Got all questions. Now answering all of them comprehensively: ─── MICROBIOLOGY PAPER II - COMPLETE ANSWERS (Apurva Shastri - Professional Exam) ─── IMMUNOLOGY INFECTION SN 1. Define: Carrier, Contact Carrier, Paradoxical Carrier, Convalescent Carrier i) Carrier: A person who harbors the pathogenic microorganism without showing overt clinical signs of disease, yet is capable of transmitting the infection to others. They serve as a reservoir of infection. ii) Contact Carrier (Healthy Carrier): A person who harbors the pathogen without ever having suffered from the disease. They acquire the organism through contact with a case/carrier but remain asymptomatic throughout. Example: Carriers of N. meningitidis in the nasopharynx (up to 10-15% of population), healthy carriers of S. typhi. iii) Paradoxical Carrier: A person who acquires the infection from a carrier, rather than from a clinical case of the disease. The source of infection is a carrier (not an overt case) - this is "paradoxical" because the carrier (who is not ill) infects another person. Example: A person who becomes a typhoid carrier after contact with another carrier. iv) Convalescent Carrier: A person who has recovered clinically from the disease but continues to harbor and shed the pathogen for a variable period. Example: Typhoid Mary (S. typhi in gallbladder), convalescent diphtheria carriers, S. typhi after enteric fever (chronic carrier if >1 year). ─── IMMUNITY SN 1. Innate Immunity - Mechanisms Definition: Non-specific, inborn resistance to infection; present from birth; does not require prior exposure to antigen; acts immediately. Mechanisms of Innate Immunity: 1. Physical/Anatomical Barriers: • Intact skin (keratin layer) - impermeable to most organisms • Mucous membranes - trap organisms in mucus • Mucociliary escalator (respiratory tract) - sweeps organisms upward • Cough, sneeze reflexes • Flushing action of urine, tears, saliva 2. Physiological/Biochemical Barriers: • pH: Gastric acid (pH 2) kills most ingested organisms; acid vaginal pH • Lysozyme: In tears, saliva, nasal secretions - cleaves NAM-NAG bonds in bacterial cell wall • Lactoferrin: Binds iron - bacteriostatic (deprives bacteria of iron) • Defensins: Antimicrobial peptides in neutrophil granules and epithelial cells • Complement (alternative pathway): Activated directly by bacterial surfaces • Interferons (IFN-α, IFN-β): Released by virus-infected cells - induce antiviral state in neighboring cells • Acute phase proteins: CRP, serum amyloid A, mannan-binding lectin (MBL) - opsonize bacteria • Temperature: Fever inhibits many pathogens; cold areas (skin surface 34°C) restrict some fungi 3. Cellular Mechanisms: • Phagocytes: ◦ Neutrophils (PMNs) - first responders; phagocytose and kill bacteria via oxidative burst (H2O2, superoxide, hypochlorite) and granule enzymes (myeloperoxidase, elastase, lactoferrin) ◦ Macrophages - tissue phagocytes; long-lived; process antigens; release cytokines (TNF, IL-1, IL-6, IL-12) ◦ Dendritic cells - bridge innate and adaptive immunity; professional antigen-presenting cells • Natural Killer (NK) cells: Lymphocytes that kill virus-infected cells and tumor cells without prior sensitization; recognize absence of MHC class I ("missing self") • Mast cells, Basophils, Eosinophils: Innate effectors in allergy and parasitic infections 4. Pattern Recognition Receptors (PRRs): • Toll-Like Receptors (TLRs): Recognize PAMPs (Pathogen-Associated Molecular Patterns) ◦ TLR4 recognizes LPS (Gram-negative endotoxin) ◦ TLR9 recognizes bacterial/viral DNA (CpG motifs) ◦ TLR3 recognizes double-stranded RNA (viral) • NOD receptors, RIG-I: Intracellular innate sensors ─── ANTIBODY - IMMUNOGLOBULIN SN 1. IgG - Structure and Function Structure of IgG: • Basic immunoglobulin structure: Two heavy chains (γ-chains) + two light chains (κ or λ), linked by disulfide bonds • MW: ~150,000 Daltons (7S) • Domains: Each heavy chain has 1 VH + 3 CH domains (CH1, CH2, CH3); each light chain has VL + CL • Fab fragment (Fragment antigen-binding): VH + VL + CH1 + CL - contains antigen-binding site • Fc fragment (Fragment crystallizable): CH2 + CH3 - mediates effector functions (complement activation, FcR binding) • Hinge region: Flexible region between CH1 and CH2 - allows Fab arm movement • Papain cleaves at hinge → 2 Fab + 1 Fc • Pepsin cleaves below hinge → F(ab')2 + pFc' • Subclasses: IgG1, IgG2, IgG3, IgG4 (differ in hinge length, complement activation) Functions of IgG: 1. Most abundant serum immunoglobulin (75-80% of total Ig) 2. Opsonization: Fc region binds Fc-γR on phagocytes → enhanced phagocytosis 3. Complement activation (classical pathway) via CH2 domain (IgG1, IgG2, IgG3) 4. Neutralization of toxins and viruses 5. ADCC (Antibody-Dependent Cell-Mediated Cytotoxicity) via NK cells 6. Placental transfer (only Ig that crosses placenta via FcRn) - provides passive immunity to neonate 7. Secondary immune response - main antibody produced in secondary/anamnestic response 8. Long half-life: ~23 days (longest of all Ig) ─── SN 2. IgM - Properties / Functions / Structure Structure of IgM: • Pentamer: 5 basic immunoglobulin units (each: 2 heavy μ-chains + 2 light chains) joined by J-chain (joining chain) and disulfide bonds • MW: ~900,000 Daltons (19S) • 10 antigen-binding sites (Fab fragments) - highest valency • J-chain: Glycoprotein that polymerizes the pentamer; also found in IgA Properties of IgM (enumerate): 1. First antibody produced in primary immune response (earliest marker of recent infection) 2. Largest immunoglobulin (pentameric, 19S, 900 kDa) 3. Confined to intravascular space (does NOT cross placenta or enter tissue fluids easily due to large size) 4. Most efficient complement activator (classical pathway - single molecule can activate C1q) 5. Best agglutinating antibody (10 binding sites - high valency) 6. Half-life: ~5 days (short) 7. Present on B-cell surface as monomer (BCR) 8. Important in early defense against bacteremia Functions: • Primary response antibody (IgM → IgG class switching) • Agglutination of bacteria, RBCs (blood group antibodies ABO are IgM) • Complement activation (most efficient) • Opsonization • Isohemagglutinins (anti-A, anti-B) are IgM ─── SN 3. Define/Classify Antibodies Definition: Antibodies (Immunoglobulins) are glycoprotein molecules produced by plasma cells (differentiated B lymphocytes) in response to antigenic stimulation, capable of specifically binding to the antigen that induced their formation. Classification: By class (isotype) - 5 classes based on heavy chain type: 1. IgG (γ chain) - 4 subclasses 2. IgA (α chain) - 2 subclasses 3. IgM (μ chain) 4. IgD (δ chain) 5. IgE (ε chain) By specificity: • Monoclonal (single clone, single specificity) vs. Polyclonal By origin: • Natural (isohemagglutinins) vs. Immune (after exposure) By function: • Agglutinins, precipitins, opsonins, antitoxins, neutralizing antibodies, complement-fixing antibodies ─── SN 4. IgA - Diagram Description Serum IgA: Monomer (MW 160 kDa, 7S); 2 heavy α-chains + 2 light chains Secretory IgA (SIgA): • Dimer: 2 IgA monomers joined by J-chain • Secretory component (SC): Poly-Ig receptor derived; protects SIgA from proteolytic digestion in mucosal secretions • Found in: Saliva, tears, colostrum, breast milk, respiratory/GI/GU secretions • First line of mucosal defense - prevents adherence of pathogens to epithelial cells ─── LAQ 1. Antibody - Immunoglobulins / IgG Structure and Function (Comprehensive - see SN 1 and SN 2 above) Five classes and subclasses: Class Heavy chain Subclasses Key feature IgG γ IgG1, IgG2, IgG3, IgG4 Most abundant; crosses placenta IgA α IgG1, IgA2 Mucosal immunity; SIgA IgM μ None Pentamer; 1st response IgD δ None B-cell surface receptor IgE ε None Allergy; antiparasitic ─── ANTIGEN-ANTIBODY REACTIONS SN 1. Agglutination vs. Precipitation - Differences with Examples Feature Agglutination Precipitation Antigen Particulate (cells, bacteria, inert particles coated with antigen) Soluble antigen Result Visible clumping/agglutination Visible precipitate (line, ring, turbidity) Mechanism Cross-linking of particulate antigens by antibody Lattice formation between soluble antigen and antibody at equivalence zone Sensitivity More sensitive Less sensitive Examples Widal test (S. typhi O/H agglutinins), ABO blood grouping, TPHA Ouchterlony double diffusion, Elek's test, Immunoelectrophoresis Two examples of Agglutination tests: Widal test, TPHA (Treponema pallidum hemagglutination) Two examples of Precipitation tests: VDRL (flocculation), Elek's immunodiffusion test ─── SN 2. ELISA with Applications ELISA (Enzyme-Linked Immunosorbent Assay): Principle: Antibody or antigen bound to solid phase (polystyrene plate