Viral Infections in Humans                     basics         Dr.T.V.Rao MD           Dr.T.V.Rao MD      1
Agents That Cause Disease.               PathogensViruses   Bacteria        Parasites   Fungi              Dr.T.V.Rao MD  ...
Source of infection:      Shedding virus           susceptible                   Man > 99%                   animals      ...
Routes of entry:                        Inhalation      ingestion                                 Bloodinoculation        ...
The Immune Response to   Infectious Disease        Dr.T.V.Rao MD    5
Important General Features of Immunity to              Pathogens.• Defense against pathogens is mediated by  both innate a...
Viruses• Obligatory intercellular  pathogens that replicate  within cells.• Use the nucleic acid and  protein synthetic  m...
Cell tropism determines disease:  Cell surface receptor:Virus         receptor              cell typeHIV           CD4    ...
Virus - Host• Virus effect Host can cause                No effect                Cell damage or Death   Polio cell death ...
How do viruses cause          disease?1. by damaging/killing cells outright2. by inducing immuno-pathology3. by transformi...
…. by damaging/killing cellse.g.Poliomyelitis• the virus is cytolytic• destroys motor neurons in the spinal  cord, and so ...
Humans react to Viral Infections             Dr.T.V.Rao MD     12
Newtons   3rd       Law works           Dr.T.V.Rao MD         13
Non Immunological            responses•   Phagocytosis•   Body Temperature.•   Hormones•   Malnutrition•   Age Young Old  ...
BARRIERS TO INFECTION• Inherent Barriers  The host has a number of barriers to infection that are  inherent to the organis...
Mucus• The mucus covering an  epithelium acts as a barrier to  prevent infection of host cells.  In some instances the muc...
Ciliated epithelium• The ciliated epithelium which drives the  mucociliary elevator can help diminish  infectivity of cert...
Herpesvirus • Classification:   – Herpesviridae (Family)   – Herpesvirus (Genus)   – Herpes simplex type 1 / type 2     (S...
Low pH• Low pH  The low pH of gastric  secretions inactivate  most viruses.  However,  enteroviruses are  resistant to gas...
The Immune System• The principal function of the immune  system is to protect the host against  pathogenic microbes.• Immu...
Components of Human Immune         System          Dr.T.V.Rao MD      21
Virus can cause Chromosomal            DamageChromosomal Injury damage Measles Mumps, Adenovirus,CMV Varicella virusDamage...
Pathogens & Disease• Pathogens are defined as microbes  capable of causing host damage.• When host damage reaches a certai...
Chronic Infections• Virus can be demonstrated at all times• Disease may or may not be present for  extended time periods o...
Evasion of Immune    Mechanisms by Viruses• Viruses can also  escape immune  attack by changing  their antigens.• A large ...
Inclusion Bodies• Inclusion bodies are  nuclear or cytoplasmic  aggregates of stainable  substances, usually  proteins. Th...
Inclusion Bodies       ( Elementary Bodies )• Inclusion bodies differ   size, shape, Location,  staining ,properties,  Som...
Negril Bodies    Dr.T.V.Rao MD   28
Staining the Virus in Inclusion            bodies• Giemsa staining can  produce Acidophilic /  Basophilic inclusions• Esin...
Pathogenesis of Viral Infection• In apparent ( Sub clinical )• Apparent     ( Clinical )  Acute Sub acute ChronicCan produ...
How Virus enter the Host• Through  Respiratory tract  Gastro Intestinal  tract  Skin,  Conjunctivae   By sex contact      ...
Respiratory tract• Small pox  Chicken pox• Influenza  Rhinovirus• Rhinovirus                Dr.T.V.Rao MD   32
Gastro Intestinal tract• Enterovirus,  Adenovirus• Reovirus• Hepatitis A E• Rota virus                  Dr.T.V.Rao MD   33
Skin• Papilloma virus• Cow pox• Molloscom  contagiosum• Animal Bite Rabies• Injections Hepatitis  B infections.           ...
Most common Viral Infections                   • Arbovirus                     Mosquito                     bite          ...
Other Routes of Entry• Conjunctiva  Adenovirus,• Genital tract  sexual contact –  HIV• Congenital  Infection  Rubella and ...
Spread of Virus• Spread from various  sources  Lymph nodes, Blood  stream, Reach target  organs.  Viremia locate to  vario...
