1. Medical Virology Medical Virology Introduction to Basics Dr.T.V.Rao MD 1

2. History Virology Smallpox was endemic in China by 1000BC. In response, the practice of variolation was developed. Recognizing that survivors of smallpox outbreaks were protected from subsequent infection, variolation involved inhalation of the dried crusts from smallpox lesions like snuff, or in later modifications, inoculation of the pus from a lesion into a scratch on the forearm of a child. Dr.T.V.Rao MD2

3. Virus infections are Universal …….

4. Introduction to Virology A virus is an obligate intracellular parasite containing genetic material surrounded by protein Virus particles can only be observed by an electron microscope Dr.T.V.Rao MD4

5. Introduction to Virology Recognizing the shape, size, and structure of different viruses is critical to the study of disease – Viruses have an inner core of nucleic acid surrounded by protein coat known as an envelope – Most viruses range in sizes from 20 – 250 nanometers Dr.T.V.Rao MD5

6. Viral Properties Viruses are inert (nucleoprotein ) filterable Agents Viruses are obligate intracellular parasites Viruses cannot make energy or proteins independent of a host cell Viral genome are RNA or DNA but not both. Viruses have a naked capsid or envelope with attached proteins Viruses do not have the genetic capability to multiply by division. Viruses are non-living entities Dr.T.V.Rao MD6

7. Viruses are Ultramicroscopic 7 Koneman et al. Color Atlas and Textbook of Microbiology 5th Ed. 1997 Dr.T.V.Rao MD

8. The size of viruses Dr.T.V.Rao MD8

9. 9 VIRAL STRUCTURE – SOME TERMINOLOGY virus particle = virion protein which coats the genome = capsid capsid usually symmetrical capsid + genome = nucleocapsid may have an envelope Dr.T.V.Rao MD

10. Virion The complete infectious unit of virus particle Structurally mature, extracellular virus particles. Dr.T.V.Rao MD10

11. Virion Capsid Viral core envelope Dr.T.V.Rao MD11

12. Virion Structure Dr.T.V.Rao MD12

13. Distinguishing characteristics of viruses Obligate intracellular parasites Extreme genetic simplicity Contain DNA or RNA Replication involves disassembly and reassembly Replicate by "one-step growth” Dr.T.V.Rao MD13

14. How are viruses named? Based on: - the disease they cause poliovirus, rabies virus - the type of disease murine leukemia virus - geographic locations Sendai virus, Coxsackie virus - their discovers Epstein-Barr virus - how they were originally thought to be contracted dengue virus (“evil spirit”), influenza virus (the “influence” of bad air) - combinations of the above Rous Sarcoma virus Dr.T.V.Rao MD14

15. 15 White, DO and Fenner, FJ. Medical Virology, 4 th Ed. 1994 Virus particle = virion Dr.T.V.Rao MD

16. 16 5 BASIC TYPES OF VIRAL STRUCTURE HELICAL ENVELOPED HELICAL ENVELOPED ICOSAHEDRAL COMPLEX ICOSAHEDRAL Adapted from Schaechter et al., Mechanisms of Microbial Disease nucleocapsid icosahedral nucleocapsid nucleocapsid helical nucleocapsid lipid bilayer glycoprotein spikes = peplomers Dr.T.V.Rao MD

18. 18

19. Icosahedral Adeno-associated Virus (AAV) Adenovirus B19 Coxsackievirus - A Coxsackievirus - B Cytomegalovirus (CMV) Eastern Equine Encephalitis Virus (EEEV) Echovirus Epstein-Barr Virus (EBV) Hepatitis A Virus (HAV) Hepatitis B Virus (HBV) Hepatitis C Virus (HCV) Hepatitis Delta Virus (HDV) Hepatitis E Virus (HEV) Herpes Simplex Virus 1 (HHV1) Herpes Simplex Virus 2 (HHV2) Human Immunodeficiency Virus (HIV) Human T-lymphotrophic Virus (HTLV) Norwalk Virus Papilloma Virus (HPV) Polio virus Rhinovirus Rubella Virus Saint Louis Encephalitis Virus Varicella-Zoster Virus (HHV3) Western Equine Encephalitis Virus (WEEV) Yellow Fever Virus Dr.T.V.Rao MD19

