host cell is nonpermissive so that viral replication does not occur or because viral replication produces viral progeny that are incapable of infecting other cells. Productive infection, which occurs in permissive cells. Results in viral replication with the production of viruses that can infect other compatible cells. Integrative interaction is typical for retroviruses and some of DNA-viruses which effect oncogenic transformation of cells.
  • We can mark out 3 period of reproduction. Early is defined as the period before genome replication. In general “early” proteins are enzymes, whereas late proteins are structural components of the virus. Within the host cell, the viral genome achieves control of the host cell’s metabolic activities. Transcription of the host cell genes is inhibited or viral mRNAs are translated more efficiently than host cell mRNAs so that viral protein synthesis dominates over synthesis of normal host cell proteins. The virus then uses the metabolic capacity of the host cell for the production of new viruses.
  • Invasion begins when the virus meet with a susceptible host cell and adsorbs specifically to receptor sites in the cell membrane. The membrane receptors that viruses attach to are usually proteins the cell requires for its normal function. For example, the rabies virus affixes to the acetylcholine receptor of nerve cells, and the human immunodeficiency virus (HIV or AIDS virus) attaches to the CD4 protein on certain white blood cells. The mode of attachment varies between the two general types of viruses. In enveloped forms (a) such as influenza virus and HIV, glycoprotein spikes bind to the cell membrane receptors. Viruses with naked nucleocapsids (b) (poliovirus, for example), use their attachment proteins for binding with cell membrane receptors. Because of this requirement to bind to specific receptors, a particular virus can only infect a limited number of cell types. This limitation is known as the host rang . Most viruses can only infect a single species, some only a single cell type within a host species, like virus hepatitis B, which infect only liver cells of human. Polioviruses can infect a intestinal, nerve and others cells but only in human and monkey.
  • The mechanism of entry of animal viruses into host cells depends upon whether the virion is enveloped or naked. In the case of enveloped viruses, 2 mechanisms exist. In one case (a), after attachment to a host cell receptor, the envelope of the virion fuses with the plasma membrane of the host. The nucleocapsid is then released directly into the cytoplasm where the nucleic acid separated from the protein coat. In the other case (b), enveloped viruses adsorb to the host cell with their protein spikes, and the virions are taken into the cell by a process termed endocytosis. In this process, the host cell plasma membrane surrounds the whole virion in a vesicle. Then, the envelope of the virion fuses with the plasma membrane, after which the nucleocapsid is released into the host’s cytoplasm. In the case of naked viruses, the virion also enters by endocytosis. The virions cause the vesicle to dissolve, resulting in their release into the cytoplasm, where the nucleic acid separates from its protein coat prior to replication.
  • The infecting parental virus particle (virion) attaches to the cell membrane and then penetrates the host cell. The viral genome is “uncoated” by removing the capsid proteins, and the genome is free to function. Early mRNA and proteins are then synthesized; the early proteins are enzymes used to replicate the viral genome. Late mRNA and proteins are then synthesized. These late proteins are the structural, capsid protein. The progeny virions are assembled from the replicated genetic material and newly made capsid proteins and are then released from the cell.
  • The free viral nucleic acid exerts control over the host’s synthetic and metabolic machinery. How this control proceeds will vary, depending on whether the virus is a DNA or an RNA virus. In general, the DNA viruses (except poxviruses) enter the host cell’s nucleus and are replicated and assembled there.
  • The most events of replication of RNA viruses occur in cytoplasm (with some exception like retroviruses). Single-stranded viruses with RNA of positive polarity serve molecule RNA as mRNA for enzymes and structural viral proteins and as template for replication of complementary minus strand RNA. This (-) strand RNA is used for viral genome replication. Enzyme for this process is virus specific and named RNA-dependent RNA polymerase or RNA replicase . RNA viruses that have RNA minus genome cannot use their nucleic acid as mRNA but only like template for synthesis of complementary mRNA which can bind with ribosomes and can be used as template for synthesis of virion genome – RNA. For viruses with negative polarity presence in virion RNA replicase as structural protein is necessary, but not for viruses with positive genome.