Incubation period• May be short,           long           Variable.Depend on site of entry and  site of lesions.Common col...
How Host Responds• Non Specific• Immunity  Humoral• Cell  mediated.                 Dr.T.V.Rao MD   39
Specific Immune Response to Viruses• Mediated by a combination of humoral and cell  mediated immune mechanisms.  • Humoral...
Dr.T.V.Rao MD   41
Specific Immune Response to              Viruses• Cell-mediated  immune responses.     • Most important in host       defe...
CD8+ T and CD4+ T     Dr.T.V.Rao MD   43
Humoral Immunity• Immunoglobulin Ig G  Ig M Ig A• Ig A Mucosal  surface secretary  immunoglobulin• Antibodies  neutralize ...
HIV destroys CD 4 cells                 • Cell Mediated                   Immunity                   Delayed              ...
Evasion of Immune   Mechanisms by Viruses• Viruses have evolved  numerous  mechanisms for  evading host  immunity.• A numb...
Evasion of Immune    Mechanisms by Viruses• Viruses can also  escape immune  attack by changing  their antigens.• A large ...
HUMORAL COMPONENTS INVOLVED IN RESISTANCE TO VIRAL INFECTIONS• Nonspecific  A number of humoral  components of the  nonspe...
…. by inducing immunopathologyThe patient’s T cells attack and destroy virus-infected cells        Inflammation and cell d...
Interferon (IFN)             • Interferon (IFN)                 IFN was discovered over 40                 years ago by Is...
Types of Interferons• Alpha• Beta• Gamma            Dr.T.V.Rao MD   51
Interferons• There are three types of interferon, IFN-  alpha (also known as leukocyte  interferon), IFN-beta (also known ...
InterferonsIsaacs Linder MannInterferon protection against viral  infection Contain Host coded proteins  Produced by viral...
Biological effects of            Interferons• Antiviral effects.• Antimicrobial effects.• Cellular effect ts Inhibition of...
Interferons• Hum Interferon alpha leukocyte interferon• Produced by leukocytes 16 subspecies• Beta Interferon Inf β Produc...
Interferons• Interferons are species specific• RNA viral infections are better inducers  than DNA virus• Potent are   Toga...
Uses of Interferons• Molecular wt. 17,000• Used in Prophylaxis  and treatment• Non toxic and Non  antigenic• Anti cancer a...
Complement• Most viruses do not fix complement by the  alternative route. However, the interaction  of a complement-fixing...
Cytokines• Cytokines other than IFN also may play a  role in resistance to virus infection. Tumor  necrosis factor alpha (...
Specific• Antibody produce by the specific immune  system is involved primarily in the  recovery from viral infection and ...
• For example, IgA will be more important in  viruses that infect the mucosa while IgG  antibodies will be more important ...
Viral persistenceAcute                                    persistenttransient    1. Inadequate immune response    2. Strat...
Immunity Notsustanbale in Influenza Infection     Need Vaccination to New strains                   Dr.T.V.Rao MD         ...
Methods of StudyMuch more expensive and difficult to study animal viruses than bacteriophages• Cultivation in host  cells ...
Laboratory Diagnosis of    Viral Diseases Different from Bacterial Infections               Dr.T.V.Rao MD           65
Virological Tests    An Overview       Dr.T.V.Rao MD   66
Why Difficult• In the past Growth of Virus was not rapid.• Diagnosis becomes routine today due to  availability of Rapid m...
Diagnostic Methods in          Virology1. Direct Examination2. Indirect  Examination (Virus  Isolation)3. Serology        ...
Microscopy• Light Microscopy –  elementary bodies• Electron Microscopy  Rota viral detection• Florescent  Microscopy Direc...
Direct Examination1. Antigen Detection            immunofluorescence, ELISA etc.2. Electron Microscopy          morphology...
Indirect Examination1. Cell Culture        cytopathic effect (CPE)                       haemabsorption                   ...
Serology      Detection of rising titers of antibody between acute and      convalescent stages of infection, or the detec...
Virus IsolationCell Cultures are most widely used for virus isolation, there are 3types of cell cultures:   1. Primary cel...
Cell CulturesGrowing virus may produce   1. Cytopathic Effect (CPE) - such as the ballooning of cells or   syncytia format...
