Lecture 8: Introduction to Virology & Immunology PDF

Lecture 8 Introduction to clinical virology & Immunology Dr. Claire Atkinson  Learning Objectives Understand what is meant by viral pathogenesis Learn about some common viral infections Learn about laboratory techniques in clinical virology Understand...

Lecture 8 Introduction to clinical virology & Immunology Dr. Claire Atkinson  Learning Objectives Understand what is meant by viral pathogenesis Learn about some common viral infections Learn about laboratory techniques in clinical virology Understand the basic principles of Immunology In the context of infection  What is Clinical Virology? The study of viruses and their impact on human health; diagnosis, treatment and prevention of viral disease Essential for public health as it enables early detection of infectious disease, development of tests, therapies and vaccines Contributes to the understanding of viral evolution, transmission and emerging pathogens  Viral pathogenesis Host cells respond to viral infections in 3 ways: No effect – asymptomatic infection Cytopathic effect and cell death – morphological changes due to viral replication Loss of control of cell replication – oncogenic viruses In the host viruses can cause 3 patterns of infection No clinical disease Localised Disseminated Viral entry spread via the bloodstream lymph nodes spread to target (viraemia) organs  Diagnostic methods in virology In general tests can be grouped into 3 categories 1. Direct Detection 2. Indirect examination (virus isolation) 3. Serology They cannot be cultivated on artificial media  Direct Detection Electron Microscope – ID viruses based on morphology; size, shape Antigen detection-immunofluorescence Histological changes in infected Cells e.g.cytomegalic inclusion bodies ‘owls eyes’  Molecular Detection of viral genomes Technique will depend on DNA or RNA genome  Molecular Detection of viral genomes Technique will depend on DNA or RNA genome Cell culture (very rarely performed now) Diagnostic methods in virology Serology  Diagnostic methods in virology - serology Following viral exposure antibodies will start to appear in most patients 1st IgM 2nd IgG It is the indirect detection of these antibodies that is used routinely to diagnose viral infection  What is the Immune Response? Key terms Immunology – the study of the genetic, biological, chemical and physical characteristics of the immune system Immunity – The body’s ability to respond to the presence of any foreign substance, a person is deemed immunocompetent if it works correctly or immuno-supressed if part of the system can’t respond to challenges Antigen- any macromolecule that can elicit an immune response. The smallest part of an antigen that can bind to an antibody is called an epitope Antibody- a protective protein produced by the immune system in response to the presence of a foreign substance, called an antigen. Immunogen – any macromolecule that can stimulate the production of antibodies Allergen – any substance that causes an allergic reaction Autoantigen- self antigens targeted by the immune system in autoimmune disease Tumour antigen – present on tumour cells Brock Biology of Microorganisms, Global Editiony Michael T. Madigan; Kelly S. Bender; Daniel H. Buckley; W. Matthew Sattley; David What is the Immune Response? Brock Biology of Microorganisms, Global Editiony Michael T. Madigan; Kelly S. Bender; Daniel H. Buckley; W. Matthew Sattley; David  What is the Immune Response? It is important to note that we are born with our innate immune system almost fully developed. This is in contrast to our adaptive immune system which develops later and grows with us as we age and conquer new infections over time. Innate Immune responses Cells are non-specific Response = FAST (minutes/hours) No memory – always the same response  Innate Immunity 1st line defence Physical barriers prevent invasion of pathogens Skin – unfavourable environment/ antimicrobial Mucosal membranes- mucus traps and immobilises pathogens/ antimicrobial Tears- lysozymes can break down the cell wall of certain bacteria Saliva – antibodies and antimicrobial Blood-Brain barrier – tightly packed cells prevent most  Innate Immunity Cells of the innate immune systems – white blood cells Monocytes - circulating WBC that migrate to infected tissue  mature into macrophages Macrophages engulf and digest pathogens through phagocytosis Mast cells – activated by immune signals to release histamine  blood vessel dilate  inflammation & attract other immune cells to site of infection Neutrophils - most abundant phagocytic (like macrophages) Dendritic cells- Found in tissues in contact with external environment = they capture antigens  migrate to lymph nodes antigen presentation to T cells Natural Killer cells – non- specific cytotoxic lymphocytes Basophils & Eosinophils – granulocyte involved in  Innate Immunity – complement system Refers to a group of proteins called the complement proteins which are produced by the liver and flow freely in the blood until they ‘bump’ into a pathogen Act collectively to destroy pathogens, clear pathogens and remove dead or dying cells ‘they complement the work of antibodies’ Complement pathway is a signal cascade where 1 protein triggers a response in another, which triggers another and so on………. 