20. Viral Structure Varies in size, shape and symmetry VIP for classification 3 types of capsid symmetry: – Cubic (icosahedral) Has 20 faces, each an equilateral triangle. Eg. adenovirus – Helical Protein binds around DNA/RNA in a helical fashion eg. Coronavirus – Complex Is neither cubic nor helical eg. poxvirus Dr.T.V.Rao MD20

21. The Baltimore classification system Based on genetic contents and replication strategies of viruses. According to the Baltimore classification, viruses are divided into the following seven classes: 1. dsDNA viruses 2. ssDNA viruses 3. dsRNA viruses 4. (+) sense ssRNA viruses (codes directly for protein) 5. (-) sense ssRNA viruses 6. RNA reverse transcribing viruses 7. DNA reverse transcribing viruses where "ds" represents "double strand" and "ss" denotes "single strand". Dr.T.V.Rao MD21

22. Virus Classification I - the Baltimore classification All viruses must produce mRNA, or (+) sense RNA A complementary strand of nucleic acid is (–) sense The Baltimore classification has + RNA as its central point Its principles are fundamental to an understanding of virus classification and genome replication, but it is rarely used as a classification system in its own right Dr.T.V.Rao MD22

23. From Principles of Virology Flint et al ASM Press Dr.T.V.Rao MD23

24. Virus classification II - the Classical system This is a based on three principles - – 1) that we are classifying the virus itself, not the host – 2) the nucleic acid genome – 3) the shared physical properties of the infectious agent (e.g capsid symmetry, dimensions, lipid envelope) Dr.T.V.Rao MD24

25. Virus classification III - the genomic system More recently a precise ordering of viruses within and between families is possible based on DNA/RNA sequence By the year 2000 there were over 4000 viruses of plants, animals and bacteria - in 71 families, 9 subfamilies and 164 genera Dr.T.V.Rao MD25

26. Viral Structure - Overview Fig 1. Schematic overview of the structure of animal viruses ** does not exist in all viruses Nucleic acid Capsid Nucleocapsid Envelope protein Membrane protein Viral envelope** Spike protein Dr.T.V.Rao MD26

27. a) Crystallographic structure of a simple icosahedral virus. b) The axes of symmetry Icosahedral capsids Dr.T.V.Rao MD27

28. Cubic or icosahedral symmetry Dr.T.V.Rao MD28

29. 29 ICOSAHEDRAL SYMMETRY Dr.T.V.Rao MD

30. 30 ICOSAHEDRAL SYMMETRY Dr.T.V.Rao MD

31. 31 ICOSAHEDRAL SYMMETRY Dr.T.V.Rao MD

32. 32 ICOSAHEDRAL SYMMETRY Dr.T.V.Rao MD

33. 33 Adenovirus Dr.T.V.Rao MD

34. 34 Adenovirus Dr.T.V.Rao MD

35. Helical symmetry Dr.T.V.Rao MD35

36. Helical California Encephalitis Virus Coronavirus Hantavirus Influenza Virus (Flu Virus) Measles Virus ( Rubeola) Mumps Virus Para influenza Virus Rabies Virus Respiratory Syncytial Virus(RSV) Dr.T.V.Rao MD36

37. Helical symmetry How to assemble Dr.T.V.Rao MD37

38. In 1955, Fraenkel, Conrat, and Williams demonstrated that tobacco mosaic virus (TMV) spontaneously formed when mixtures of purified coat protein and its genomic RNA were incubated together. Helical symmetry TMV, a filamentous virus Dr.T.V.Rao MD38