well) captures corresponding antigen/antibody from test sample; detected by enzyme-conjugated antibody; enzyme substrate produces color proportional to concentration. Types: 1. Direct ELISA: Antigen coated on plate → enzyme-labeled antibody added directly → substrate → color 2. Indirect ELISA: Antigen on plate → primary antibody from patient → enzyme-labeled secondary antibody → substrate → color (detects patient antibody) 3. Sandwich ELISA: Capture antibody on plate → antigen → detection antibody (labeled) → substrate; most sensitive; for antigen detection 4. Competitive ELISA: Patient antigen competes with labeled antigen for antibody binding; inversely proportional signal Applications: 1. Serology: HIV diagnosis (anti-HIV antibody), HBsAg detection, Dengue NS1 antigen, anti-HCV, anti-Toxoplasma 2. Blood bank screening: HIV, HBV, HCV, syphilis in donated blood 3. Drug detection: Therapeutic drug monitoring, drugs of abuse 4. Hormone assays: Pregnancy test (hCG), thyroid hormones, insulin 5. Food testing: Allergens, pesticide residues, food adulteration 6. Research: Cytokine quantification, protein assays ─── SN 3. Agglutination Reactions - Enumerate / Widal Test / Tube Agglutination Types of Agglutination Reactions: 1. Direct (slide/tube) agglutination 2. Passive (indirect) agglutination - see SN 4 3. Reverse passive agglutination 4. Co-agglutination 5. Inhibition of agglutination (haemagglutination inhibition - HAI) 6. Antiglobulin (Coombs) test Widal Test: • Principle: Tube agglutination; detects agglutinating antibodies (O and H) against S. typhi in patient serum • Procedure: Serial doubling dilutions of patient serum (1:20 to 1:640+) mixed with standardized Salmonella antigen suspensions (TO, TH, AO, AH, BO, BH); incubate 37°C/24 hrs; read agglutination • Interpretation: O titre ≥1:80, H titre ≥1:160 (endemic area); fourfold rise in paired sera = diagnostic • O agglutination: Granular clumps (active infection); H agglutination: Large fluffy clumps (past infection/vaccination) Tube Agglutination - Principle: Patient serum diluted serially in tubes; antigen added; tube with highest dilution showing visible agglutination = titre. Advantage: quantitative; used for Brucella (SAT - standard agglutination test), Widal, Weil-Felix ─── SN 4. Passive Agglutination Tests Definition: Agglutination test in which soluble antigens are artificially attached ("passively") to carrier particles, which then agglutinate in the presence of specific antibody. Carrier particles used: • Sheep/human RBCs → Indirect Hemagglutination (IHA) • Latex beads → Latex Agglutination Test (LAT) • Bentonite, charcoal, Staphylococcus (protein A) → co-agglutination Examples: 1. TPHA/MHA-TP (syphilis) - T. pallidum antigen on sheep RBCs 2. RA Latex test - IgG on latex for Rheumatoid Factor detection 3. Latex agglutination for CSF antigens (meningococcal/pneumococcal meningitis) 4. RPR (Rapid Plasma Reagin) - cardiolipin on carbon particles 5. Wuchereria bancrofti - IHA for filarial antibodies ─── LAQ 1. Antigen-Antibody Reactions / Prozone Phenomenon / Agglutination General Features of Ag-Ab Reactions: 1. Specificity (key-lock complementarity) 2. Reversibility (non-covalent bonds: hydrogen, hydrophobic, van der Waals, ionic) 3. Proportionality (optimal ratio for visible reaction) 4. Two stages: Primary binding (rapid, invisible) → Secondary effect (visible: precipitation, agglutination) Prozone Phenomenon: • When antibody concentration is very high (excess) relative to antigen, each antigen molecule is saturated with antibody - no cross-linking - NO visible agglutination/precipitation • False-NEGATIVE result despite high antibody titre • Solution: Dilute the serum (test beyond prozone zone) • Seen in: Widal test, VDRL (undiluted serum in secondary syphilis), Brucella SAT Named Reactions: (See SN 1, 2, 3 above) ─── LAQ 2 & 4. Precipitation Reactions - Principle / Applications Precipitation Principle: • Soluble antigen + antibody → insoluble lattice network → visible precipitate • Requires optimal antigen-antibody ratio (equivalence zone); excess antibody (prozone) or antigen (postzone) gives no precipitate Types: 1. Ring (interface) test: Antigen layered over antibody in tube; white precipitate ring at interface 2. Ouchterlony (Double Diffusion in Agar): Both antigen and antibody diffuse toward each other in agar; precipitin line