Basics of Virology - Introduction, culture techniques.pptx

Basics of viruses

1. BASIC CONCEPTS OF VIROLOGY

2. Smallest unicellular organisms that are obligate intracellular. Viruses are the most primitive microorganisms infecting man.

3. CHARACTERISTICS OF VIRUSES Viruses do not have cellular organisation They range in size from 20–300 nm They contain only one type of nucleic acid They are obligate intracellular parasites They lack the enzymes necessary for protein and nucleic acid synthesis They multiply by a complex process and not by binary fission They are unaffected by antibacterial antibiotics

4. Important differentiating characteristics of virus and bacteria

5. MORPHOLOGY OF VIRUS 5 The entire virus particle called as virion , comprises of a nucleic acid (DNA or RNA) surrounded by a protein coat called as capsid , together known as the nucleocapsid . Some viruses also have an outer envelope.

6. Nucleic Acid 6 Viruses have only one type of nucleic acid, either DNA or RNA. They are classified as DNA viruses and RNA viruses. Nucleic acid - single or double stranded, circular or linear, segmented or unsegmented.

7. Nucleic Acid (Cont..) 7 Most DNA viruses possess dsDNA, except parvoviruses, which have ssDNA. RNA viruses possess ssRNA , except: Reoviruses (e.g. rotaviurs )–possess dsRNA Retrovirsues –possess two copies of ssRNA

8. Capsid 8 Composed of a number of repeated protein subunits (polypeptides) called capsomeres . Functions: Protects the nucleic acid core from the external environment In non-enveloped viruses - initiates the first step of viral replication. Antigenic and specific for each virus.

9. Symmetry 9 Based on arrangement of capsomeres : Type of symmetry Explanation Examples Icosahedral (cubical) symmetry Capsomeres are arranged as if they lay on the faces of an icosahedron 20 triangular facets and 12 corners or vertices Rigid structure. All DNA viruses (except poxviruses) Most of the RNA viruses have icosahedral symmetry

10. Symmetry (Cont..) 10 Based on arrangement of capsomeres : Type of symmetry Explanation Examples Helical symmetry Capsomeres are coiled surrounding the nucleic acid in the form of a helix or spiral. Flexible structure. RNA viruses such as- myxoviruses , rhabdoviruses , filoviruses, bunyaviruses , etc.

11. Symmetry (Cont..) 11 Based on arrangement of capsomeres : Type of symmetry Explanation Examples Complex symmetry Do not have either of the above symmetry. Poxviruses

12. Envelope 12 Envelope surrounding the nucleocapsid . Lipoprotein in nature. Lipid part is derived from host cell membrane Protein part is virus coded, made up of subunits called peplomers . Peplomers - project as spikes on the surface of the envelope.

13. Envelope (Cont..) 13 Most Viruses are Enveloped Except: Non-enveloped DNA viruses - parvovirus, adenovirus and papovavirus Non-enveloped RNA viruses - picornavirus, reovirus , calicivirus , hepatitis A virus and hepatitis E virus.

14. SIZES Relative sizes of representative viruses, bacteriophages (bacterial viruses) and bacteria, including Chlamydia Universities Press Pvt Ltd.

15. Shapes of the Viruses 15 Most of the animal viruses are roughly spherical with some exceptions: Rabies virus: Bullet shaped Ebola virus: Filamentous Poxvirus: Brick shaped Adenovirus: Space vehicle shaped Rotavirus: wheel shaped Tobacco mosaic virus: Rod shaped

16. VIROIDS Subviral agents Show an apparent absence of an extracellular dormant phase (virion) and a genome much smaller than those of known viruses Protein-free Low-molecular-weight RNA Resistant to heat and organic solvents but sensitive to nucleases Universities Press Pvt Ltd.

17. PRIONS Proteinaceous infectious particles Discovered by Stanley B Prusiner in 1982 May be responsible for chronic neurological degenerative diseases of humans, which result in fatal dementia Causative agents of scrapie, Kuru and Creutzfeldt–Jakob disease Lack any detectable nucleic acid Resistant to heat (90°C for three minutes), UV rays and nucleases S ensitive to proteases Universities Press Pvt Ltd.

