1. VIRUSES Fahareen-Binta- Mosharraf MIC 301 1

2. General characteristics of viruses What is a virus? Ultramicroscopic, obligate intracellular parasite Alive inside the cell but inert extracellularly Cannot generate energy for to carryout any biochemical processes Completely dependent on the host cell for its macromolecule synthesis 2

3. Why are viruses unique?   Virus particles are produced from the assembly of preformed components.   A virus particle cannot grow or under go division.   Lack genetic information for the generation of metabolic energy. 3

4. Pathogenic agents smaller than viruses   Viroids: very small (200-400nm) circular RNA molecules but have no proteins.   Virusoids: satellite, viroids like molecules, depends on presence and replication of another plant virus.   Prions: infectious agents consists of a single type of protein with no nucleic acid components. 4

5. Basic structures of Viruses   Virion- a complete infectious virus particle composed of NA and protein 1. 1. Nucleic acid- either DNA or RNA, but never both. 1. 1. DNA – may be single stranded or ds 2. 2. RNA - may be ss or ds or segmented. 2. 2. Capsid- a protein coat that surrounds the NA 3. 3. Envelope- outer shell consisting of lipid, proteins and carbohydrates 5

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7. CAPSID   In a virion the virus genome is enclosed in a protein coat, known as a capsid. .  Capsids are constructed from many molecules of one or a few species of protein. The individual protein molecules are asymmetrical, but they are organized to form symmetrical structures 7

8. Capsomeres Subunit structure is important Subunit structure is important - The capsomere is a basic subunit of the capsid an outer covering of protein that protects the genetic material of a virus. Capsomeres self-assemble to form the capsid. - The capsomere is a basic subunit of the capsid an outer covering of protein that protects the genetic material of a virus. Capsomeres self-assemble to form the capsid. - the viral coat is made up of repeating units of a single protein – Allows for construction of the virus particles by a process of self assembly into structures - held together by non-covalent bonds in the process of crystallization - held together by non-covalent bonds in the process of crystallization – no need for enzyme-catalyzed reactions for coat assembly – Intracellular release of the viral genome only requires dissociation of non-covalent bonds rather than degradation of a protein coat – Intracellular release of the viral genome only requires dissociation of non-covalent bonds rather than degradation of a protein coat 8

9. Viral lipid envelopes:   Many animal viruses are enveloped, including all those with helical symmetry, e.g. influenza viruses, and a significant number of those with icosahedral symmetry, e.g. herpesviruses.   “Nucleic acids plus protein covering plus perhaps lipids”. --These can be in assembled into very complex structures, e.g. T even bacteriophages 9

10. Virus: Morphology   Helical symmetry- long rods, rigid or flexible   Icosahedral symmetry- polyhedron with 20 triangular faces and 12 corners.   Enveloped viruses- contain lipid cover.   Complex viruses- contain tail, tail fiber, plates and pins etc. 10

11. Virus Morphology 11

12. Structures compared 12

13. 13 Basic virus structure Capsid protein Nucleocapsid Naked capsid virus DNA RNA or = + Nucleocapsid Lipid membrane, glycoprotein Enveloped virus +

14. 14 Capsid symmetry Icosahedral Helical Naked capsid Enveloped Lipid Glycoprotein Matrix

15. 15 Icosahedral naked capsid viruses Adenovirus Electron micrograph Foot and mouth disease virus Crystallographic model

16. 16 Helical naked capsid viruses Tobacco mosaic virus Electron micrograph Tobacco mosaic virus Model RNAProtein

17. 17 Icosahedral enveloped viruses Herpes simplex virus Electron micrograph Herpes simplex virus Nucleocapsid cryoEM model

18. 18 Properties of enveloped viruses   Envelope is sensitive to Drying Heat Detergents Acid   Consequences Transmission in large droplets and secretions Cannot survive in the gastrointestinal tract May require both a humoral and a cellular immune response

19. 19 Properties of naked capsid viruses   Capsid is resistant to Drying Heat Detergents Acids Proteases   Consequences Can survive in the gastrointestinal tract Survive well on environmental surfaces Spread easily via fomites Humoral antibody response may be sufficient to neutralize infection

20. Viral Proteins   Virus proteins may have additional roles, some of which may be carried out by structural proteins, and some by non- structural proteins (proteins synthesized by the virus in an infected cell but they are not virion components). These additional roles include   enzymes, e.g. protease, reverse transcriptase   transcription factors   primers for nucleic acid replication   interference with the immune response of the host. 20