  • Assembly involves the packaging of the nucleic acid genome into the capsid to form the nucleocapsid. In case of naked viruses there are completed viral particles. Surprisingly, certain viruses can be assembled in the test tube by using only purified RNA and purified protein. This indicates that the specificity of the interaction resides within the RNA and protein and that the action does not require special enzymes and energy. Since it is a self-assembly process . There are 2 general pathways assemble of nucleocapsid. For viruses with helical symmetry of nucleocapsid (as tobacco mosaic virus), many identical protein structural subunits (capsomeres) are first formed and then are added one by one to the growing coat structure that surrounds the viral RNA. For viruses with cubic symmetry (as polioviruses) In the case of picornaviruses, copies each of virion proteins assemble in the cytoplasm into a procapsid which similar to empty shell. Viral RNA is then packaged into the procapsid, and form nucleocapsid.
  • After assemble viruses can form masse, s often in crystalline packets, in so-called “viral factory “
  • To complete the cycle, assembled viruses leave their host in one of two ways. Lyses . Naked and complex viruses that reach maturation in the cell nucleus or cytoplasm are released when the cell died and lyses spontaneously. Non-enveloped viruses egress from infected cell using mechanism of “explosion”. Nucleocapsids are accumulated in the cell and egress into the extracellular environment all together with disintegrating cell. Budding . Enveloped viruses are liberated by budding from the membranes of the cytoplasm, nucleus, endoplasmic reticulum, or vesicles.
  • In the enveloped viruses the virus undergoes further modification, including the addition of the envelope from the host cell. This process is called maturation .
  • As s first step in the budding process, the region of the host cell plasma membrane where budding is going, is modified by specific viral proteins (spikes). Simultaneously these proteins displace cell protein from this patch. Then the inside of the membrane becomes coated with the virion protein, termed matrix protein . Next, viral nucleocapsids bind to this modified places of membrane with spikes and matrix protein and become wrapped up by the patch. So, viruses do not have cellular proteins, only virus-specific proteins.
  • Inasmuch as viruses are obligate intracellular parasite s, they are demand live cells and can not be cultivate in nutrient medium like bacteria. There are three appropriate systems for virus isolation and cultivation:
  • The exact tissue that is inoculated is guided by the type of viruses being cultivated.
  • Chicken, duck and turkey eggs are the most common choices for inoculation. Strict sterile condition must be used when inoculation to prevent contamination by bacteria and fungi.
  • As a rule, cell culture is grown like monolayer. Monolayer is a single layer of attached cells.
  • Virus-infected animal cells often develop abnormally, with visible changes in their appearance, known as the cytopathic effect . CPEs are secondary effects of the viral doing what is necessary to replicate and are not simply toxic effects of viral gene products on the host cell.
  • Some viruses, such as herpesviruses, measles virus, cause infected cells to fuse together, forming multinuclear giant cells called syncytia .
  • Inclusions sometimes occur within the nucleus or cytoplasm of infected cells. These inclusions may be stained with basic or acid dyes and viewed with a microscope. Cells infected with rabies virus develop acidophilic inclusion within the cytoplasm (Negry bodies_, cells infected with adenoviruses develop basopholic inclusions within the nucleus, cells infected with small pox viruses form inclusion in cytoplasm (Gvarniery’s bodies).
  • One way to detect the growth of a virus in culture is to observe degeneration and lysis of infected cells in the monolayer of cell. The areas where virus-infected cells have been destroyed show up a clear, well defined patches in the cell sheet called plaques. A plaque develops when the viruses released by an infected host cell radiate out to nearby host cells. As new cells become infected, they die and release more viruses, and so on. These clean spaces – plaques- correspond to areas of dead cells that may be observed by naked eye.
  • Clear spaces in culture indicate sites of virus growth (plaques). Microscopic views of normal, undisturbed cell layer and plaques, which consist of cells disrupted by viral infection.
