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Dr.T.V.Rao MD 2The Immune Response to Infectious Disease: The Immune Response to Infectious Disease Dr.T.V.Rao MD 3 Important General Features of Immunity to Pathogens. : 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 4Viruses: 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 5 Virus - Host: Virus - Host Virus effect Host can cause No effect Cell damage or Death Polio cell death paralysis proliferation in Moll scum contagiosum Malignant transformation oncogenic virus Dr.T.V.Rao MD 6Humans react to Viral Infections: Humans react to Viral Infections Dr.T.V.Rao MD 7Newton's 3rd Law works: Newton's 3 rd Law works Dr.T.V.Rao MD 8Slide 9: Dr.T.V.Rao MD 9Non Immunological responses: Non Immunological responses Phagocytosis Body Temperature. Hormones Malnutrition Age Young Old are more prone for viral Infections Dr.T.V.Rao MD 10BARRIERS TO INFECTION: 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 11Mucus: 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. Sialic acid-containing glycoproteins in mucus can thus compete with the cell receptors and diminish or prevent binding of virus to the cells. Dr.T.V.Rao MD 12Ciliated epithelium : 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 13Low pH : 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 14The Immune System: 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 15Components of Human Immune System : Components of Human Immune System Dr.T.V.Rao MD 16Virus can cause Chromosomal Damage: Virus can cause Chromosomal Damage Chromosomal Injury damage Measles Mumps, Adenovirus,CMV Varicella virus Damage to chromosomes of the Host C 17 Dr.T.V.Rao MD 17Pathogens & Disease: 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 18Evasion of Immune Mechanisms by Viruses: 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 19Inclusion Bodies: 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 20Inclusion Bodies ( Elementary Bodies ): Inclusion Bodies ( Elementary Bodies ) Inclusion bodies differ size, shape, Location, staining ,properties, Some are seen under microscope In Cytoplasm Pox virus Nucleus is affected Herpes virus Dr.T.V.Rao MD 21Negril Bodies: Negril Bodies Dr.T.V.Rao MD 22Staining the Virus in Inclusion bodies : 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 23Pathogenesis of Viral Infection: Pathogenesis of Viral Infection In apparent ( Sub clinical ) Apparent ( Clinical ) Acute Sub acute Chronic Can produce latency Herpes zoster – virus in nerve toot ganglion, Kuru Human slow virus infection. Dr.T.V.Rao MD 24How Virus enter the Host: How Virus enter the Host Through Respiratory tract Gastro Intestinal tract Skin, Conjunctivae By sex contact Dr.T.V.Rao MD 25Respiratory tract: Respiratory tract Small pox Chicken pox Influenza Rhinovirus Rhinovirus Dr.T.V.Rao MD 26Gastro Intestinal tract: Gastro Intestinal tract Enterovirus, Adenovirus Reovirus Hepatitis A E Rota virus Dr.T.V.Rao MD 27Skin : Skin Papilloma virus Cow pox Molloscom contagiosum Animal Bite Rabies Injections Hepatitis B infections. Dr.T.V.Rao MD 28Most common Viral Infections: Most common Viral Infections Arbovirus Mosquito bite Dengue Chikungunya Dr.T.V.Rao MD 29Other Routes of Entry: Other Routes of Entry Conjunctiva Adenovirus, Genital tract sexual contact – HIV Congenital Infection Rubella and CMV Dr.T.V.Rao MD 30Spread of Virus: Spread of Virus Spread from various sources Lymph nodes, Blood stream, Reach target organs. Viremia locate to various organs. Dr.T.V.Rao MD 31Incubation period: 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 days Arbovirus 5 – 6 days Hepatitis B 2 – 6 months AIDS ? Slow Virus many years Dr.T.V.Rao MD 32How Host Responds: How Host Responds Non Specific Immunity Humoral Cell mediated. Dr.T.V.Rao MD 33Specific Immune Response to Viruses: 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 34Slide 35: Dr.T.V.Rao MD 35Specific Immune Response to Viruses: Specific Immune Response to Viruses Cell-mediated immune responses. Most important in host defense, once a viral infection is established. CD8 + T c cells (Cytotoxic T lymphocytes; CTLs) and CD4 + t h 1 cells (helper T lymphocytes) are the main components of cell mediated antiviral defense. Dr.T.V.Rao MD 36CD8+ T and CD4+ T: CD8 + T and CD4 + T Dr.T.V.Rao MD 37Humoral Immunity: 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 38HIV destroys CD 4 cells: HIV destroys CD 4 cells Cell Mediated Immunity Delayed Hypersensitivity HIV destroys CD 4 Lymphocytes . Dr.T.V.Rao MD 39Evasion of Immune Mechanisms by Viruses: 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 40Evasion of Immune Mechanisms by Viruses: 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 41HUMORAL COMPONENTS INVOLVED IN RESISTANCE TO VIRAL INFECTIONS: 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. 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 42Immunity Notsustanbale in Influenza Infection Need Vaccination to New strains: Immunity Notsustanbale in Influenza Infection Need Vaccination to New strains Dr.T.V.Rao MD 43Types of Interferon's: Types of Interferon's Alpha Beta Gamma Dr.T.V.Rao MD 44Interferon's: Interferon's 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. 