9 proteins in total named C1-C9 3 pathways - Classic (discovered 1st) – triggered by antibody bound to antigen - alternative (discovered 2nd) – triggered by cell surface molecules - Lectin – triggered by mannose binding lectin (MBL on cell surface of pathogen)  Innate Immunity – complement system Refers to a group of proteins called the complement proteins which are produced by the liver and flow freely in the blood until they ‘bump’ into a pathogen Act collectively to destroy pathogens, clear pathogens and remove dead or dying cells ‘they complement the work of antibodies’ Complement pathway is a signal cascade where 1 protein triggers a response in another, which triggers another and so on………. 9 proteins in total named C1-C9 3 pathways - Classic (discovered 1st) – triggered by antibody bound to antigen - alternative (discovered 2nd) – triggered by cell surface molecules The goal is to form a membrane - Lectin – triggered by mannose binding lectin (MBL on attack complex (MAC) that cell surface of pathogen) compromises the pathogen's cell  Innate Immunity – other key proteins Toll-like receptors (TLRs) are an important family of transmembrane receptors that constitute the first line of defence system against microbes. They recognize specific ligands known as Pathogen Associated Molecular Patterns (PAMPS) and endogenous danger molecules released from dying cells and damaged tissues (Damage associated molecular patterns DAMPS) inflammation When TLRs are activated, they provide information about the type of pathogen and instruct lymphocytes to respond They play a key role in linking innate and adaptive immunity.  Cytokines Small proteins - Chemical mediators of the immune response (growth, activation and function) 1. Colony-Stimulating Factors (CSF) - cell development and differentiation. 2. Interferons – a group of signalling proteins produced in response to viral infections and other microbial threats. Play a crucial role in activating antiviral defences and enhance the immune response. They help neighbouring cells resist viral replication and limit the spread of infection. Type I interferons - antiviral immune responses, and type II interferon - antibacterial responses 3. Interleukins (IL) - activate or inhibit immune responses. 4. Chemokines - formed in the site of infection to attract immune cells. Different chemokines will recruit different immune cells to the site needed. 5. Tumour necrosis factor (TNF) - stimulate immune-cell proliferation and activation and critical in activating inflammatory responses - and as such, TNF blockers are used to treat a variety of disorders, including some autoimmune diseases.  Acute Inflammation The inflammatory reaction occurs is a protective mechanism that occurs after injury or when microbes enter damaged tissue. Skin is broken releasing PAMPS Bradykinin released from injured cells  pain Pain and DAMPS recruit mast cells and basophils which release histamine Bradykinin + histamine = capillary dilation redness and swelling allowing entry of leukocytes Leukocytes (monocytes and neutrophils)migrate to site of injury and release cytokines Neutrophils & monocytes phagocytose bacteria and cell debris Platelets start to repair damage The influx of blood and fluid  swelling, redness, pain and heat that is associated with inflammation  Acute vs Chronic Inflammation Chronic inflammation occurs when immune cells try to do their job of healing But ongoing stimulus results in more cells recruitment , increased Inflammation, changes to cells and can result in increased disease Autoimmune disease, neurological disease, rheumatoid arthritis What is the Immune Response? Brock Biology of Microorganisms, Global Editiony Michael T. Madigan; Kelly S. Bender; Daniel H. Buckley; W. Matthew Sattley; David  What is the Adaptive immunity? Adaptive immunity is extremely advantageous because it targets specific pathogens and can store a ‘memory’ of that pathogen for many years. This immune memory is