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22. Chemical composition of viruses  Structural proteins: - - Provide structural symmetry of the virus particles. - transfer NA from one host to another. - Protect viral genome. - Participate attachment to the host cell. - Major antigen against which neutralizing Abs are produced. 22

23. Chemical composition of viruses  Non- structural proteins: - some viruses carry enzymes within the virion- without which the virus cannot be viable. e.g. Retrovirus, influenza virus. VIRAL ENZYMES: Lysozyme – entry and exit from bacteria Reverse transcriptase RNA → DNA Neuraminidase – tissue breakdown 23

24. Chemical composition of viruses   Viral proteins can be also grouped into two broad categories 1. 1. Proteins synthesized soon after infection, which are necessary for viral replication- EARLY proteins 2. 2. Proteins synthesized later which include the coat proteins-LATE proteins 24

25. Chemical composition of viruses   Viral nucleic acid: DNA- ds or ss, circular or linear, size of DNA ranges from 3.2 kb to 375 kb. RNA- ss or ds, ‘+’ sense or ‘-’ sense; segmented or non-segmented; size of RNA ranges from 7 kb to 30 kb. ‘+’ sense RNA genome serve as mRNA ‘-’ sense RNA genome is non-infectious 25

26. Chemical composition of viruses   Viral lipid envelopes: Acquired during budding through cell membrane. Consisting of phospholipids, specificity of which depends on the type of membrane involve in budding Composed of one or more species of protein. Most of these proteins are integral membrane proteins and most are O- and/or N-glycosylated Glycosylated proteins are exposed on the outer surface. Unglycosylated proteins are underneath the envelope. 26

27. Classification of Viruses Basis for classification A. Primary Characteristics 1. Chemical nature of NA 2. Structure of virion, helical or icosahedral, naked or enveloped. B. Secondary Characteristics 1. Host range 2. Mode of transmission, 3. Specific surface structure 27

28. Classification system  ICTV-  ICTV- International Committee on Taxonomy of Viruses- authority for classification of viruses. Family Name -viridae; e.g. retroviridae Sub family Name- virinae; e.g. lentivirinae Genera- virus; e.g. Human immunodeficiency virus 28

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31. Multiplication   Lytic cycle Phage causes lysis and death of host cell   Lysogenic cycle Prophage DNA incorporated in host DNA Phage conversion 31

32. Multiplication cycle of Viruses be permissive for that virus A host cell the allows the complete replication cycle of a virus to take place is said to be permissive for that virus Five steps 1. Attachment 2. Penetration 3. Genome multiplication and Gene expression 4. Assembly and Maturation 5 Release 32

33. Lytic Cycle of a T-Even Bacteriophage 33 1 2 3 Figure 13.11

34. 4 Lytic Cycle of a T-Even Bacteriophage 34

35. The Lysogenic Cycle 35

36. VIRAL LIFE CYCLE VIRAL LIFE CYCLE 36 VIRAL LIFE CYCLE ATTACHMENT PENETRATION HOST FUNCTIONS ASSEMBLY (MATURATION) Transcription REPLICATION RELEASE UNCOATING Translation MULTIPLICATION

37. Multiplication of Viruses (cont.) Attachment Attachment  consist of specific binding of viral protein to cellular receptor molecule. .  Receptors are proteins,carbohydrates, glycoprotein lipid, lipoproteins and complex of these. HIV---------------------- CD 4 receptor Rhinoviruses ----------ICAM EBV --------------------- CD21 No. of receptor site may be 100,000 per cell. No. of receptor site may be 100,000 per cell. 37

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39.   If receptor site is altered by any mutation the host may become resistant to viruses   Some animal viruses may able to use more than one host,so loss of one receptor may not necessarily prevent attachment   In the absence of specific receptor viruses cant adsorb the host cell, hence cant infect 39

40. Multiplication of Viruses (cont.) Penetration   Attachment of viruses change the surface structure of both viruses and cell surfaces   Translocation of the entire virus particle   Endocytosis into intracellular vacuole   Fusion of the virus envelope 40

41. Strategies of penetration   Some enveloped virus uncoat in cytoplasmic membrane and release virion content in cytoplasm   The entire virion of naked virus and some enveloped virus enter the cell via endocytosis. In this case virus uncoated inside the cell and genome is exposed and replication can proceed 41

42. Sites of Releases   At the cell surface capsid remaining exterior to surface   Within nucleus   Within cytoplasm   At nuclear pore 42