Detection of Viral antigen• Use of  Immunofluorescence Imunoelectrphoresis Radio immunoassay RIA ELISA                   D...
Serology• Since the isolation and identification of viruses is  not commonly done in the clinical laboratory, the  clinica...
Serology          • The development of            antibodies to different            components of the            virus is...
Serological Diagnosis• Detection of  Immunologlublins Ig  G. Ig M Ig A• Raise of titers Ist  sample later sample  (convale...
ELISA for HIV antibodyMicroplate ELISA for HIV antibody: coloured wells indicate reactivity                            Dr....
Western BlotHIV-1 Western Blot•   Lane1: Positive Control•   Lane 2: Negative Control•   Sample A: Negative•   Sample B: I...
Polymerase Chain Reaction• Advantages of PCR:      – Extremely high sensitivity, may detect down to one viral genome      ...
Schematic of PCR                 Dr.T.V.Rao MD                     82Each cycle doubles the copy number of the target
Growing Technologies• Molecular  methods• Probes• Polymerase  chain reaction  Can produce  Rapid Highly  scientific Specif...
Regular Methods in Use• Egg inoculation  Pox virus,  Influenza• Into tissue  culture               Dr.T.V.Rao MD   84
Advantages of Molecular          Methods• Increases  Sensitivity  and  Specificity.• PCR• RT PCR         Dr.T.V.Rao MD   85
Antiviral Agents• Restricted spectrum• No standardized in-vitro susceptibility tests• Most inhibit replication. Cure depen...
Nucleoside Analogues          General Mechanism of Action1. Taken up by cells2. Converted by viral and cellular enzymes to...
Ganciclovir• Mechanism like Acyclovir• Active against all Herpes viruses including CMV• Low oral bioavailability given I.V...
Anti-retroviral Agents• Zidovudine (AZT)• Cellular enzyme phosphorylate to the triphosphate form  which inhibits RT and ca...
Protease Inhibitors• Produce non-infectious particles or virions• Reduces the number of new rounds of infection  in suscep...
Drug ExamplesTamiflu-• Prevents the mature viruses  from leaving the cell• It is a neuraminidase  inhibitor, it works on b...
Other Drugs        Amantadine                          Interferons• Prevents uncoating (?)           • Antiviral, anticanc...
BEWARE VIRAL INFECTIONS  CAN INFECT ANY ONE         Dr.T.V.Rao MD   93
• Programme Created by Dr.T.V.Rao   MD for Medical and Paramedical             Graduates              • Email     • doctot...
Upcoming SlideShare
Loading in …5
×

Viral infections in humans, BASICS

14,253 views

Published on

Viral infections in humans BASICS

Published in: Health & Medicine, Technology

Viral infections in humans, BASICS

  1. 1. Viral Infections in Humans basics Dr.T.V.Rao MD Dr.T.V.Rao MD 1
  2. 2. Agents That Cause Disease. PathogensViruses Bacteria Parasites Fungi Dr.T.V.Rao MD 2
  3. 3. Source of infection: Shedding virus susceptible Man > 99% animals zoonosis
  4. 4. Routes of entry: Inhalation ingestion Bloodinoculation organ t/plant Congenital / vertical sexual
  5. 5. The Immune Response to Infectious Disease Dr.T.V.Rao MD 5
  6. 6. Important General Features of Immunity to Pathogens.• Defense against pathogens is mediated by both innate and specific immunity.• The innate immune response to pathogens plays an important role in determining the nature of the specific immune response.• The immune response is capable of responding in distinct and specialized ways to different pathogens in order to combat these infectious agents most effectively. Dr.T.V.Rao MD 6
  7. 7. Viruses• Obligatory intercellular pathogens that replicate within cells.• Use the nucleic acid and protein synthetic machineries of the host cell.• Infect a variety of cell populations by utilizing normal cell surface molecules as receptors to enter cell. Dr.T.V.Rao MD 7