the principle behind vaccination The most critical job of the immune system is to distinguish self from non-self Any macromolecule that is detected as non-self is called an antigen B cells develop in the bone marrow and are responsible for antigen interaction, production of antibodies and immune  Antibodies IgG One of the main functions of Major antibody in the circulation the adaptive immune system Can cross the placenta is to produce antibodies IgA They are proteins— In secretions—tears, saliva, etc. immunoglobulins (Ig) Provides local protection Produced by B-lymphocytes IgM Recognize and bind to foreign Largest of the Ig antigens IgD Form antigen-antibody high Surface of B cells (BCR) affinity complex Plays role in B-cell activation In circulation and specific receptors on B cells (BCR) IgE Implicated in allergic reactions e Igs—first response Brock Biology of Microorganisms, Global Editiony Michael T. Madigan; Kelly S. Bender; Daniel H. Buckley; W. Matthew Sattley; David A. Stahl 2022 Once an innate immune antigen presenting cell (APC) such as a macrophages engulfs a bacteria they travel to a lymph node and present an antigen from that pathogen on their cell surface to a T-cell They present the antigen using specific receptors on their cell surface called major histocompatibility complexes (MHC) T cells detect this unique antigen/receptor presentation via T-Cell receptors (TCRs) 2 major types of T cell - helper T cells (Th) –activate other cells including B cells - cytotoxic T cell – specialised killer cells that target infected or cancerous cells Both release cytokines when activated which induce the maturation of immature T  Although they rely on T cells for optimum function, B cells can be activated without help from T cells through B-cell receptors (BCRs) This is called T cell-independent activation and occurs when BCRs interact with T-independent antigens e.g., polysaccharide capsules, lipopolysaccharide. Because T cells are not involved, the second signal has to come from other sources, such as interactions of toll-like receptors with PAMPs or interactions with factors from the complement system. But ….it doesn’t generate strong immune response (no memory cells, IgM is the only antibody class produced, and the immunity doesn’t last long). Once activated, the B cell proliferates and differentiates into antibody-secreting  T Cell-Dependent Activation of B cells T cell-dependent activation of B cells is a process that occurs when a helper T cell activates a B cell to produce antibodies in response to an antigen: 1. Antigen presentation B cell absorbs an antigen and presents pieces of it on its surface using a major histocompatibility complex (MHC). 2. Helper T cell activation A CD4 T cell recognizes the antigen on the B cell and becomes activated. 3. Helper T cell activation of B cell The activated helper T cell activates the B cell through a cell-to-cell interaction. The helper T cell also secretes cytokines that further stimulate the B cell. 4. B cell activation The B cell multiplies into clones of immunoglobulin-secreting cells. These cells can become either effector B cells or memory B cells. 5. Immune response The actions of the helper T cells and B cells work together to create a specific and intense immune response. The immune response to infection  Primary and Secondary Antibody Response Primary immune response - Latent period—no specific antibodies present Recognition of antigen when the epitope fits into the antigen-binding site Cloning of B cells/ IgM Secondary immune response -Occurs when the system is exposed to the same immunogen after weeks, months, or years Antibody titer increases Class switch to IgG Brock Biology of Microorganisms, Global Editiony Michael T. Madigan; Kelly S. Bender; Daniel H. Buckley; W. Matthew Sattley; David  Immunity (Humoral): Active /Passive Naturally acquired active immunity Naturally acquired passive immunity Individual becomes ill with pathogens and Maternal immunoglobulins—IgG recovers Produced specific immunity to that antigen Long-term protection Artificially acquired passive immunity Artificially acquired active immunity Administration of exogenous Controlled, intentional immunity antibodies Vaccination Brock Biology of Microorganisms, Global Editiony Michael T. Madigan; Kelly S. Bender; Daniel H. Buckley; W. Matthew Sattley; David  Vaccination Attenuated microbe Living nonvirulent strains of a microorganism Protein fragments Killed or fragmented microbe DNA/RNA immunization Recombinant DNA—genes of viral antigen Toxoids Altered toxins (“nontoxic” toxins) injected —provide protection from toxin and not the antigen itself