18. RESISTANCE OF VIRUSES TO CHEMICAL AND PHYSICAL AGENTS Viruses are resistant to Temperature — very heat-labile For long-term storage, they are frozen at –70°C pH: Viruses vary greatly in their resistance to acidity Radiations: Viruses are inactivated by sunlight, UV rays and ionising radiation Disinfectants: In general, viruses are more resistant than bacteria to chemical disinfectants Most active antiviral disinfectants are oxidising agents such as hydrogen peroxide, potassium permanganate and hypochlorites Lipid solvents: Enveloped viruses are sensitive and naked viruses resistant to them Antibiotics: Absolutely ineffective Universities Press Pvt Ltd.

19. CLASSIFICATION OF VIRUSES DNA viruses 1 . Poxviridae family 2. Herpesviridae family 3. Adenoviridae family 4. Papovaviridae family 5 . Parvoviridae family 6. Hepadnaviridae family RNA viruses 1. Picornaviridae family 2. Orthomyxoviridae family 3. Paramyxoviridae family 4. Togaviridae family 5. Flaviviridae family 6. Bunyaviridae family 7. Arenaviridae family 8. Rhabdoviridae family 9. Reoviridae family 10. Coronaviridae family 11. Retroviridae (re = reverse; tr = transcriptase) family 12. Caliciviridae family 13. Filoviridae family Universities Press Pvt Ltd.

20. RESISTANCE OF VIRUSES TO CHEMICAL AND PHYSICAL AGENTS Viruses are resistant to Temperature — very heat-labile For long-term storage, they are frozen at –70°C pH: Viruses vary greatly in their resistance to acidity Radiations: Viruses are inactivated by sunlight, UV rays and ionising radiation Disinfectants: In general, viruses are more resistant than bacteria to chemical disinfectants Most active antiviral disinfectants are oxidising agents such as hydrogen peroxide, potassium permanganate and hypochlorites Lipid solvents: Enveloped viruses are sensitive and naked viruses resistant to them Antibiotics: Absolutely ineffective Universities Press Pvt Ltd.

21. REPLICATION OF VIRUSES Six sequential phases E arly events in replication: Attachment → Penetration → Uncoating Stages in the infection of a host cell and replication of a virus Universities Press Pvt Ltd.

22. REPLICATION OF VIRUSES I. Attachment and adsorption Host c ell surface should bear specific receptor sites to which the virus can gain attachment : C ell specificity D ifferences in susceptibility to viral infection are based on presence/absence of receptors on cells Influenza virus—glycoprotein receptor sites on the surface of the respiratory epithelium HIV—the CD4 receptor on host cells Polioviruses—the lipoprotein present on the surface of primate cells Universities Press Pvt Ltd.

23. REPLICATION OF VIRUSES II. Penetration Virus particles may be engulfed : P inocytosis Viral envelope may fuse with the plasma membrane of the host cell and release the nucleocapsid into the cytoplasm III . Uncoating Stripping the virus of its outer layers and capsid Nucleic acid is released into the cell The next phase of viral replication is gene expression and genome replication Universities Press Pvt Ltd.

24. REPLICATION OF VIRUSES IV . (a) Synthesis of DNA viruses Synthesis of viral nucleic acid and capsid protein, enzymes DNA viruses synthesise their nucleic acid in the host cell nucleus Exceptions to this are the poxviruses Replication of a DNA (herpes) virus Universities Press Pvt Ltd.

25. REPLICATION OF VIRUSES I V . (b) Synthesis of RNA viruses They synthesise all their components in the cytoplasm Exceptions are orthomyxoviruses, some paramyxoviruses and retroviruses Steps involved in viral genome biosynthesis 1) Early transcription of messenger RNA (mRNA) from the viral nucleic acid 2) Early translation of the mRNA into ‘early’ or ‘non-structural’ proteins 3) Replication of viral nucleic acid 4) Late synthesis of ‘late’ or structural proteins, which are the components of daughter virion capsids Replication of RNA viruses Universities Press Pvt Ltd.