17,000 Dr.T.V.Rao MD 45Interferon's: Interferon's Isaacs Linder Mann Interferon 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 46Biological effects of Interferon's: Biological effects of Interferon's 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 47Interferon's: Interferon's 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 - 60 0 c for 30 – 6o mt Dr.T.V.Rao MD 48Interferon's: Interferon's Interferon's 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 49Uses of Interferon's: Uses of Interferon's 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 50Complement : 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 51Cytokines: 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 52Specific : 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. 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 53Laboratory Diagnosis of Viral Diseases: Laboratory Diagnosis of Viral Diseases Different from Bacterial Infections Dr.T.V.Rao MD 54Virological Tests: Virological Tests An Overview Dr.T.V.Rao MD 55Why Difficult: 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 56Diagnostic Methods in Virology: Diagnostic Methods in Virology 1. Direct Examination 2. Indirect Examination (Virus Isolation) 3. Serology Dr.T.V.Rao MD 57Microscopy: Microscopy Light Microscopy – elementary bodies Electron Microscopy Rota viral detection Florescent Microscopy Direct / Indirect Dr.T.V.Rao MD 58Direct Examination: Direct Examination 1. Antigen Detection immunofluorescence, ELISA etc. 2. Electron Microscopy morphology of virus particles immune electron microscopy 3. Light Microscopy histological appearance inclusion bodies 4. Viral Genome Detection hybridization with specific nucleic acid probes polymerase chain reaction (PCR) Dr.T.V.Rao MD 59Indirect Examination: Indirect Examination 1. Cell Culture cytopathic effect (CPE) haemabsorption immunofluorescence 2. Eggs pocks on CAM haemagglutination inclusion bodies 3. Animals disease or death Dr.T.V.Rao MD 60Serology: Serology Detection of rising titers of antibody between acute and convalescent stages of infection, or the detection of IgM in primary infection. Dr.T.V.Rao MD 61Virus Isolation: Virus Isolation Cell Cultures are most widely used for virus isolation, there are 3 types 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, MDCK Primary cell culture are widely acknowledged as the best cell culture systems 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 viruses supported is often limited. Dr.T.V.Rao MD 62Cell Cultures: Cell Cultures Growing 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 using neutralization, haemabsorption-inhibition or immunofluorescence tests. Dr.T.V.Rao MD 63Detection of Viral antigen: Detection of Viral antigen Use of Immunofluorescence Imunoelectrphoresis Radio immunoassay RIA ELISA Dr.T.V.Rao MD 64Serology : 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 65Serology : 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 66Serological Diagnosis: 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 67ELISA for HIV antibody: ELISA for HIV antibody Microplate ELISA for HIV antibody: coloured wells indicate reactivity Dr.T.V.Rao MD 68Western Blot: Western Blot HIV-1 Western Blot Lane1: Positive Control Lane 2: Negative Control Sample A: Negative Sample B: Indeterminate Sample C: Positive Dr.T.V.Rao MD 69Polymerase Chain Reaction : 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 70Slide 71: Schematic of PCR Each cycle doubles the copy number of the target Dr.T.V.Rao MD 71Growing Technologies: Growing Technologies Molecular methods Probes Polymerase chain reaction Can produce Rapid Highly scientific Specific Dr.T.V.Rao MD 72Regular Methods in Use: Regular Methods in Use Egg inoculation Pox virus, Influenza Into tissue culture Dr.T.V.Rao MD 73Advantages of Molecular Methods: Advantages of Molecular Methods Increases Sensitivity and Specificity. PCR RT PCR Dr.T.V.Rao MD 74Antiviral Agents: 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 75Nucleoside Analogues General Mechanism of Action: Nucleoside Analogues General Mechanism of Action Taken up by cells Converted by viral and cellular enzymes to the triphosphate form The triphosphate form inhibits: DNA polymerase Reverse transcriptase RNA polymerase Or it may get incorporated into growing DNA leading to abnormal proteins or breakage. Dr.T.V.Rao MD 76Ganciclovir: 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). 1/3 of patients have to stop because of adverse effects Drug of choice for CMV infections: retinitis, pneumonia, colitis… Dr.T.V.Rao MD 77Anti-retroviral Agents: 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 78Protease Inhibitors: 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 79Drug Examples: Dr.T.V.Rao MD Drug Examples Tamiflu- Recently sold to 40 countries to battle avian flu 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. mechanismOther Drugs: Other Drugs Amantadine Prevents uncoating (?) &/or assembly CNS Toxicity due to dopaminergic action Prophylaxis of Influenza A during epidemics. If used within 48 hours may help cure Influenza infection Rimantadine: analog with less CNS toxicity Interferon's Antiviral, anticancer and immunomodulating Several sites of action in viral cycle but mainly inhibit translation of viral proteins Toxicity: flu-like syndrome, BM suppression; CNS Hepatitis B and C Dr.T.V.Rao MD 81Created by Dr.T.V.Rao MD for Basic Learning of Medical Virology email doctortvrao@gmail: Created by Dr.T.V.Rao MD for Basic Learning of Medical Virology email doctortvrao@gmail Dr.T.V.Rao MD 82 You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.