  8. 8. Cell tropism determines disease: Cell surface receptor:Virus receptor cell typeHIV CD4 Th cellsEBV CR2 B cellsInfluenza sialic acid many cell typesRhinovirus ICAM-1 many cell typesPoliovirus poliovirus receptor neuronsMeasles CD46 many cell typesHHV6 CD46 many cell typesFactors required for virus replication
  9. 9. Virus - Host• Virus effect Host can cause No effect Cell damage or Death Polio cell death paralysis proliferation in Moll scumcontagiosum Malignant transformation oncogenic virus Dr.T.V.Rao MD 9
  10. 10. How do viruses cause disease?1. by damaging/killing cells outright2. by inducing immuno-pathology3. by transforming cells cancer
  11. 11. …. by damaging/killing cellse.g.Poliomyelitis• the virus is cytolytic• destroys motor neurons in the spinal cord, and so causes paralysis X-section of the spinal cord showing severe inflammation of anterior horn cells
  12. 12. Humans react to Viral Infections Dr.T.V.Rao MD 12
  13. 13. Newtons 3rd Law works Dr.T.V.Rao MD 13
  14. 14. Non Immunological responses• Phagocytosis• Body Temperature.• Hormones• Malnutrition• Age Young Old are more prone for viral Infections Dr.T.V.Rao MD 14
  15. 15. BARRIERS TO INFECTION• Inherent Barriers The host has a number of barriers to infection that are inherent to the organism. These represent the first line of defense which function to prevent or limit infection.• Skin The skin acts a formidable barrier to most viruses and only after this barrier is breached will viruses be able to infect the host.• Lack of Membrane Receptors Viruses gain entry into host cells by first binding to specific receptors on cells Dr.T.V.Rao MD 15
  16. 16. Mucus• The mucus covering an epithelium acts as a barrier to prevent infection of host cells. In some instances the mucus simply acts as a barrier but in other cases the mucus can prevent infection by competing with virus receptors on cells. For example, orthomyxo- and paramyxovirus families infect the host cells by binding to sialic acid receptors. Dr.T.V.Rao MD 16
  17. 17. Ciliated epithelium• The ciliated epithelium which drives the mucociliary elevator can help diminish infectivity of certain viruses. This system has been shown to be important in respiratory infections since, when the activity of this system is inhibited by drugs or infection, there is an increased infection rate with a given inoculum of virus. Dr.T.V.Rao MD 17
  18. 18. Herpesvirus • Classification: – Herpesviridae (Family) – Herpesvirus (Genus) – Herpes simplex type 1 / type 2 (Species) • Structure: – non-seg., lin., dsDNA, helical, env.
  19. 19. Low pH• Low pH The low pH of gastric secretions inactivate most viruses. However, enteroviruses are resistant to gastric secretions and thus can survive and replicate in the gut. Dr.T.V.Rao MD 19
  20. 20. The Immune System• The principal function of the immune system is to protect the host against pathogenic microbes.• Immunity may be innate or specific. Dr.T.V.Rao MD 20
  21. 21. Components of Human Immune System Dr.T.V.Rao MD 21
  22. 22. Virus can cause Chromosomal DamageChromosomal Injury damage Measles Mumps, Adenovirus,CMV Varicella virusDamage to chromosomes of the Host C 17 Dr.T.V.Rao MD 22
  23. 23. Pathogens & Disease• Pathogens are defined as microbes capable of causing host damage.• When host damage reaches a certain threshold, it can manifest itself as a disease. – The evolution of an infectious disease in an individual involves complex interactions between the pathogen and the host. Dr.T.V.Rao MD 23
  24. 24. Chronic Infections• Virus can be demonstrated at all times• Disease may or may not be present for extended time periods or show up late (carriers!)