Brock Biology of Microorganisms, Global Editiony Michael T. Madigan; Kelly S. Bender; Daniel H. Buckley; W. Matthew Sattley; David A. Stahl 2022  Immune assays: timing Antibodies against the pathogen (blood) Antigen (pathogen or parts of it) Brock Biology of Microorganisms, Global Editiony Michael T. Madigan; Kelly S. Bender; Daniel H. Buckley; W. Matthew Sattley; David A. Stahl 2022  Antigen detection – agglutination test Brock Biology of Microorganisms, Global Editiony Michael T. Madigan; Kelly S. Bender; Daniel H. Buckley; W. Matthew Sattley; David A. Stahl 2022 Immune assays timing Immune assays –ELISA enzyme linked immunosorbent Assay  In clinical practice- what test should I use for HIV? Window period = time between infection and when the test can reliably detect infection In clinical practice- what test should I use for HIV? Questions Viruses can be cultivated on artificial media? True False What is an antigen? The study of the immune system Any substance that can cause an allergic reaction A chemical stimulus Any macromolecule that can elicit an immune response Innate immune responses are NOT? FAST Always the same Non specific Specific to an antigen Which is NOT an example of a physical barrier of the innate response? Blood brain barrier Skin Saliva fingernail Which is NOT a class of antibody IgA IgM IgE IgH B –cells attack non-self antigens…………… Inside the cell Outside the cell Antibodies are produced by T cells B cells Phagocytic cells Antigen presenting cells Complement is involved in Specific defence Non specific defence Both None of these Inflammation is the body’s natural reaction against injury and infection True False Questions BREAK  Case study – outbreak of D & V A young child aged 5 years and two neighbouring children become ill with D&V within 12 hours of each other. The 1st child complained of nausea, followed by vomiting and diarrhoea. A stool sample was given to the GP and was negative for the ‘usual bacterial pathogens.’ The two other children had similar symptoms, both had returned to school the following day but did not play together. All 3 children attended a birthday party the day before the sickness, where they shared food and ice cream. The birthday party was a t the local swimming pool and one parent had raised concerns about the cleanliness of the swimming pool. 1) What is the most likely cause of the D&V?  Case study – outbreak of D & V A young child aged 5 years and two neighbouring children become ill with D&V within 12 hours of each other. The 1st child complained of nausea, followed by vomiting and diarrhoea. A stool sample was given to the GP and was negative for the ‘usual bacterial pathogens.’ The two other children had similar symptoms, both had returned to school the following day but did not play together. All 3 children attended a birthday party the day before the sickness, where they shared food and ice cream. The birthday party was a t the local swimming pool and one parent had raised concerns about the cleanliness of the swimming pool. 1) What is the most likely cause of the D&V? Suspicion around food poisoning? Ice cream? However all the usual bacterial pathogens had been rules out (Salmonella, Shigella, E.Coli) Points to a viral cause  Case study – outbreak of D & V A young child aged 5 years and two neighbouring children become ill with D&V within 12 hours of each other. The 1st child complained of nausea, followed by vomiting and diarrhoea. A stool sample was given to the GP and was negative for the ‘usual bacterial pathogens.’ The two other children had similar symptoms, both had returned to school the following day but did not play together. All 3 children attended a birthday party the day before the sickness, where they shared food and ice cream. The birthday party was a t the local swimming pool and one parent had raised concerns about the cleanliness of the swimming pool. 2) What samples should be taken to find the likely cause?  Case study – outbreak of D & V A young child aged 5 years and two neighbouring children become ill with D&V within 12 hours of each other. The 1st child complained of nausea, followed by vomiting and diarrhoea. A stool sample was given to the GP and was negative for the ‘usual bacterial pathogens.’ The two other children had similar symptoms, both had returned to school the following day but did not play together. All 3 children attended a birthday party the day before the sickness, where they shared food and ice cream. The birthday party was a t the local swimming pool and one parent had raised concerns about the cleanliness of the swimming pool. 2) What samples should be taken to find the likely cause? Viral investigation of faeces should always be performed in a suspected outbreak Reverse- transcriptase real time PCR for norovirus, sapovirus, calicivirus, adenovirus, rotavirus  Case study – outbreak of D & V A young child aged 5 years and two neighbouring children become ill with D&V within 12 hours of each other. The 1st child complained of nausea, followed by vomiting and diarrhoea. A stool sample was given to the GP and was negative for the ‘usual bacterial pathogens.’ The two other children had similar symptoms, both had returned to school the following day but did not play together. All 3 children attended a birthday party the day before the sickness, where they shared food and ice cream. The birthday party was a t the local swimming pool and one parent had raised concerns about the cleanliness of the swimming pool. 2) What samples should be taken to find the likely cause? Viral investigation of faeces should always be performed in a suspected outbreak Reverse- transcriptase real time PCR fo r norovirus, sapovirus, calicivirus, adenovirus, rotavirus The causative agent was found to be Norovirus  Case study – outbreak of D & V HISTORY A 23 year-old male security guard working in a small department store was stabbed with a needle on a syringe of an intravenous drug addict he was arresting for shoplifting. Two months later he developed general malaise, with nausea and vomiting. He lost his appetite and began to lose weight. When he noticed darkening of the urine and a yellowish colour to his sclerae, he went to his GP. The patient denied a history of hepatitis or liver disease. He denied drug abuse, and had no history of blood transfusions, surgery or exposure to jaundiced individuals. He never travelled outside the US.  Case study 2 HISTORY A 23 year-old male security guard working in a small department store was stabbed with a needle on a syringe of an intravenous drug addict he was arresting for shoplifting. Two months later he developed general malaise, with nausea and vomiting. He lost his appetite and began to lose weight. When he noticed darkening of the urine and a yellowish colour to his sclerae, he went to his GP. The patient denied a history of hepatitis or liver disease. He denied drug abuse, and had no history of blood transfusions, surgery or exposure to jaundiced individuals. He never travelled outside the US. 1. The security guard developed hepatitis 2 months after the incident involving the hypodermic syringe (a) given the length of time that has elapsed, is it likely that the incident has anything to do with the disease? Since the incubation time for hepatitis A-E is from about 15 days to 180 days, respectively, therefore possible If one were to acquire hepatitis via a needle stick from the syringe of an intravenous drug addict, what kind(s) of hepatitis would be most likely? The most likely kinds for parenteral transmission would be hepatitis B, hepatitis C and possibly hepatitis D (which would require the presence of HBV). Hepatitis A is usually transmitted via the fecal-oral route; there is a viremia with HAV but it is transient (a chronic carrier state does not develop) and although there have been cases of parenteral transmission, they are very rare; so it seems very unlikely that the guard would have gotten HAV from the needle stick. 3. Laboratory serology tests showed: HAV Ab(total) Positive HAV Ab(IgM) Positive HBsAg Negative total anti-HBcAg Negative anti-HBsAg Positive HCVAb(total) Negative If one were to acquire hepatitis via a needle stick from the syringe of an intravenous drug addict, what kind(s) of hepatitis would be most likely? The most likely kinds for parenteral transmission would be hepatitis B, hepatitis C and possibly hepatitis D (which would require the presence of HBV). Hepatitis A is usually transmitted via the fecal-oral route; there is a viremia with HAV but it is transient (a chronic carrier state does not develop) and although there have been cases of parenteral transmission, they are very rare; so it seems very unlikely that the guard would have gotten HAV from the needle stick. 3. Laboratory serology tests showed: The guard is apparently suffering HAV Ab(total) Positive from HAV (by a process of elimination combined with the serology HAV Ab(IgM) Positive suggesting recent exposure to HAV). Since he has developed IgG HBsAg Negative antibodies, he is past the period of peak infectivity (furthermore, total anti-HBcAg Negative jaundice with HAV infections typically occurs when patients are past the anti-HBsAg Positive peak of viral shedding) but there may still be some virus in the faeces. HCVAb(total) Negative