43. Attachment, Penetration, Uncoating  By pinocytosis 43

44. Figure 13.14b Attachment, Penetration, Uncoating  By fusion 44

45. Viral genome   The virion contains the genome of the virus. DNA, there are four possibilities for a virus genome: 1. 1. double-stranded DNA 2. 2. single-stranded DNA 3. 3. double-stranded RNA 4. 4. single-stranded RNA. 45

46. Genome multiplication and Gene expression   genome contains RNA or DNA.   mRNA is said to be in the plus (+) configuration. Its complement is said to be in the minus (–) configuration.   a virus that has a single-stranded RNA genome with the same orientation as its mRNA is said to be a positive strand RNA virus.   A virus whose single-stranded RNA genome is complementary to its mRNA is said to be a negative-strand RNA virus. 46

47. Baltimore’s system   Proposed by David Baltimore   Classify all viruses into six groups  Based on mode of gene expression  ‘plus’  mRNA is the ‘plus’ strand RNA  ‘minus’  Complementary to mRNA is considered as a ‘minus’ strand  ‘plus’  Strand have the same sequence as mRNA is also a ‘plus’ strand. 47

48. Baltimore’s system 48 ss ‘+’ DNA Class-II a ss ‘–’ DNA Class- II b ± DNA Class- I m RNA ss ‘+’ RNA Class- VI ± RNA Class-III ss ‘-’ RNA Class- V ss ‘+’ RNA Class-IV

49. dsDNA virus replication 49 dsDNA (+)mRNA

50. ssDNA virus replication 50 (+)mRNA dsDNA ssDNA (+)

51. (+)ssRNA virus replication 51 (+)RNA (-)RNA (+)RNA

52. (-)ssRNA virus replication 52 (+)mRNA (-)RNA (+)RNA (-)RNA

53. dsRNA virus replication 53 (+)mRNA dsRNA

54. (+)ssRNA retrovirus replication 54 (+)RNA dsDNA ssDNA (+)mRNA (+)RNA

55. dsDNA retrovirus replication 55 dsDNA ssDNA (+)mRNA (+)RNA dsDNA

56. Virus Multiplication Cycle   Assembly   Occurs at a particular site of the cell   Assembly site depend upon the replication site and the release of the virus.   Picorna, pox, reo viruses assemble in the cytoplasm.   Adeno, polyoma, and parvo- viruses assemble in the nucleus. 56

57. Cont.   Maturation and release   Maturation involves structural changes in the virus capsid protein.   Lytic viruses release by breaking of the cell.   Most enveloped viruses release by budding.   Enveloped viruses acquire their lipid membrane during bud out. 57

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60. Budding of an Enveloped Virus 60

61. Non integrative lysogeny   Less common   Phage DNA not integrated into host genome becomes plasmid and replicate independently   Eg.E.coli phage p1

62. Virus Existance   Virus exists in three ways 1. 1. Extracellular virion: complete virus particle outside the cell 2. 2. Vegetative Phage: Intracellular free NA, autonomous replication 3. 3. Prophage: insert viral NA in bacterial chromosome; no autonomous replication, replication with host genome

63. Growth and quantification of viruses Viruses need to be grown in their host cell – animal, plant, bacteria   Animal viruses – cell culture – cells from   animals grown artificially in the laboratory   Often in monolayers on glass or plastic overlaid with suitable liquid media for the cells to grow 63

64. Cultivation of Viruses   First started in early 1900’s   Successfully done in 1949 in case of poliovirus.  3 main methods:   Embryonated chicken eggs   Tissue Culture   Animal model 64

65. Virus cultivation (Cont) Embryonated Chicken   Most economical and convenient method   Fertile eggs of 5-14 days inoculated through shell aseptically.   Opening sealed with paraffin wax   Incubated at 36 o C until the virus grow   Different types of viruses are grown in different tissues of chick embryo   Can be used for vaccine production 65

66.   Embryonated egg inoculation 66

67. Figure 13.7 Growing Viruses   Animal viruses may be grown in living animals or in embryonated eggs 67

68. Virus cultivation (Cont) Tissue Culture Method   Most common method for propagation of virus   Convenient   Economical for maintenance   Choice of cells for their susceptibility to particular viruses 68

69. Virus cultivation (Cont) Preparation of tissue culture   Dissociate tissue into single cell   mechanical disruption   Proteolytic enzymes   Cell suspensions are placed into plastic flask or plates   Cells grow in monolayer in presence of chemically defined media, with vitamins, coenzymes, and amino acids. 69