forms where they meet at equivalence; demonstrates: identity, partial identity, non-identity 3. Single Radial Immunodiffusion (SRID/Mancini): Antigen diffuses into antibody-containing agar; ring diameter² ∝ antigen concentration; used to quantify Ig levels 4. Immunoelectrophoresis: Serum proteins separated by electrophoresis, then precipitated with antiserum; identifies M-band in myeloma 5. Elek's test (immunodiffusion for toxin): See Paper I answers 6. Counterimmunoelectrophoresis (CIE): Electrophoresis drives antigen and antibody toward each other; faster than Ouchterlony; CSF antigen detection 7. Immunoturbidimetry/Nephelometry: Light scattering by immune complexes; used for quantification of CRP, complement, Ig ─── COMPLEMENT SYSTEM SN 1. Classical Pathway of Complement Activation: IgG or IgM antibody bound to antigen activates C1q Sequence: 1. Recognition: C1q binds Fc of IgM (1 molecule) or 2 IgG molecules → C1r and C1s activated → C1 complex 2. C1s cleaves C4 → C4a (anaphylatoxin) + C4b (binds to surface) 3. C4b + C2 → C1s cleaves C2 → C2a + C2b → C3 convertase (C4b2a) 4. C3 convertase cleaves C3 → C3a (anaphylatoxin, chemotaxis) + C3b (opsonin - binds CR1 on phagocytes; also joins C4b2a) 5. C5 convertase (C4b2a3b) cleaves C5 → C5a (most potent anaphylatoxin + chemotactic) + C5b 6. Terminal Complement (MAC): C5b + C6 + C7 + C8 + C9 (poly-C9) → Membrane Attack Complex (MAC) → pore in bacterial membrane → lysis Biological Effects of Complement: 1. Lysis of bacteria (MAC) 2. Opsonization (C3b, C4b bind to CR1 on phagocytes → enhanced phagocytosis) 3. Anaphylatoxins (C3a, C4a, C5a) → mast cell degranulation → histamine → vasodilation, increased permeability 4. Chemotaxis (C5a, C3a) → attracts neutrophils and monocytes to infection site 5. Immune complex solubilization (C3b prevents precipitation of immune complexes) 6. Enhancement of adaptive immunity (C3d on antigen binds CR2 on B cells → lowers activation threshold) SN 2. Complement Cascade and Biological Effects (See SN 1 above for full cascade) Additional - Alternative Pathway: • Spontaneous hydrolysis of C3 → C3(H2O) → factor B binds → factor D cleaves B → C3 convertase (C3bBb, stabilized by properdin) → amplification loop • Activated by: LPS, zymosan (fungal), cobra venom factor; does NOT require antibody Lectin Pathway: • MBL (Mannose-Binding Lectin) binds mannose on bacteria → MASP-1, MASP-2 activated → cleave C4 and C2 (like classical) ─── IMMUNE RESPONSE SN 1. Cell-Mediated Immunity (CMI) - Tests for Detection In Vivo Tests: 1. Tuberculin test (Mantoux test): 0.1 mL PPD intradermally; read induration at 48-72 hrs; tests CMI to M. tuberculosis 2. Lepromin test (Mitsuda): Tests CMI to M. leprae 3. Dinitrochlorobenzene (DNCB) test: Contact sensitization test for general CMI competence 4. Candida/Mumps/Trichophyton skin tests: Recall antigen tests for general CMI In Vitro Tests: 1. Lymphocyte Transformation Test (LTT) / Lymphocyte Blastogenesis: PBMCs + specific antigen → T cells proliferate → measured by ³H-thymidine incorporation or BrdU 2. Leukocyte Migration Inhibition Test (LMIT): T cells sensitized to antigen → release MIF (Migration Inhibitory Factor) → inhibits macrophage migration in capillary tube; positive = CMI present 3. Cytotoxic T Lymphocyte (CTL) assay: CD8+ T cells kill target cells; measured by ⁵¹Cr release 4. Flow cytometry: CD4/CD8 ratio, activation markers (CD25, CD69) 5. ELISPOT: Detects individual cytokine-secreting cells (IFN-γ); basis of QuantiFERON-TB Gold for latent TB 6. Interferon-gamma release assays (IGRA) ─── HYPERSENSITIVITY SN 1. Classify Hypersensitivity / Type III - Describe Gell and Coombs Classification: Type Name Mechanism Antibody/Cell Time Examples I Immediate/Anaphylactic IgE-mediated mast cell degranulation IgE Minutes Anaphylaxis, asthma, urticaria, hay fever II Cytotoxic/Antibody-mediated IgG/IgM + complement; ADCC against cell-surface antigens IgG, IgM Hours Hemolytic anemia, HDN, Goodpasture's, myasthenia gravis III Immune Complex-mediated IgG immune complexes deposited in tissues → complement activation → inflammation IgG (soluble immune complexes) 4-8 hrs SLE, post-streptococcal GN, serum sickness, Farmer's lung (Arthus reaction) IV Delayed-type/Cell-mediated T cell (CD4+ Th1, CD8+ CTL) mediated T cells (no antibody) 48-72 hrs TB, contact dermatitis, transplant rejection, Mantoux test