  • Many animal viruses, but not all, are able to clump (agglutinate) red blood cells because they interact with the surfaces of the red cells. This phenomenon is called hemagghutination. In this process, a virion attaches to two red cells simultaneously and causes them to clump. Hemagglutination may be used for detection of viruses that have hemagglutinating spikes on surface
  • After adsorption, a phage is still on the outside of its host cell. The strong, rigid bacterial cell wall is quite an impenetrable barrier, and the entire virus particle is unable to cross it. After attachment, phage plate becomes embedded in the cell wall, and the tail contracts, pushing the tube through the cell wall and releasing the nucleic acid into the interior of the cell. Once inside, the viral nucleic acid alone can complete the multiplication cycle.
  • Within a few minutes, the bacterium stops synthesizing its own molecules, and its metabolism shifts to the expression of genes on the viral nucleic acid strand. In T-even bacteriophages, the viral DNA redirects the genetic and metabolic activity of the cell, blocking the utilization of host DNA and ensuring that viral DNA is copied and used to synthesize new viral components. The early stages of viral replication are known as eclipse, a period during which no mature virions can be detected within the host cell. This corresponds with the period of penetration, replication, and early assembly, and if the cell were disrupted at this time, no infective virus would be released. As the host cell rapidly produces new phage parts, these parts spontaneously assemble into bacteriophages, similar to a mass production assembly line. The first parts to assemble are the capsid and tail. Viral DNA is inserted into the capsid before the capsomers are completely joined. An average-sized Escherichia coli cell can contain up to 200 new phage units at the end of this period.
  • Eventually, the host cell becomes so packed with viruses that is lysis thereby liberating the mature virions. This process is hastened by viral enzymes released late in the infection cycle.
  • Not all bacteriophages complete the lytic cycle, however. Special DNA phages, called temperate phages, undergo adsorption and penetration in the bacterial host but are not replicated or released. Instead, the viral DNA enters an inactive prophage state, in which it is inserted into the bacterial chromosome. This viral DNA will be retained by the bacterial cell and copied during its normal cell division so that the cell’s progeny will also have the temperate phage DNA. This condition of the host chromosome’s carrying bacteriophage DNA is termed lysogeny . Because the viral genome is not expressed, the bacterial cells carrying temperate phages do not lyse, and they appear entirely normal. On occasion, the prophage is a lysogenic cell will be activated and progress directly into viral replication and the lytic cycle.
  • Under ordinary conditions of growth of a lysogenic cell, the phage DNA is released from the bacterial chromosome only about once in 10.000 divisions. If a lysogenic culture is treated with an agent that damages the DNA (such as ultraviolet light), all of the prophages enter the lytic cycle and a productive infection results. This process termed induction , results in complete lysis of the culture. Lysogenic culture becomes immune to infection by the same type of phage but not to infection by other phages.
  • Characteristics used for placement in a particular family include type of capsid, nucleic acid strand number, presence and type of envelope, overall viral size, and area of the host cell in which the virus multiplies.
  • Some virus families are named for their mi­croscopic appearance (shape and size). Examples include rhabdoviruses,* which have a bullet-shaped envelope, and togaviruses, which have a cloaklike envelope. Anatomical or geographic areas have also been used in naming. For instance, adenoviruses were first discovered in adenoides, and bunyaviruses were originally isolated in an area in Africa called Bunyamwera.
  • Viruses can also be named for their effects on the host. Lentiviruses tend to cause slow, chronic infections. Acronyms made from blending several characteristics include picornaviruses,* which are tiny RNA viruses, and reoviruses (or respiratory enteric orphan viruses), which inhabit the respiratory tract and the intestine and are not yet associated with any known disease state.