  25. 25. Evasion of Immune Mechanisms by Viruses• Viruses can also escape immune attack by changing their antigens.• A large number of viruses evade the immune response by causing generalized immunosuppression. Dr.T.V.Rao MD 25
  26. 26. Inclusion Bodies• Inclusion bodies are nuclear or cytoplasmic aggregates of stainable substances, usually proteins. They typically represent sites of viral multiplication in a bacterium or a eukaryotic cell and usually consist of viral capsid proteins. Dr.T.V.Rao MD 26
  27. 27. Inclusion Bodies ( Elementary Bodies )• Inclusion bodies differ size, shape, Location, staining ,properties, Some are seen under microscopeIn Cytoplasm Pox virus Nucleus is affected Herpes virus Dr.T.V.Rao MD 27
  28. 28. Negril Bodies Dr.T.V.Rao MD 28
  29. 29. Staining the Virus in Inclusion bodies• Giemsa staining can produce Acidophilic / Basophilic inclusions• Esinophilic inclusions Negril bodies in brain cells in Rabies• Mollusca Bodies – moll scum contagiosum Dr.T.V.Rao MD 29
  30. 30. Pathogenesis of Viral Infection• In apparent ( Sub clinical )• Apparent ( Clinical ) Acute Sub acute ChronicCan produce latency Herpes zoster – virus in nerve toot ganglion, Kuru Human slow virus infection. Dr.T.V.Rao MD 30
  31. 31. How Virus enter the Host• Through Respiratory tract Gastro Intestinal tract Skin, Conjunctivae By sex contact Dr.T.V.Rao MD 31
  32. 32. Respiratory tract• Small pox Chicken pox• Influenza Rhinovirus• Rhinovirus Dr.T.V.Rao MD 32
  33. 33. Gastro Intestinal tract• Enterovirus, Adenovirus• Reovirus• Hepatitis A E• Rota virus Dr.T.V.Rao MD 33
  34. 34. Skin• Papilloma virus• Cow pox• Molloscom contagiosum• Animal Bite Rabies• Injections Hepatitis B infections. Dr.T.V.Rao MD 34
  35. 35. Most common Viral Infections • Arbovirus Mosquito bite Dengue Chikungunya Dr.T.V.Rao MD 35
  36. 36. Other Routes of Entry• Conjunctiva Adenovirus,• Genital tract sexual contact – HIV• Congenital Infection Rubella and CMV Dr.T.V.Rao MD 36
  37. 37. Spread of Virus• Spread from various sources Lymph nodes, Blood stream, Reach target organs. Viremia locate to various organs. Dr.T.V.Rao MD 37
  38. 38. Incubation period• May be short, long Variable.Depend on site of entry and site of lesions.Common cold very rapid onset.Chicken pox and Poliomyelitis 10 – 20 daysArbovirus 5 – 6 daysHepatitis B 2 – 6 monthsAIDS ? Dr.T.V.Rao MD 38Slow Virus many years
  39. 39. How Host Responds• Non Specific• Immunity Humoral• Cell mediated. Dr.T.V.Rao MD 39
  40. 40. Specific Immune Response to Viruses• Mediated by a combination of humoral and cell mediated immune mechanisms. • Humoral mediated immune response. • Antibodies specific for viral surface antigens are often crucial in containing the spread of a virus during acute infection and in protecting against re- infection. • Specific antibodies are important in defense against viruses early in the course of infection and in defense against cytopathic viruses that are liberated from lysed infected cells. Dr.T.V.Rao MD 40
  41. 41. Dr.T.V.Rao MD 41
  42. 42. Specific Immune Response to Viruses• Cell-mediated immune responses. • Most important in host defense, once a viral infection is established. • CD8+ Tc cells (Cytotoxic T lymphocytes; CTLs) and CD4+ th1 cells (helper T lymphocytes) are the main components of cell mediated antiviral defense. Dr.T.V.Rao MD 42
  43. 43. CD8+ T and CD4+ T Dr.T.V.Rao MD 43
  44. 44. Humoral Immunity• Immunoglobulin Ig G Ig M Ig A• Ig A Mucosal surface secretary immunoglobulin• Antibodies neutralize with help of complement. Dr.T.V.Rao MD 44
  45. 45. HIV destroys CD 4 cells • Cell Mediated Immunity Delayed Hypersensitivity • HIV destroys CD 4 Lymphocytes. Dr.T.V.Rao MD 45
  46. 46. Evasion of Immune Mechanisms by Viruses• Viruses have evolved numerous mechanisms for evading host immunity.• A number of viruses have strategies to evade complement- mediated destruction. Dr.T.V.Rao MD 46
  47. 47. Evasion of Immune Mechanisms by Viruses• Viruses can also escape immune attack by changing their antigens.• A large number of viruses evade the immune response by causing generalized immunosuppression. Dr.T.V.Rao MD 47
  48. 48. HUMORAL COMPONENTS INVOLVED IN RESISTANCE TO VIRAL INFECTIONS• Nonspecific A number of humoral components of the nonspecific immune system function in resistance to viral infection. Some of theses are constitutively present while others are induced by infection. Dr.T.V.Rao MD 48
  49. 49. …. by inducing immunopathologyThe patient’s T cells attack and destroy virus-infected cells Inflammation and cell death “Help!