26. REPLICATION OF VIRUSES REPLICATION MECHANISMS Class 1: In fully double-stranded DNA viruses DNA enters the host cell nucleus and uses the host cell enzymes for transcription Class 2: In single-stranded DNA viruses DNA molecule moves into the host cell nucleus and is converted into the duplex form Class 3: In reoviruses, the double-stranded RNA is transcribed to mRNA by viral polymerases Class 4: In positive-strand (plus strand, positive sense) RNA viruses, the viral RNA itself acts as the mRNA Viral RNA is infectious by itself and is translated directly into viral proteins in the host cell cytoplasm (e.g., picorna and togaviruses) Universities Press Pvt Ltd.

27. REPLICATION OF VIRUSES Class 5: In negative-strand (minus sense) RNA viruses (e.g., rhabdo , orthomyxo and paramyxoviridae ), the RNA is ‘antisense’, with polarity opposite to mRNA Such viruses possess their own RNA polymerases for mRNA transcription Class 6: Retroviridae — single-stranded RNA genome is converted into an RNA:DNA hybrid by the viral reverse transcriptase Double-stranded DNA is then synthesised from the RNA:DNA hybrid The double-stranded DNA form of the virus (provirus) is integrated into the host cell chromosome Universities Press Pvt Ltd.

28. REPLICATION OF VIRUSES V. Maturation Virion assembly Viral envelopes are derived from the host cell membrane Release: U sually occurs without cell lysis Eclipse phase From the stage of penetration until the appearance of mature progeny virions, the virus cannot be demonstrated inside the host cell Universities Press Pvt Ltd.

29. ABNORMAL REPLICATION CYCLES Daughter virions produced are not i nfective due to defective assembly: ‘incomplete viruses ’ The influenza virus — von Magnus phenomenon Viruses that are genetically deficient; incapable of producing infectious virions without the helper activity of another virus: defective viruses In some cells, virus infection does not lead to the production of infectious progeny: non-permissive cells Some viruses are genetically defective in that when they infect cells, they are unable to produce fully formed progeny E.g., Rous sarcoma virus (RSV), hepatitis D virus and adeno-associated satellite viruses (AAV) Universities Press Pvt Ltd.

30. VIRAL GENETICS The two main mechanisms for genetic modification in viruses are: Mutation Recombination Variations are due to gene product interactions MUTATION Occurs spontaneously or may be induced by mutagens, physical agents such as irradiation or chemical agents such as 5-fluorouracil Universities Press Pvt Ltd.

31. MUTATION Types of mutants Conditional lethal mutants Can grow under certain conditions (called permissive conditions ) Do not grow in certain other specified conditions, which are lethal (called non-permissive or restrictive conditions ) Ts or temperature-sensitive mutants Host-dependent mutants Universities Press Pvt Ltd.

32. GENETIC INTERACTIONS i ) Recombination Two different but related viruses infect a cell simultaneously They exchange segments of their nucleic acids so that a hybrid results ii) Reassortment Two different strains of the same virus are grown together; recombinants or reassortant viruses may be produced iii) Reactivation Universities Press Pvt Ltd.

33. VIRAL INTERACTIONS Phenotypic mixing Genotypic mixing Complementation Interference: T he infection of a cell by one virus inhibits the simultaneous or subsequent infection by another The most important mediator of interference is the interferon Viral interference has been applied in the field in controlling poliomyelitis outbreaks Universities Press Pvt Ltd.

34. PATHOGENESIS OF VIRAL INFECTIONS ROUTES OF ENTRY Inhalation (droplet infection—influenza, SARS- CoV ) Ingestion (hepatitis A, rotavirus) Direct transfer from an infected host Sexual contact (HIV, hepatitis C) Transplacental (rubella, HIV) Direct inoculation (bite of dogs—rabies, through sexual contact—HIV) Bites of vectors (arbovirus) Blood transfusion (hepatitis B and C, HIV) Organ-specific infections by some common viral agents Universities Press Pvt Ltd.