39. Enveloped helical virusEnveloped icosahedral virus Dr.T.V.Rao MD39

40. Properties of naked viruses Stable in hostile environment Not damaged by drying, acid, detergent, and heat Released by lysis of host cells Can sustain in dry environment Can infect the GI tract and survive the acid and bile Can spread easily via hands, dust, fomites, etc Can stay dry and still retain infectivity Neutralizing mucosal and systemic antibodies are needed to control the establishment of infection

41. Naked viruses( Non Enveloped ) Adeno-associated Virus (AAV) Adenovirus B19 Coxsackievirus - A Coxsackievirus - B Echovirus Hepatitis A Virus (HAV) Hepatitis E Virus (HEV) Norwalk Virus

42. 42 COMPLEX SYMMETRY POXVIRUS FAMILY surface viewcross section White, DO and Fenner, FJ. Medical Virology, 4 th Ed. 1994 Dr.T.V.Rao MD

43. 43 ENVELOPE OBTAINED BY BUDDING THROUGH A CELLULAR MEMBRANE (except poxviruses) POSSIBILITY OF EXITING CELL WITHOUT KILLING IT CONTAINS AT LEAST ONE VIRALLY CODED PROTEIN – ATTACHMENT PROTEIN LOSS OF ENVELOPE RESULTS IN LOSS OF INFECTIVITY Dr.T.V.Rao MD

44. 44 RNA or DNA segmented or non-segmented linear or circular single-stranded or double-stranded if single-stranded RNA – is genome mRNA (+) sense or complementary to mRNA (-) sense CLASSIFICATION NUCLEIC ACID Dr.T.V.Rao MD

45. Genome The genome of a virus can be either DNA or RNA DNA-double stranded (ds): linear or circular Single stranded (ss) : linear or circular RNA- ss:segmented or non-segmented ss:polarity+(sense) or polarity – (non- sense) ds: linear (only reovirus family) Dr.T.V.Rao MD45

46. DNARNA double-strandedsingle-stranded double- stranded single-stranded line ar circular line ar circularlinearlinear (circular)** singl e multip le singl e multip le singl e multipl e (+)sense(-)sense sing le multip le sing le multip le Dr.T.V.Rao MD46

47. Viral genome strategies dsDNA (herpes, papova, adeno, pox) ssDNA (parvo) dsRNA (reo, rota) ssRNA (+) (picorna, toga, flavi, corona) ssRNA (-) (rhabdo, paramyxo, orthomyxo, bunya, filo) ssRNA (+/-) (arena, bunya) ssRNA (+RTase) (retro, lenti) Dr.T.V.Rao MD47

48. 48 HERPESVIRIDAE HEPADNAVIRIDAE ENVELOPED PAPILLOMAVIRIDAE POLYOMAVIRIDAE (formerly grouped together as the PAPOVAVIRIDAE) CIRCULAR ADENOVIRIDAE LINEAR NON-ENVELOPED DOUBLE STRANDED PARVOVIRIDAE SINGLE STRANDED NON-ENVELOPED POXVIRIDAE COMPLEX ENVELOPED DNA VIRUSES Modified from Volk et al., Essentials of Medical Microbiology, 4th Ed. 1991 All families shown are icosahedral except for poxviruses Dr.T.V.Rao MD

49. DNA viruses From Principles of Virology Flint et al ASM Press Dr.T.V.Rao MD49

50. 50 FLAVIVIRIDAE TOGAVIRIDAE RETROVIRIDAE ICOSAHEDRAL CORONAVIRIDAE HELICAL ENVELOPED ICOSAHEDRAL PICORNAVIRIDAE CALICIVIRIDAE ASTROVIRIDAE NONENVELOPED SINGLE STRANDED positive sense BUNYAVIRIDAE ARENAVIRIDAE ORTHOMYXOVIRIDAE PARAMYXOVIRIDAE RHABDOVIRIDAE FILOVIRIDAE SINGLE STRANDED negative sense REOVIRIDAE DOUBLE STRANDED RNA VIRUSES ENVELOPED HELICALICOSAHEDRAL NONENVELOPED Modified from Volk et al., Essentials of Medical Microbiology, 4th Ed. 1991 Dr.T.V.Rao MD