  50. 50. Interferon (IFN) • Interferon (IFN) IFN was discovered over 40 years ago by Issacs and Lindemann who showed that supernatant fractions from virus-infected cells contained a protein that could confer resistance to infection to other cells. This substance did not act directly on the virus, rather it acted on the cells to make them resistant to infection (Figure 1). Dr.T.V.Rao MD 50
  51. 51. Types of Interferons• Alpha• Beta• Gamma Dr.T.V.Rao MD 51
  52. 52. Interferons• There are three types of interferon, IFN- alpha (also known as leukocyte interferon), IFN-beta (also known as fibroblast interferon) and IFN-gamma (also known as immune interferon). IFN-alpha and IFN-beta are also referred to as Type I interferon and IFN-gamma as Type II. There are approximately 20 subtypes of IFN-alpha but only one IFN-beta and IFN- gamma. Molecular wt. MD Dr.T.V.Rao 17,000 52
  53. 53. InterferonsIsaacs Linder MannInterferon protection against viral infection Contain Host coded proteins Produced by viral and Non viral inducer On exposure to Interferon cell produce translation inhibiting proteins TIP Inhibits translation of m RNA But no effect on cell mRNA Viral transcription inhibited . Dr.T.V.Rao MD 53
  54. 54. Biological effects of Interferons• Antiviral effects.• Antimicrobial effects.• Cellular effect ts Inhibition of cell growth and proliferation. and of DNA and protein synthesis Expansion MHC antigens• Immunoregulatory effects Enhanced cytotoxic activity of NK, K and T• Suppression of DTH. Dr.T.V.Rao MD 54
  55. 55. Interferons• Hum Interferon alpha leukocyte interferon• Produced by leukocytes 16 subspecies• Beta Interferon Inf β Produced by Fibroblast and epithelial cells.• Gamma Interferon Inf Ɣ produce by lymphocytes,• Produce Immuno modulation and ant proliferative function• Resist heating at 56 - 600 c for 30 – 6o mt Dr.T.V.Rao MD 55
  56. 56. Interferons• Interferons are species specific• RNA viral infections are better inducers than DNA virus• Potent are Toga viridae Vesicular stomatitis virus and Sendai virus. Production starts in > 1 hour and increases in 6 – 12 hours. Dr.T.V.Rao MD 56
  57. 57. Uses of Interferons• Molecular wt. 17,000• Used in Prophylaxis and treatment• Non toxic and Non antigenic• Anti cancer agent lymphomas• Used in Hepatitis B and C infections. Dr.T.V.Rao MD 57
  58. 58. Complement• Most viruses do not fix complement by the alternative route. However, the interaction of a complement-fixing antibody with a virus infected cell or with an enveloped virus can result in the lysis of the cell or virus. Thus, by interfacing with the specific immune system, complement also plays a role in resistance to viral infections. Dr.T.V.Rao MD 58
  59. 59. Cytokines• Cytokines other than IFN also may play a role in resistance to virus infection. Tumor necrosis factor alpha (TNF-α), interleukin- 1 (IL-1) and IL-6 have been shown to have antiviral activities in vitro. These cytokines are produced by activated macrophages but their contribution to resistance in vivo has not been fully elucidated. Dr.T.V.Rao MD 59
  60. 60. Specific• Antibody produce by the specific immune system is involved primarily in the recovery from viral infection and in resistance to subsequent challenge with the virus. IgG, IgM and IgA antibodies can all play a role in immunity to virus infection but the relative contributions of the different classes depends on the virus and the portal of entry. Dr.T.V.Rao MD 60
  61. 61. • For example, IgA will be more important in viruses that infect the mucosa while IgG antibodies will be more important in infections in which viremia is a prominent feature. Antibodies can have both beneficial and harmful effects for the host. Dr.T.V.Rao MD 61
  62. 62. Viral persistenceAcute persistenttransient 1. Inadequate immune response 2. Strategies Sanctuary sites latency brain skin Antibody Persistence enhancement Interference with antigen Antigenic variation presentation
  63. 63. Immunity Notsustanbale in Influenza Infection Need Vaccination to New strains Dr.T.V.Rao MD 63
  64. 64. Methods of StudyMuch more expensive and difficult to study animal viruses than bacteriophages• Cultivation in host cells – Living animal – Embryonated chicken eggs – Cell or tissue culture (= in vitro)
  65. 65. Laboratory Diagnosis of Viral Diseases Different from Bacterial Infections Dr.T.V.Rao MD 65
  66. 66. Virological Tests An Overview Dr.T.V.Rao MD 66