35. Cell tropism of human viruses 35 Cell Type Associated DNA Viruses RNA Viruses Lymphocytes Epstein-Barr virus Cytomegalovirus Hepatitis B JC virus, BK virus Mumps, Measles, Rubella, HIV Monocytes-macrophages Cytomegalovirus Poliovirus HIV Measles Neutrophils  - Influenza virus Red blood cells Parvovirus B19 Colorado tick fever virus None (free in plasma)  - Togavirus , Picornavirus

36. Virus Shedding 36 Stages Explanation Examples Portal of entry For those viruses that produce local infection. Influenza virus is shed in respiratory secretions Blood Viruses that spread through vector bite or blood transfusion or needle pricks Arboviruses, h epatitis B Near the target tissue /organ Skin, salivary gland and kidney Varicella zoster , mumps, cytomegalovirus No shedding Humans are the dead end for certain viruses infecting CNS, such as Rabies.

37. ACTION OF VIRUSES ON CELLS Shutdown of host protein and DNA synthesis Distort the cellular architecture and lead to autolysis Polykaryocytosis or syncytium formation Virus-coded antigens may appear on the surface of infected cells Inclusion bodies Histological feature of virus-infected cells is the presence of inclusion bodies Distinct size, shape, location and staining properties : D emonstrated in virus-infected cells with the help of light microscopy Universities Press Pvt Ltd.

38. PATHOGENESIS OF VIRAL INFECTIONS Types of infections Full-blown acute infection Persistent (chronic) infection Latent infection Virus–cell interactions 1) Cytocidal effect or lysis (cytolysis) 2) Cellular proliferation 3) Steady-state infection Fig. 4.8 Outcomes of viral infection Universities Press Pvt Ltd.

39. INCLUSION BODIES Crystalline aggregates of virions or made up of virus antigens present at the site of virus synthesis Confer altered staining properties on the cell Situated in the cytoplasm (poxviruses), nucleus (herpesviruses) or both (measles virus) Fig. 4.9 Negri bodies (intracytoplasmic inclusion bodies) in a case of rabies virus infection seen in the pyramidal cells of Ammon’s horn ( Source: PHIL, Image ID 3372/ CDC) Universities Press Pvt Ltd.

40. INCLUSION BODIES Table 4.3 Some viral inclusion bodies, their staining characteristics and sites in the cell Universities Press Pvt Ltd.

41. LABORATORY DIAGNOSIS OF VIRAL DISEASES 41

42. LABORATORY DIAGNOSIS OF VIRAL DISEASES 42 Laboratory diagnosis of viral infections is useful for the following purposes: To start antiviral drugs Screening of blood donors for HIV, hepatitis B and hepatitis C- Surveillance purpose For outbreak or epidemic investigation To start post-exposure prophylaxis

43. LABORATORY DIAGNOSIS OF VIRAL DISEASES (Cont..) 43 Direct Demonstration of Virus Electron microscopy Immunoelectron microscopy Fluorescent microscopy Light microscopy Detection of Viral Antigens By various formats such as ELISA, direct IF, ICT, flow through assays.

44. LABORATORY DIAGNOSIS OF VIRAL DISEASES (Cont..) 44 Detection of Specific Antibodies Conventional techniques such as HAI, neutralization test and CFT Newer diagnostic formats such as ELISA, ICT, flow through assays. Molecular Methods to Detect Viral Genes Nucleic acid probe—for detection of DNA or RNA by hybridization PCR—for DNA detection by amplification Reverse transcriptase-PCR—for RNA detection Real time PCR—for DNA quantification Real time RT-PCR—for RNA quantification.

45. LABORATORY DIAGNOSIS OF VIRAL DISEASES (Cont..) 45 Isolation of Virus by Animal inoculation Embryonated egg inoculation Tissue cultures: Organ culture, explant culture, cell line culture (primary, secondary and continuous cell lines).