Type III Hypersensitivity: • Mechanism: Soluble antigen-antibody complexes (immune complexes) form in antigen excess → not cleared by phagocytes → deposit in vessel walls, glomeruli, synovial membranes, choroid plexus • Complement activation → C3a, C5a → mast cell degranulation + neutrophil chemotaxis → neutrophils release lysosomal enzymes → tissue damage • Arthus Reaction (local Type III): Intradermal antigen injection in immunized individual → local immune complex deposition → edema, hemorrhage, necrosis (6-8 hrs) • Serum sickness (systemic Type III): After injection of foreign serum (heterologous antisera, e.g., antitetanus horse serum) → fever, urticaria, arthralgia, proteinuria (7-10 days) • Diseases: SLE (anti-dsDNA), Post-streptococcal GN, Rheumatoid arthritis, Hypersensitivity pneumonitis (Farmer's lung, Bird fancier's lung) ─── SN 2. Type IV Hypersensitivity Definition: Delayed-type hypersensitivity (DTH); cell-mediated; T-lymphocyte dependent; peaks at 48-72 hours after antigen exposure. Mechanism: 1. Sensitization: First exposure → APCs present antigen to CD4+ Th1 cells → Th1 cells sensitized (clonal expansion + memory) 2. Elicitation (Challenge): Re-exposure → Th1 cells recognize antigen-MHC II on APCs → release cytokines: IFN-γ (activates macrophages), TNF-β (lymphotoxin - cytotoxic), IL-2 (T cell proliferation), MIF (migration inhibitory factor) 3. Effector: Activated macrophages → release proteases, reactive oxygen species → tissue damage; granuloma formation in chronic DTH Examples: 1. Tuberculin reaction (Mantoux test) - prototype 2. Contact dermatitis (nickel, formaldehyde, poison ivy - urushiol) 3. Granulomatous hypersensitivity (TB, leprosy, sarcoidosis, schistosomiasis) 4. Transplant rejection (allograft) 5. Graft-versus-host disease ─── SN 3. Anaphylaxis Definition: Acute, severe, potentially fatal systemic Type I hypersensitivity reaction mediated by IgE and mast cell degranulation. Mechanism: 1. Sensitization: Antigen → IgE produced → IgE binds to FcεRI on mast cells and basophils (primed state) 2. Re-exposure: Antigen cross-links 2 IgE molecules on mast cell → immediate degranulation 3. Mediators released: Histamine, prostaglandins, leukotrienes (LTC4, LTD4), PAF, tryptase 4. Effects: Vasodilation, increased vascular permeability → oedema; bronchospasm; urticaria; cardiovascular collapse Common triggers: Penicillin, bee venom, peanuts, shellfish, latex, blood products Clinical features: Urticaria, angioedema, bronchospasm, hypotension, shock, cardiac arrest Treatment: Adrenaline (epinephrine) 0.5 mg IM (first line); antihistamines; corticosteroids; IV fluids; oxygen ─── LAQ 1 & 2. Hypersensitivity - Three Types of Immediate Hypersensitivity / Type I Three Types of Immediate Hypersensitivity (Types I, II, III): (See table in SN 1 above and detailed Type III description) Type I Hypersensitivity (IgE-mediated) in Detail: Mechanism: 1. Primary exposure: Antigen (allergen) processed by APCs → Th2 cells activated → IL-4, IL-5, IL-13 → B cells class switch to IgE 2. Sensitization: IgE binds to FcεRI on mast cells (skin, mucosa, lungs) and basophils - no symptoms yet 3. Re-exposure: Allergen cross-links adjacent IgE-FcεRI complexes on mast cell → signal transduction → Ca²⁺ influx → degranulation Mediators: • Preformed (immediate, 0-30 min): Histamine, heparin, tryptase, chemotactic factors ◦ Histamine: H1 → vasodilation, bronchospasm, itch; H2 → gastric acid • Newly synthesized (late, 4-6 hrs): Prostaglandin D2, Leukotriene C4, D4, E4 (SRS-A - slow-reacting substance of anaphylaxis); PAF ◦ LTC4/LTD4: Bronchospasm (1000x more potent than histamine), mucus secretion Clinical Manifestations: • Anaphylaxis (systemic, life-threatening) • Allergic asthma (bronchospasm) • Allergic rhinitis/hay fever (sneezing, rhinorrhea) • Urticaria and angioedema (skin) • Food allergy (peanuts, milk, eggs) • Atopic dermatitis (eczema) Diagnosis: Total IgE elevated; allergen-specific IgE (RAST/ImmunoCAP); skin prick test Treatment: Avoid allergen; antihistamines; beta-2 agonists (asthma); oral steroids; allergen immunotherapy (desensitization); anti-IgE (Omalizumab) ─── LAQ 3. Hypersensitivity - Classify / Pathogenesis / Type IV (See SN 1 and SN 2 above for complete detail) ─── AUTOIMMUNITY SN 1. Autoimmune Diseases - Four Features 1. Presence of autoantibodies or autoreactive T cells directed