  67. 67. Why Difficult• In the past Growth of Virus was not rapid.• Diagnosis becomes routine today due to availability of Rapid method.• Important in HBV and HIV infections.• Rubella in pregnant women.• Simple methods – Microscopy and detection of inclusion bodies. Dr.T.V.Rao MD 67
  68. 68. Diagnostic Methods in Virology1. Direct Examination2. Indirect Examination (Virus Isolation)3. Serology Dr.T.V.Rao MD 68
  69. 69. Microscopy• Light Microscopy – elementary bodies• Electron Microscopy Rota viral detection• Florescent Microscopy Direct / Indirect Dr.T.V.Rao MD 69
  70. 70. Direct Examination1. Antigen Detection immunofluorescence, ELISA etc.2. Electron Microscopy morphology of virus particles immune electron microscopy3. Light Microscopy histological appearance inclusion bodies4. Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR) Dr.T.V.Rao MD 70
  71. 71. Indirect Examination1. Cell Culture cytopathic effect (CPE) haemabsorption immunofluorescence2. Eggs pocks on CAM haemagglutination inclusion bodies3. Animals disease or death Dr.T.V.Rao MD 71
  72. 72. Serology Detection of rising titers of antibody between acute and convalescent stages of infection, or the detection of IgM in primary infection.Classical Techniques Newer Techniques1. Complement fixation tests (CFT) 1. Radioimmunoassay (RIA)2. Haemagglutination inhibition tests 2. Enzyme linked immunosorbent assay (EIA)3. Immunofluorescence techniques (IF) 3. Particle agglutination4. Neutralization tests 4. Western Blot (WB)5. Counter-immunoelectrophoresis 5. RIBA, Line immunoassay Dr.T.V.Rao MD 72
  73. 73. Virus IsolationCell Cultures are most widely used for virus isolation, there are 3types of cell cultures: 1. Primary cells - Monkey Kidney 2. Semi-continuous cells - Human embryonic kidney and skin fibroblasts 3. Continuous cells - HeLa, Vero, Hep2, LLC-MK2, MDCKPrimary cell culture are widely acknowledged as the best cell culturesystems available since they support the widest range of viruses. However,they are very expensive and it is often difficult to obtain a reliable supply.Continuous cells are the most easy to handle but the range of virusessupported is often limited. Dr.T.V.Rao MD 73
  74. 74. Cell CulturesGrowing virus may produce 1. Cytopathic Effect (CPE) - such as the ballooning of cells or syncytia formation, may be specific or non-specific. 2. Haemabsorption - cells acquire the ability to stick to mammalian red blood cells.Confirmation of the identity of the virus may be carried out usingneutralization, haemabsorption-inhibition or immunofluorescencetests. Dr.T.V.Rao MD 74
  75. 75. Detection of Viral antigen• Use of Immunofluorescence Imunoelectrphoresis Radio immunoassay RIA ELISA Dr.T.V.Rao MD 75
  76. 76. Serology• Since the isolation and identification of viruses is not commonly done in the clinical laboratory, the clinical picture and serology plays a greater role in the diagnosis of viral disease. The major types of antibodies that are assayed for are neutralizing, haemagglutination inhibiting and complement fixing antibodies. Complement fixing antibodies follow the kinetics of IgM and are most useful in indicating a current or recent infection Dr.T.V.Rao MD 76
  77. 77. Serology • The development of antibodies to different components of the virus is used in staging the disease. For example in hepatitis B and HIV infections this approach is used. Dr.T.V.Rao MD 77
  78. 78. Serological Diagnosis• Detection of Immunologlublins Ig G. Ig M Ig A• Raise of titers Ist sample later sample (convalescent sample) tested after 10 – 14 days Raise of titer is diagnostic Dr.T.V.Rao MD 78
  79. 79. ELISA for HIV antibodyMicroplate ELISA for HIV antibody: coloured wells indicate reactivity Dr.T.V.Rao MD 79
  80. 80. Western BlotHIV-1 Western Blot• Lane1: Positive Control• Lane 2: Negative Control• Sample A: Negative• Sample B: Indeterminate• Sample C: Positive Dr.T.V.Rao MD 80
  81. 81. Polymerase Chain Reaction• Advantages of PCR: – Extremely high sensitivity, may detect down to one viral genome per sample volume – Easy to set up – Fast turnaround time• Disadvantages of PCR – Extremely liable to contamination – High degree of operator skill required – Not easy to set up a quantitative assay. – A positive result may be difficult to interpret, especially with latent viruses such as CMV, where any seropositive person will have virus present in their blood irrespective whether they have disease or not. Dr.T.V.Rao MD 81• .