46. Electron Microscopy 46 Specimens are negatively stained by potassium phosphotungstate and scanned under EM. Shape- Viruses can be identified based on their distinct appearances: Rabies- bullet shaped Rotavirus- wheel shaped Corona virus- petal shaped peplomers Adenovirus- space vehicle shaped

47. Electron Microscopy (Cont..) 47 Direct detection from specimens: For viruses that are difficult to cultivate, EM can be used as primary tool for diagnosis. As an alternative to tissue culture

48. Electron Microscopy (Cont..) 48 Virus detection from tissue culture Drawbacks: Highly expensive Has low sensitivity with a detection limit of at least 10 7 virions /ml. The specificity is also low.

49. Immuno-electron Microscopy 49 Sensitivity and specificity of EM can be improved by adding specific antibody to the specimen to aggregate the virus particles which can be centrifuged. Sediment is negatively stained and viewed under EM.

50. Fluorescent Microscopy 50 Direct immunofluorescence technique is employed to detect viral particles in the clinical samples.

51. Fluorescent Microscopy (Cont..) 51 Clinical applications - Diagnosis of rabies in skin biopsies, corneal smear of infected patients. Syndromic approach- Rapid diagnosis of respiratory infections caused by influenza, rhinoviruses, respiratory syncytial virus, adenoviruses and herpesviruses can be carried out by adding specific antibodies to each of these viruses Detection of adenovirus from conjunctival smears

52. Light Microscopy 52 Inclusion bodies: Histopathological staining of tissue sections may be useful for detection of inclusion bodies which helps in the diagnosis of certain viral infections.

53. Light Microscopy (Cont..) 53 Immunoperoxidase staining: Tissue sections or cells coated with viral antigens are stained using antibodies tagged with horse radish peroxidise following which hydrogen peroxide and a coloring agent ( benzidine derivative) are added. The color complex formed can be viewed under light microscope.

54. Detection of Viral Antigens 54 Various formats are available for detection of viral antigens in serum and other samples Some important antigen detection tests include: HBsAg and HBeAg antigen detection for hepatitis Binfection from serum. NS1 antigen detection for dengue virus infection from serum p24 antigen detection for HIV from serum Rotavirus antigen detection from diarrheic stool CMV specific pp65 antigen detection (serum)

55. Detection of Viral Antibodies 55 Antibody detection from serum is one of the most commonly used method in diagnostic virology. Appearance of IgM antibody or a four-fold rise of titer of IgG antibody indicates recent infection. Presence of IgG antibody (without a recent rise) indicates chronic or past infection.

56. Detection of Viral Antibodies (Cont..) 56 Newer diagnostic formats such as ELISA, ICT, flow through assays are widely used for antibody detection against most of the viral infections such as: Anti- HBc , Anti-HBs and Anti- HBe antibodies for Hepatitis B infection Anti-Hepatitis C antibodies Antibodies against HIV-1 and HIV-2 antigens from serum Anti-Dengue IgM/IgG antibodies from serum.

57. Molecular Methods 57 More sensitive, specific and yield quicker results than culture. Polymerase chain reaction (PCR) Reverse transcriptase-PCR (RT-PCR) Multiplex PCR formats BioFire FilmArray Real time-PCR ( rt -PCR)

58. Isolation of Virus 58 Viruses cannot be grown on artificial culture media. Cultivated by animal inoculation, egg inoculation or tissue cultures. Being labor intensive, technically demanding and time consuming, virus isolation is not routinely used in diagnostic virology. The specimen should be collected properly and immediately transported to the laboratory. Refrigeration is essential during transportation as most viruses are heat labile.

59. Isolation of Virus 59 Because of the ethical issues related to use of animals, animal inoculation is largely restricted only for research use. Research use- To study viral pathogenesis or viral oncogenesis or for viral vaccine trials Diagnostic use- Primary isolation of certain viruses which are difficult to cultivate otherwise; such as arboviruses and coxsackie viruses.

60. Egg inoculation 60 Embryonated eggs - first used for viral cultivation by Good pasture in 1931. Embryonated hen’s egg has four sites which are specific for the growth of certain viruses.