against self-antigens (e.g., anti-dsDNA in SLE, anti-acetylcholine receptor in myasthenia gravis) 2. Association with MHC (HLA) alleles - genetic predisposition (e.g., HLA-DR3/DR4 in Type 1 diabetes, HLA-B27 in ankylosing spondylitis) 3. Female predominance (most autoimmune diseases affect women more - hormonal influence) 4. Chronicity and remission-relapse pattern - autoimmune diseases tend to be lifelong with episodic flares LAQ 1. Autoimmunity - Define / Mechanisms Definition: Immune response (humoral or cellular) directed against self (autologous) antigens, causing tissue damage and disease. Mechanisms of Autoimmunity: 1. Release of sequestered antigens: Normally hidden antigens (lens protein, myelin, sperm) released by infection/trauma → treated as foreign → autoimmune response (sympathetic ophthalmia, orchitis) 1. Molecular mimicry: Microbial antigens structurally similar to self-antigens → antibodies/T cells cross-react with self tissues ◦ Example: Streptococcal M protein → cross-reacts with cardiac myosin → rheumatic fever ◦ Campylobacter jejuni ganglioside mimicry → Guillain-Barré syndrome 1. Polyclonal B cell activation: Some pathogens (EBV, LPS) activate B cells non-specifically → production of autoantibodies 1. Epitope spreading: Initial autoimmune response → tissue damage → release of more self-antigens → activation of additional autoreactive clones → self-perpetuating cycle 1. Loss of peripheral tolerance: ◦ Regulatory T cell (Treg) failure: FoxP3+ Tregs suppress autoreactive cells; deficiency → autoimmunity (IPEX syndrome) ◦ Defective Fas/FasL apoptosis: Autoreactive cells not deleted (ALPS - autoimmune lymphoproliferative syndrome) 1. Bystander activation: Inflammation near self-tissues activates APCs that present self-antigens → autoreactive T cells activated 1. Altered self-antigens: Drug-modified antigens (e.g., penicillin binding to red cell surface → autoimmune hemolysis), viral modification of self-proteins 1. Superantigen stimulation: Superantigens activate large numbers of T cells non-specifically → some autoreactive ─── VIROLOGY GENERAL PROPERTIES OF VIRUS SN 1. Egg Inoculation Method for Viral Culture Embryonated Hen's Egg: • 8-14 day old fertile hen's egg used • Contains 5 sites for inoculation Routes of Inoculation and Examples: Route Cavity/Membrane Example viruses Chorioallantoic membrane (CAM) Surface of CAM HSV (pocks), Vaccinia, Poxviruses Allantoic cavity Allantoic fluid Influenza, Mumps, Newcastle disease Amniotic cavity Amniotic fluid Influenza (primary isolation), Mumps Yolk sac Yolk sac Chlamydia, Rickettsia, Arboviruses, HSV Intravenous (vitelline vein) Blood Yellow fever virus Procedure (CAM inoculation as example): 1. Candle the egg (7-12 days) to identify air sac and embryo 2. Mark and make a small hole over air sac; second hole over CAM 3. Suction air → CAM drops → forms artificial air sac 4. Inoculate 0.1-0.2 mL virus suspension onto CAM through 2nd hole 5. Seal holes with paraffin; incubate at 37°C for 48-72 hrs 6. Observe pock lesions on CAM Uses of egg culture: • Virus isolation and propagation • Vaccine production (influenza vaccine - allantoic fluid) • Virus titration (pock counting) ─── SN 2. Methods of Detecting Viral Growth in Cell Cultures 1. Cytopathic Effect (CPE): • Most common method; observed by inverted microscope • Cells show: rounding, swelling, shrinkage, syncytia formation (giant multinucleated cells), cell lysis • Examples: HSV → rapid CPE (24-48 hrs); Adenovirus → "bunch of grapes" cluster; Respiratory syncytial virus (RSV) → syncytia; CMV → "owl eye" cells 2. Haemadsorption: • Viruses with haemagglutinin on infected cell surface adsorb guinea pig RBCs • Add RBCs to cell monolayer; wash; infected cells hold RBCs • Used for: Influenza, Parainfluenza, Mumps 3. Haemagglutination: • Virus in cell culture supernatant agglutinates RBCs in wells • Used for: Influenza, Mumps 4. Interference (Heterologous interference): • Non-cytopathic virus grows in cells → prevents CPE by cytopathic virus added later • Used for: Rubella (infects cells without CPE; detected by blocking CPE of challenge Echovirus) 5. Immunofluorescence (IF): • Fluorescent-labeled antibodies react with viral antigens in infected cells • Direct IF: Fluorescent antibody directly; Indirect IF: Unlabeled antibody + fluorescent anti-antibody • Used for: Rabies (negri