  82. 82. Schematic of PCR Dr.T.V.Rao MD 82Each cycle doubles the copy number of the target
  83. 83. Growing Technologies• Molecular methods• Probes• Polymerase chain reaction Can produce Rapid Highly scientific Specific Dr.T.V.Rao MD 83
  84. 84. Regular Methods in Use• Egg inoculation Pox virus, Influenza• Into tissue culture Dr.T.V.Rao MD 84
  85. 85. Advantages of Molecular Methods• Increases Sensitivity and Specificity.• PCR• RT PCR Dr.T.V.Rao MD 85
  86. 86. Antiviral Agents• Restricted spectrum• No standardized in-vitro susceptibility tests• Most inhibit replication. Cure depends on host immune system to eradicate. If patients are immunocompromized, may have recurrences.• Many need to be activated by viral and cellular enzymes before exerting antiviral effect. Activity of enzymes and concentration of substrates will influence the efficacy. Dr.T.V.Rao MD 86
  87. 87. Nucleoside Analogues General Mechanism of Action1. Taken up by cells2. Converted by viral and cellular enzymes to the triphosphate form3. The triphosphate form inhibits: 1. DNA polymerase 2. Reverse transcriptase 3. RNA polymerase4. Or it may get incorporated into growing DNA leading to abnormal proteins or breakage. Dr.T.V.Rao MD 87
  88. 88. Ganciclovir• Mechanism like Acyclovir• Active against all Herpes viruses including CMV• Low oral bioavailability given I.V.• Most common adverse effect: bone marrow suppression (leukopenia 40%, thrombocytopenia (20%) and CNS effects (headache, behavioral, psychosis, coma, convulsions).• Drug of choice for CMV infections: retinitis, pneumonia, colitis… Dr.T.V.Rao MD 88
  89. 89. Anti-retroviral Agents• Zidovudine (AZT)• Cellular enzyme phosphorylate to the triphosphate form which inhibits RT and causes chain termination• Adverse effect: – Granulocytopenia and anemia: 45% in AIDS but 5% if asymptomatic HIV – Severe headache, nausea, insomnia, myalgia• ↓mortality & opportunistic infections, gain weight, better quality of life, delays signs and symptoms of AIDS Dr.T.V.Rao MD 89
  90. 90. Protease Inhibitors• Produce non-infectious particles or virions• Reduces the number of new rounds of infection in susceptible cells• To be effective must be prolonged, profound and constant.• Pharmacokinetics important to maintain constant concentrations within the effective range.• Metabolic adverse effects (DM, hyperglycemia) and GI (diarrhea, pain vomiting). Dr.T.V.Rao MD 90
  91. 91. Drug ExamplesTamiflu-• Prevents the mature viruses from leaving the cell• It is a neuraminidase inhibitor, it works on both influenza A and B• Neuraminidase is an enzyme found on the virus which cleaves sialic acid from cell membrane, leading to a more effective release of viruses. mechanism Dr.T.V.Rao MD
  92. 92. Other Drugs Amantadine Interferons• Prevents uncoating (?) • Antiviral, anticancer and &/or assembly immunomodulating• CNS Toxicity due to • Several sites of action in dopaminergic action viral cycle but mainly• Prophylaxis of Influenza A inhibit translation of viral during epidemics. proteins• If used within 48 hours may help cure Influenza • Toxicity: flu-like infection syndrome, BM• Rimantadine: analog with suppression; CNS less CNS toxicity • Hepatitis B and C Dr.T.V.Rao MD 92
  93. 93. BEWARE VIRAL INFECTIONS CAN INFECT ANY ONE Dr.T.V.Rao MD 93
  94. 94. • Programme Created by Dr.T.V.Rao MD for Medical and Paramedical Graduates • Email • doctotvrao@gmail.com Dr.T.V.Rao MD 94

×