70. Cell Culture Some cells will grow indefinitely – permanent cell lines; others will remain alive only for a short period – primary cell lines. 70

71. Tissue Culture Method- 3 types   Primary culture-derived from normal tissues of animal or human, have limited life span.   Diploid cell strain- establish from developing embryo, possess diploid karyotype, can divide up to 100 times.   Continuous cell line- propagate for indefinite time, derived from tumor tissues or cells treating with tumor virus. 71

72. Purification of Virus particles   Concentration of Virus Particles by: -(NH 4 ) 2 SO 4 -PEG -Ultracentrifugation -Haemagglutination   Virus particles can be separated by: -Differential centrifugation -Column chromatography 72

73. Differential centrifugation 73

74. Measurement of Infectious Virus   Quantitation: Based on bacterial plaques but applicable to monolayer culture of animal and plant cultures .  Done by determining the titer in a sample.   Plaque assay   Fluorescent Focus assay   Infection center assay   Transformation assay   End point dilution assay 74

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76. Plaque Assay   Discovered by Renato Dulbecco, 1952.   Cell monolayer is incubated by virus and covered with nutrient media and agar.   Each infectious virus produce a zone of infected cells, known as plaque.   Plaques can be distinguished from other cells as the cells in plaque are lysed.   No. of plaques in a given dilution is proportional to the no. of infectious particles.   Can be done only for cytopathic viruses 76

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79. Plaque assay (cont.) 79 Time in hr Titer of virus, p.f.u./ml One -hit- kinetics: when one infectious particle is sufficient to initiate infection. And the curve is linear. Two- hit- kinetics: two different types of virus particles must infect a single cell to ensure infection. The curve is parabolic. One-hit-kinetics Two-hit kinetics

80. Fluorescent Focus assay   useful for measurement of non-cytopathic virus.   an antibody to the virus is added   a fluorescin conjugated secondary Ab is added   a foci of infected cells fluoresce under UV microscope.   each focus is initiated by a single virus particle. 80

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82. Measurement of Virus particles and their components 1. 1. Electron Microscopy 2. 2. Hemagglutination 3. 3. Measurements of viral enzymes 4. 4. Serological methods 1. 1. Neutralization 2. 2. Heamagglutination inhibition 3. 3. Complement fixation 4. 4. Immunoblotting 5. 5. EIA 5. 5. NA detection 82

83. one-step growth curve   The graph which displays the results of a single round of viral replication in a population of cell. 83

84. Stages of one step growth curve 1. 1. Eclipse :   in the first few minutes of infection as soon as the infectious virus particles are removed from environment by adsorbing to host cell   the infectious virus particles cannot be detected in culture medium   Once attached the virions are no longer available to infect the other cells   Followed by entry of viral NA (or intact virion) into host cell 84

85. 2. Maturation:   Begins with newly synthesized NA molecules become packaged inside protein coat.   The titer of active virions within the cell rises dramatically   These new virus particles cannot be detected unless the cells are artificially lysed to release them As the newly synthesized virions have not yet appeared in eclipse and maturation stages so these stages are together called LATENT PERIOD. 85

86. 3. Assembly and release   NA and proteins are assembled into,mature virions   Release may be 100 virions/cell. (BURST SIZE)   Release is through cell lysis, budding &   Occasionally continuous secretion Over all virus replication cycle- 20-60 minutes (bacterial virus) to 8-40h (animal viruses) 86

87. Figure 13.10 A Viral One-Step Growth Curve 87

88. Virus growth From Schaechter’s Mechanisms of Microbial Disease; 4 th ed.; Engleberg, DiRita & Dermody; Lippincott, Williams & Wilkins; 2007; Fig. 31-8 1,000 – 100,000 viruses/cell, 5 – 24 hours

89. INTERFERONS   Interferons (IFN) are host encoded proteins of that inhibit viral replication.   Belongs to cytokine family   Produced by live animals or cultured cells in response to viral infection or other inducers. 89

90. Induction of interferons   Categorize into 3 general groups: IFN α, IFNβ, and IFNγ   IFN α and IFNβ are type I interferons; IFNγ is a type II interferon   Normal cells do not synthesize interferons unless they are induced   Antiviral activity 90 Virus Titer Interferon titer Ab titer

91. Types of antiviral drugs   Nucleoside analogs   Nucleotide analogs   Protease inhibitors   Others 91