61. Egg inoculation (Cont..) 61 Yolk sac inoculation Amniotic sac Allantoic sac Chorioallantoic Membrane (CAM)

62. Tissue Culture 62 Types Explanation Examples Organ culture (obsolete ) Used for certain fastidious viruses that have affinity to specific organs. Tracheal ring culture for isolation of corona virus Explant culture (obsolete ) Fragments of minced tissue can be grown as 'explants’ Adenoid explants used for adenoviruses. Cell line culture This is the only isolation method which is in use now.

63. Preparation of the Cell Lines 63 Tissues digested – by treatment with proteolytic enzymes ( trypsinor collagenase) followed by mechanical shaking. Viral growth medium: Cells are then washed, counted, and suspended in viral growth medium containing balanced salt solution added with essential amino acids and vitamins, salts and glucose supplemented by 5-10% of fetal calf serum and antibiotics.

64. Preparation of the Cell Lines (Cont..) 64 Tissue culture flasks

65. Preparation of the Cell Lines (Cont..) 65 Incubation: Tissue culture flasks are incubated horizontally in presence of CO 2 , either as a stationery culture or as a roller drum culture. Rolling of the culture bottle in roller drums provides better aeration which is useful for isolation of fastidious viruses (e.g. rotavirus)

66. Types of Cell Lines 66 Type Explanation Examples Primary cell line Derived from normal cells. Freshly taken from the organs and cultured Capable of very limited growth in culture, maximum up to 5-10 divisions. aintain a diploid karyosome Useful for both primary isolation as well as growth of the viruses for vaccine production. Monkey kidney cell line- useful for isolation of myxoviruses , enteroviruses& adenoviruses Human amnion cell line Chick embryo cell line

67. Types of Cell Lines (Cont..) 67 Type Explanation Examples Secondary or diploid cell lines Can divide maximum up to 10-50 divisions before they undergo senescence (death). Also derived from the normal host cells. Maintain the diploid karyosome . Human fibroblast cell line- excellent for the recovery of cytomegalovirus. MRC-5 &WI-38 (human embryonic lung cell strain)

68. Types of Cell Lines (Cont..) 68 Type Explanation Examples Continuous cell lines Derived from cancerous cell lines, hence are immortal. They also possess altered haploid chromosome. Easy to maintain HeLa cell line (Human carcinoma of cervix cell line) Hep-2 cell line (Human epithelioma of larynx cell line)- widely used for respiratory syncytial viruses, adenoviruses and HSV KB cell line (Human carcinoma of nasopharynx cell line) McCoy cell line (Human synovial carcinoma cell line)- useful for isolation of viruses as well as Chlamydia Vero cell line ( Vervet monkey kidney cell line)-used for rabies vaccine production.

69. Types of Cell Lines (Cont..) 69 Human lung fibroblast cell line (Normal) HeLa cell line (normal, uninfected) Vero cell line (normal, uninfected) HEp-2 cell line (normal, uninfected)

70. Detection of Viral Growth in Cell Cultures 70 Two methods are used to detect the growth of the virus in cell cultures. Cytopathic Effect (CPE) Shell Vial Technique

71. Cytopathic Effect (CPE) 71 Defined as the morphological change produced by the virus in the cell line detected by light microscope. Type of Cytopathic effect (CPE) Virus Rapid crenation and degeneration of the entire cell sheet Enteroviruses Syncytium or multinucleated giant cellformation Measles, RSV, HSV Diffuse roundening and ballooning of the cell line HSV Cytoplasmic vacuolations SV 40 (Simian vacuolating virus-40) Large granular clumps resembling bunches of grapes Adenovirus

72. Shell Vial Technique 72 Involves centrifugation of cell culture (mixed with the specimen) to enhance the cell contact and viral replication, followed by Detection of early viral antigen in the infected cells by direct fluorescence technique.