  • Bohomolets Microbiology Lecture #5

    1. 1. Virology Reproduction and cultivation
    2. 2. Types of virus interaction with cell <ul><li>Abortive. When abortive infection replication of virus progeny is interrupted in someone stage. </li></ul><ul><li>Productive . Productive infection results in viral replication with the production of virus progeny and death of the host cel. </li></ul><ul><li>Integrative (latent, persistent). Integrative infection occur when virus genome integrate into cell chromosome. . </li></ul>
    3. 3. Stages of viral replication <ul><li>Adsorption (attachment) </li></ul><ul><li>Penetration </li></ul><ul><li>Uncoating of the viral genome </li></ul><ul><li>Gene expression, transcription, early and late viral protein synthesis </li></ul><ul><li>Genome replication </li></ul><ul><li>Assembly of progeny virions </li></ul><ul><li>Release virions from cell </li></ul>Early events Middle events Late events
    4. 4. Adsorption of animal viruses
    5. 5. Entry of enveloped viruses into host cell a) b)
    6. 6. General scheme replication of poxviruses
    7. 7. Replication in DNA viruses
    8. 8. Replication in RNA viruses
    9. 9. Maturation of virions Assembly of viruses is a self-assembly process Tobacco mosaic virus maturation
    10. 10. Nucleus of a cell, containing a crystalline mass of adenoviruses
    11. 11. Release of mature viruses from host cell
    12. 12. Budding release of retroviruses
    13. 13. Maturation of an enveloped virus
    14. 14. Основні ефекти, що викликають віруси в клітинах: <ul><li>Морфологічні (ЦПД, вірусні включення) </li></ul><ul><li>Фізіологічні (зміна клітинних мембран – плинність, ліпідний склад, мембранний потенціал) </li></ul><ul><li>Біохімічні (переключення генетичної інформації, зміни у обмінних процесах, зміни у цитоскелеті клітин, поява нових ферментів) </li></ul><ul><li>Генотоксичні (руйнуваня або зміни геному клітини, хромосомні аберації) </li></ul><ul><li>Біологічні (поява нових антигенів, малігнізація) </li></ul>
    15. 15. Methods cultivating of animal viruses <ul><li>Live animal organism </li></ul><ul><li>Chicken embryo </li></ul><ul><li>Cell (or tissue) culture </li></ul>
    16. 16. Cultivation viruses in newborn mice <ul><li>Advantages: </li></ul><ul><li>Study of pathogenesis </li></ul><ul><li>Examination of vaccines and antiviral drags </li></ul><ul><li>Disadvantages: </li></ul><ul><li>Not-universal method </li></ul><ul><li>Presence own viruses and bacteria in animal organism </li></ul>
    17. 17. Sign of viral infection in animals Mouse with paralysis of back extremities <ul><li>Death </li></ul><ul><li>Symptoms of illness </li></ul>Newborn mice that are behind in development
    18. 18. Virus cultivation in bird embryos <ul><li>Advantages: </li></ul><ul><li>Standardized system </li></ul><ul><li>Sterile system </li></ul><ul><li>Possibility to cultivate viruses and make viral vaccines </li></ul><ul><li>Disadvantages: </li></ul><ul><li>Not-universal method </li></ul><ul><li>Possible presence own latent viruses </li></ul>
    19. 19. Virus cultivation in bird embryos <ul><li>The signs of viral growth in bird embryos are: </li></ul><ul><li>death of the embryo </li></ul><ul><li>defects in embryonic development </li></ul><ul><li>pocks ( localized areas of damage in the membranes ) </li></ul><ul><li>Other methods for detection of viruses that do not produce visible changes in embryo: </li></ul><ul><li>direct examination with electron microscope </li></ul><ul><li>hemagglutination test </li></ul><ul><li>serological test </li></ul>
    20. 20. Pocks developing on a chorioallantoic membrane caused by herpes simplex viruses I and II types
    21. 21. Cell culture <ul><li>there is the aggregate of human, animal or plant cells that are cultivated in vitro with using of complex nutrient mediums and conditions of cultivation </li></ul>
    22. 22. Cell cultures Cells of macaque kidneys Cells of human embryo HeLa Cells of pig embryo ( RES )