bodies), RSV, CMV, HSV 6. Metabolic Inhibition / pH change: • Virus replication → cell death → no glucose metabolism → no acid production → medium stays red/alkaline • Used for: Enteroviruses, polioviruses 7. Transformation: • Oncogenic viruses transform cells → loss of contact inhibition → foci of piled-up cells visible ─── SN 3. Stages of Viral Multiplication (Replication Cycle) 1. Adsorption (Attachment): ◦ Viral surface protein binds to specific host cell receptor ◦ Example: HIV gp120 → CD4 + CCR5/CXCR4; Influenza HA → sialic acid; Rabies → acetylcholine receptor 1. Penetration (Entry): ◦ Receptor-mediated endocytosis (most viruses) ◦ Membrane fusion (enveloped viruses - HIV, Influenza) ◦ Direct injection (bacteriophages - only DNA injected) ◦ Viropexis (engulfment) 1. Uncoating (Eclipse phase): ◦ Viral capsid removed; nucleic acid released into cytoplasm ◦ Viral enzymes or lysosomal enzymes responsible 1. Biosynthesis (Replication): ◦ DNA viruses (most replicate in nucleus): DNA → mRNA → viral proteins; new DNA synthesized ◦ RNA viruses (replicate in cytoplasm): +ssRNA acts as mRNA; -ssRNA requires RNA-dependent RNA polymerase (RdRp); Retroviruses use reverse transcriptase (RNA → DNA → RNA) 1. Assembly (Maturation): ◦ Viral components assembled into new virions ◦ Capsid proteins surround nucleic acid 1. Release: ◦ Lysis (non-enveloped viruses) - cell bursts → virus released ◦ Budding (enveloped viruses - HIV, Influenza) - cell membrane wraps around nucleocapsid; cell may survive ◦ Exocytosis (some viruses) ─── SN 4. Embryonated Hen's Egg - Cross Section / Routes / Uses (See SN 1 above for routes and examples) Cross Section of Embryonated Egg (12-day): • Shell → Shell membrane → Air sac → Chorioallantoic membrane (CAM) → Allantoic cavity (allantoic fluid) → Amniotic cavity (amnion, amniotic fluid) → Embryo → Yolk sac Methods of Cultivation of Viruses: 1. Embryonated hen's egg 2. Cell (tissue) culture - most widely used today 3. Animal inoculation (laboratory animals - mice, guinea pigs, rabbits) ─── VIRUS-HOST INTERACTIONS SN 1. Negri Bodies • Definition: Intracytoplasmic inclusion bodies found in neurons infected with Rabies virus • Location: Hippocampal neurons (Ammon's horn/CA1-CA4), Purkinje cells of cerebellum • Nature: Aggregates of rabies viral nucleocapsids (RNP - ribonucleoprotein) within cytoplasm • Appearance: Eosinophilic (pink), round to oval, sharply defined, 2-10 µm; contain basophilic inner granules • Demonstration: ◦ Seller's stain (magenta/red Negri bodies against blue-grey neurons) - on brain impression smears ◦ H&E stain - eosinophilic intracytoplasmic inclusions ◦ Immunofluorescence (most sensitive and specific) • Diagnostic significance: Presence of Negri bodies in brain = pathognomonic of rabies • Note: Absent in ~20% of rabies cases; hence immunofluorescence is preferred ─── SN 2. Inclusion Bodies Definition: Abnormal intracellular structures (aggregates of viral particles, viral antigens, or cellular response products) visible by light microscopy in virus-infected cells. Two Intracytoplasmic Inclusion Bodies: 1. Negri bodies - Rabies virus; neurons; eosinophilic (Seller's stain) 2. Bollinger bodies - Fowlpox virus; cytoplasmic; contain Borrel bodies (individual virions) 3. Henderson-Patterson (Molluscum) bodies - Molluscum contagiosum virus; keratinocytes 4. Guarnieri bodies - Vaccinia/Variola virus; cytoplasmic Two Intranuclear Inclusion Bodies: 1. Cowdry type A - HSV, VZV, CMV, Yellow fever; eosinophilic, surrounded by clear halo, marginated chromatin ("owl eye" in CMV) 2. Cowdry type B - Poliovirus, Rabies (older classification); multiple small, no halo 3. Torres bodies - Yellow fever; intranuclear eosinophilic 4. Intranuclear inclusions of Adenovirus - basophilic, fill nucleus ("smudge cells") ─── VIRUSES SN 1. Hepatitis B - Laboratory Diagnosis Serological Markers (Timeline): Marker Significance When appears HBsAg Surface antigen; first marker; active infection 4-12 weeks Anti-HBc IgM Acute/recent infection 6-14 weeks HBeAg High infectivity, active replication With HBsAg HBV DNA Most sensitive marker of replication Early Anti-HBe Seroconversion; lower infectivity After HBeAg Anti-HBs Recovery; immunity (vaccination) >20 weeks Anti-HBc IgG Past exposure (lifelong) Persists Window period: HBsAg gone but anti-HBs not yet appeared → only Anti-HBc IgM positive Tests: 