73. TREATMENT OF VIRAL DISEASES 73

74. TREATMENT OF VIRAL DISEASES 74 Anti-Viral drugs Mechanism of action Active against Anti-herpesvirus drugs Acyclovir , Valacyclovir , Penciclovir Inhibit Viral DNA polymerase HSV1>HSV2>VZV&EBV Famciclovir Inhibit Viral DNA polymerase HSV,VZV,HBV Ganciclovir Inhibit Viral DNA polymerase CMV,EBV, HSV, VZV Cidofovir Inhibit Viral DNA polymerase HSV,CMV Foscarnet Inhibit Viral DNA polymerase HSV& CMV Fomivirsen Inhibit mRNA of CMV CMV(including resistant strains) Docosanol (topical) Inhibit the fusion of the human host cell with envelope of herpes virus HSV (recurrent herpes labialis ) Trifluridine (topical) Inhibits viral DNA polymerase Herpes keratitis (eye drops)

75. TREATMENT OF VIRAL DISEASES (Cont..) 75 Anti-Viral drugs Mechanism of action Active against Anti- Influenza virus drugs Oseltamivir, Zanamivir Neuraminidase Inhibitor H1N1flu Avian flu Seasonal flu Amantadine, Rimantadine Matrix protein inhibitor Seasonal flu

76. TREATMENT OF VIRAL DISEASES (Cont..) 76 Anti-Viral drugs Mechanism of action Active against Anti-Hepatitis drugs Telbivudine , tenofovir , lamivudine, adefovir , entecavir Nucleoside analogues Primarily for Hepatitis B Interferon alfa Indirectly inhibits viral protein synthesis For hepatitis B and C infection Grazoprevir Paritaprevir , Simeprevir NS3/4A inhibitors (proteases) For hepatitis C infection Dasabuvir , Sofosbuvir NS5B inhibitors (polymerases) For hepatitis C infection Daclatasvir , Ledipasvir , Velpatasvir NS5A inhibitors For hepatitis C infection Ribavirin Nucleoside inhibitor For For hepatitis C infection

77. IMMUNOPROPHYLAXIS FOR VIRAL DISEASES 77

78. Viral Vaccines (Active Immunization) 78 Since viral antigens are potent immunogens, viral vaccines confer prolonged and effective immunity. Vaccines for viral infections may be available either in live, killed or in subunit forms.

79. Killed Viral Vaccines 79 Preparation: By inactivating viruses with heat, phenol, formalin or beta propiolactone . Ultraviolet irradiation is not recommended because of the risk of multiplicity reactivation. E.g., Rabies vaccine . Advantages- They are more stable and are safely when given in immunodeficiency or in pregnancy. Disadvantages- Killed vaccines are associated with more adverse side effects due to reactogenicity , which can be reduced to some extent by purification of viruses.

80. Subunit Vaccines 80 Only a particular antigen of the virus is incorporated in the subunit vaccine. Preparation - DNA recombinant technology. The gene coding for the desired antigen is integrated into bacteria or yeast chromosome. Replication of the bacteria or yeast yields a large quantity of desired antigens, e.g. Hepatitis B vaccine Unlike killed vaccines, there is no local side effects associated with subunit vaccines

81. Live Vaccines 81 Preparation- Most of the live vaccines are prepared by attenuation by serial passages. (Exception is small pox vaccinewhere the naturally occurring vaccinia viruses were used for vaccination). Advantage- Live vaccines provide a stronger and long lasting immunity, mimickingimmunity produced after natural infection. They are administered as a single dose (except OPV). Disadvantages - Live vaccines are risky in immunodeficiency or pregnancy. They are less stable than killed vaccines

82. Passive Immunization (Immunoglobulin) 82 Passive immunization is indicated when an individual is immunodeficient or when an early protection is needed (i.e. for post-exposure prophylaxis). However, as there is no Passive immunization has no role in prevention of subsequent infections. Human immunoglobulins are available for many viral infections such as mumps, measles, hepatitis B, rabies and varicella-zoster.

83. Combined Immunization 83 Simultaneous administration of vaccine and immunoglobulin in post exposure prophylaxis is extremely useful. It is recommended for- Hepatitis B (neonates born to HBsAg positive mothers or for unvaccinated people following exposure ) Rabies (for exposures to severe class III bites)

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