51. RNA viruses From Principles of Virology Flint et al ASM Press Dr.T.V.Rao MD51

52. 52 BASIC STEPS IN VIRAL LIFE CYCLE ABSORPTION PENETRATION UNCOATING AND ECLIPSE SYNTHESIS OF VIRAL NUCLEIC ACID AND PROTEIN ASSEMBLY (maturation) RELEASE Dr.T.V.Rao MD

53. RECEPTORVIRUS ICAM-1polio CD4HIV acetylcholinerabies EGFvaccinia CR2/CD21Epstein- Barr HVEMherpes Sialic acidInfluenza, reo, corona

54. Virus Replication 1 Virus attachment and entry 1 2 Uncoating of virion2 3 Migration of genome nucleic acid to nucleus 3 4 Transcription 5 Genome replication 45 6 Translation of virus mRNAs 6 7 Virion assembly 7 8 Release of new virus particles 8 Dr.T.V.Rao MD54

55. 55 ADSORPTION TEMPERATURE INDEPENDENT REQUIRES VIRAL ATTACHMENT PROTEIN CELLULAR RECEPTORS Dr.T.V.Rao MD

56. 56 PENETRATION - ENVELOPED VIRUSES FUSION WITH PLASMA MEMBRANEFUSION WITH PLASMA MEMBRANE ENTRY VIA ENDOSOMES Dr.T.V.Rao MD

57. 57 PENETRATION herpesviruses, paramyxoviruses, HIV Dr.T.V.Rao MD

58. 58 PENETRATION - ENVELOPED VIRUSES FUSION WITH PLASMA MEMBRANE ENTRY VIA ENDOSOMES, FUSION WITH ACIDIC ENDOSOME MEMBRANE Dr.T.V.Rao MD

59. 59 Dr.T.V.Rao MD

60. 60 Dr.T.V.Rao MD

61. 61 VIRUS UPTAKE VIA ENDOSOMES CALLED – VIROPEXIS / ENDOCYTOSIS / PINOCYTOSIS Dr.T.V.Rao MD

62. 62 PENETRATION NON-ENVELOPED VIRUSES entry directly across plasma membrane: Dr.T.V.Rao MD

63. 63 Dr.T.V.Rao MD

64. Replicative cycle As obligate intracellular parasites, Virus must enter and replicate in living cells in order to “ reproduce ” themselves. This “ growth cycle ” involves specific attachment of virus, penetration and uncoating, nucleic acid transcription, protein synthesis, maturation and assembly of the virions and their subsequent release from the cell by budding or lysis Dr.T.V.Rao MD64

65. Dr.T.V.Rao MD65

66. Dr.T.V.Rao MD66

67. 67 UNCOATING NEED TO MAKE GENOME AVAILABLE ONCE UNCOATING OCCURS, ENTER ECLIPSE PHASE ECLIPSE PHASE LASTS UNTIL FIRST NEW VIRUS PARTICLE FORMED Dr.T.V.Rao MD

68. 68 SYNTHESIS OF VIRAL NUCLEIC ACID AND PROTEIN MANY STRATEGIES NUCLEIC ACID MAY BE MADE IN NUCLEUS OR CYTOPLASM PROTEIN SYNTHESIS IS ALWAYS IN THE CYTOPLASM Dr.T.V.Rao MD

69. 69 ASSEMBLY AND MATURATION NUCLEUS CYTOPLASM AT MEMBRANE Dr.T.V.Rao MD

70. 70 RELEASE LYSIS BUDDING THROUGH PLASMA MEMBRANE NOT EVERY RELEASED VIRION IS INFECTIOUS Dr.T.V.Rao MD

71. Transmission of Viruses Respiratory transmission – Influenza A virus Faecal-oral transmission – Enterovirus Blood-borne transmission – Hepatitis B virus Sexual Transmission – HIV Animal or insect vectors – Rabies virus Dr.T.V.Rao MD71