    23. 23. Methods detection of viruses in cell culture <ul><li>Cytopathic effect (CPE) </li></ul><ul><li>Inclusion body formation </li></ul><ul><li>Coloured test </li></ul><ul><li>Plaque formation under agar or bentonit </li></ul><ul><li>Hemadsorbtion </li></ul><ul><li>Hemagglutination </li></ul><ul><li>Detection viral antigens in infected cell with serological test </li></ul><ul><li>Detection of virions under electron microscope </li></ul>
    24. 24. Types of viral CPE Respiratory syncytial virus, herpes simplex viruses Syncytium formation (a mass of fused cells) Oncogenic viruses as retroviruses, some herpesviruses Transformation and proliferation. Polioviruses, togavirus Cell lysis Smallpox virus, influenza virus Cell rounding and detachment from the substrate Instance Type of CPE
    25. 25. Cell lysis caused by enterovirus Normal cells Vero Infected cells Vero after poliovirus reproduction
    26. 26. Syncytium formation caused by herpesvirus
    27. 27. CPE caused by adenovirus Intact cells HeLa Clumping of infected cells
    28. 28. Inclusion body formation Gvarniery bodies Negry bodies
    29. 29. Plaque formation Plaques under agar Plaques under bentonit
    30. 30. Normal cells Infected cells Plagues
    31. 31. Detection viruses that have hemagglutinins Hemagglutination Hemadsorbtion Red cells Viruses Hemagglutination
    32. 32. Attachment phages on bacterial surface Phages that attached to bacterial flagellum
    33. 33. Penetration of tailed phage
    34. 34. Multiplication of bacteriophages
    35. 35. Releasing phages from ruptured bacterium A bacterial cell, crowded with viruses, has ruptured and released numerous virions that can attack nearby susceptible cells.
    36. 36. The lysogenic state in bacteria Lysogeny is a condition in which viral DNA is inserted into the bacterial chromosome and remains inactive for an extended period. Virulent phage propagates in bacteria and induces its death Temperate phage after entering in the bacterium integrates their DNA in the chromosome of the host cell and turn into prophage.
    37. 37. Life cycle of phage lambda – lytic and reproductive infection Lysogenic cell is the bacterial cell carrying a prophage
    38. 38. Animal DNA viruses families Parvovirus B19 - 18-26 Icosahedral Single Parvo-viridae Hepatitis B virus + 42 Icosahedral double Hepadna-viridae Human papillomavirus - 45-55 Icosahedral Double Papova-viridae Human adenoviruses - 70-90 Icosahedral Double Adeno-viridae Herpes simplex virus, Varicella zoster virus, Epstein-Barr virus + 150-200 Icosahedral Double Herpes-viridae Smallpox virus; complex virus; brick-shaped + 130-300 None Double Poxviridae Common Name of Important Members Envelope Size Capsid type Strand type Family
    39. 39. Animal RNA viruses families (1) Parainfluenza virus, mumps virus, measles virus + 125-250 Helical Single Paramyxo-viridae Yellow fever virus, Japanese encephalitis virus + 40-70 Icosahedral Single Flaviviridae Rubella virus, western equine encephalitis + 45-70 Icosahedral Single Togaviridae Norwalk virus - 35-40 Icosahedral Single Calciviridae Hepatitis A virus, poliovirus, coxsackieviruses, rhinoviruses - 20-30 Icosahedral Single Picorna-viridae Common Name of Important Members Envelope Size (nm) Capsid Type Strand type Family
    40. 40. Animal RNA viruses families (2) Influenza viruses + 80-120 Helical Single Orthomyxo-viridae Human rotavirus, Colorado tick fever virus - 60-80 Icosahedral Double Reoviridae Bunyamwera virus, Hanta virus + 90-100 Helical Single Bunyaviridae Ebola and Marburg viruses + 790-970 Helical Single Filoviridae Common Name of Important Members Envelope Size (nm) Capsid Type Strand type Family
    41. 41. Animal RNA viruses families (3) Human infectious bronchitis and corona viruses + 80-130 Helical Single Corona-viridae Lassa virus; lymphocytic choriomeningitis virus + 50-300 ? Single Arenaviridae Human immunodeficiency virus (AIDS), oncoviruses + 100 Icosahedral Single Retroviridae Rabies virus + 60-75 Helical Single Rhabdo-viridae Common Name of Important Members Envelope Size (nm) Capsid Type Strand type Family