72. Viruses enter the body of the host in a variety of ways, for example... Dr.T.V.Rao MD72

73. The commonest forms of transmission are via... INHALED DROPLETS in sneezing of coughing for example the COMMON COLD or INFLUENZA VIRUSES. Dr.T.V.Rao MD73

74. or by... drinking water or eating raw food, for example, HEPATITIS A and POLIOVIRUS. Dr.T.V.Rao MD74

75. The commonest forms of transmission are also via... sexual intercourse for example HIV and HEPATITIS B and... Dr.T.V.Rao MD75

76. also... vertical transmission - from mother to baby for example HIV, HEPATITIS B and RUBELLA... Dr.T.V.Rao MD76

77. also... bites of vector arthropods such as mosquitoes for example YELLOW FEVER, RIFT VALLEY FEVER and DENGUE. Dr.T.V.Rao MD77

78. Most viral infections... do not lead to such serious complications and the host... Dr.T.V.Rao MD78

79. get well after a period of sickness to be immune for the rest of their lives. Examples are MEASLES INFECTION, RUBELLA or German measles, MUMPS and many others... Dr.T.V.Rao MD79

80. A bacteriophage A bacteriophage is any one of a number of viruses that infect bacteria. They do this by injecting genetic material, which they carry enclosed in an outer protein capsid. The genetic material can be ssRNA, dsRNA, ssDNA, or dsDNA ('ss-' or 'ds-' prefix denotes single- strand or double-strand) along with either circular or linear arrangement. Dr.T.V.Rao MD80

81. Structure of Bacteriophage Dr.T.V.Rao MD81

82. Classification of Bacteriophages The dsDNA tailed phages, or Caudovirales, account for 95% of all the phages reported in the scientific literature, and possibly make up the majority of phages on the planet. However, other phages occur abundantly in the biosphere, with different virions, genomes and lifestyles. Phages are classified by the International Committee on Taxonomy of Viruses (ICTV) according to morphology and nucleic acid. Dr.T.V.Rao MD82

83. Sub-viral agents Satellites – Contain nucleic acid – Depend on co-infection with a helper virus – May be encapsidated (satellite virus) – Mostly in plants, can be human e.g. hepatitis delta virus – If nucleic acid only = virusoid Viroids – Unencapsidated, small circular ssRNA molecules that replicate autonomously – Only in plants, e.g. potato spindle tuber viroid – Depend on host cell polII for replication, no protein or mRNA Prions – No nucleic acid – Infectious protein e.g. BSE Dr.T.V.Rao MD83

84. Viroids & Prions Viroids – ss RNA genome and the smallest known pathogens. – Affects plants Prions – Infectious particles that are entirely protein. – No nucleic acid – Highly heat resistant – Animal disease that affects nervous tissue – Affects nervous tissue and results in Bovine spongiform encepahltits (BSE) “mad cow disease”, scrapie in sheep kuru & Creutzfeld-Jakob Disease (CJD) in humans Dr.T.V.Rao MD84

85. Viroids Viroids are small (200-400nt), circular RNA molecules with a rod- like secondary structure which possess no capsid or envelope which are associated with certain plant diseases. Their replication strategy like that of viruses - they are obligate intracellular parasites.

86. Dependovirus /Virusoids Viroids are small (200-400nt), circular RNA molecules with a rod-like secondary structure which possess no capsid or envelope which are associated with certain plant diseases. Their replication strategy like that of viruses - they are obligate intracellular parasites.

87. (Prions) Prions are rather ill-defined infectious agents believed to consist of a single type of protein molecule with no nucleic acid component. Confusion arises from the fact that the prion protein & the gene which encodes it are also found in normal 'uninfected' cells. These agents are associated with diseases such as Creutzfeldt-Jakob disease in humans, scrapie in sheep & bovine spongiform encephalopathy (BSE) in cattle.

88. Programme Created by Dr.T.V.Rao MD for Medical and Paramedical Students in the Developing World Programme Created by Dr.T.V.Rao MD for Medical and Paramedical Students in the Developing World Email doctortvrao@gmail.com Dr.T.V.Rao MD88