1. ELISA for HBsAg, Anti-HBc, HBeAg, Anti-HBs 2. HBV DNA by PCR (quantitative - viral load; qualitative for occult HBV) 3. Liver function tests (ALT, AST elevated) 4. Liver biopsy for histology, fibrosis staging 5. HBsAg by CLIA/RIA (blood bank screening) ─── SN 2. Rhabdoviruses - Immunoprophylaxis / Schedule / Non-neural Vaccines Rhabdoviruses = Rabies virus (Lyssavirus genus) Non-Neural (Modern) Vaccines: 1. HDCV (Human Diploid Cell Vaccine) - MRC-5 cells; gold standard; 1 mL IM 2. PCECV (Purified Chick Embryo Cell Vaccine) - Rabipur; 1 mL IM 3. PVRV (Purified Vero Rabies Vaccine) - Verobrab; 0.5 mL IM Post-Exposure Prophylaxis (PEP) Schedule: • Wound care (most important): Wash wound with soap and water 15 min + povidone iodine • RIG (Rabies Immune Globulin): Given on Day 0 only; infiltrate into wound; HRIG 20 IU/kg or ERIG 40 IU/kg • Vaccine (Essen schedule): Days 0, 3, 7, 14, 28 - 5 doses IM deltoid • Zagreb (2-1-1) schedule: Days 0(×2 sites), 7, 21 - 4 doses; WHO-approved; saves doses Pre-Exposure Prophylaxis (PrEP): Days 0, 7, 21/28 - 3 doses (for vets, lab workers, travelers) Intradermal (ID) regimen (cost-saving): 0.1 mL ID (vs 1.0 mL IM); requires trained staff; same schedule ─── SN 3. Influenza Virus and HIV - Diagrams (Described) Influenza Virus Structure: • Enveloped, segmented -ssRNA virus (8 segments) • Outer spikes: ◦ Haemagglutinin (HA) - triangular trimer; attaches to sialic acid receptors; fusion protein; vaccine target ◦ Neuraminidase (NA) - mushroom-shaped tetramer; cleaves sialic acid → allows release of new virions; target of oseltamivir/zanamivir • M2 ion channel in envelope (target of amantadine) • M1 protein (matrix protein) beneath envelope • Nucleoprotein (NP) around RNA segments • PB1, PB2, PA proteins (RNA polymerase) HIV Structure: • Enveloped, +ssRNA retrovirus (diploid - 2 copies of RNA) • Outer envelope: Lipid bilayer + gp120 (SU - surface unit; binds CD4) + gp41 (TM - transmembrane; mediates fusion) • gp120 + gp41 = gp160 (processed by furin) • Matrix (MA/p17): Between envelope and core • Core (capsid, p24): Conical; contains 2 RNA strands + RT + IN + PR • Enzymes: Reverse transcriptase (p66/p51), Integrase (p32), Protease (p11) • Genome: gag, pol, env + accessory genes (tat, rev, vif, vpr, vpu, nef) ─── SN 4. Type-A Influenza Virus - Antigenic Variations and Significance Two Types of Antigenic Variation: 1. Antigenic Drift (Minor variation): • Mechanism: Accumulation of point mutations in the genes for HA and NA due to error-prone RNA polymerase (no proofreading) • Effect: Gradual, stepwise change in antigenic structure of HA/NA; old antibodies partially effective • Result: Annual seasonal epidemics (partial immunity in population); new vaccine strain needed each year • Affects all influenza types (A, B) 2. Antigenic Shift (Major variation): • Mechanism: Reassortment of gene segments between human influenza virus and animal (avian/swine) influenza virus in a mixed-infected host (pig as "mixing vessel") • Effect: Sudden appearance of new HA and/or NA subtype (novel virus); no pre-existing immunity in population • Result: Pandemic (worldwide epidemic); high morbidity and mortality • Only affects Type A (has animal reservoirs); 18 HA and 11 NA subtypes known Significance of Antigenic Variation: • Necessitates annual influenza vaccine reformulation (WHO recommends strains twice yearly) • Basis of pandemics (1918 H1N1 "Spanish flu"; 1957 H2N2; 1968 H3N2; 2009 H1N1 "Swine flu") ─── SN 5. Merits and Demerits of Salk's and Sabin's Vaccines Feature Salk's Vaccine (IPV - Inactivated Polio Vaccine) Sabin's Vaccine (OPV - Oral Polio Vaccine) Type Killed/inactivated (formaldehyde) Live attenuated Route IM injection Oral (2 drops) Types covered All 3 serotypes All 3 serotypes (trivalent/bivalent) Immunity induced Humoral (IgG) - good; NO mucosal IgA Both humoral AND mucosal (sIgA in gut) Herd immunity Poor (no gut immunity; virus can still replicate in gut) Excellent - immunized person spreads virus to contacts (passive immunization of community) Stability Stable; no cold chain issues Requires cold chain (heat labile) VAPP risk None Vaccine-Associated Paralytic Poliomyelitis (VAPP) - 1 per 2.4 million doses Safety Safe in immunocompromised Contraindicated in immunocompromised Cost More expensive Cheaper; easier to administer Use today Used in polio-free countries (UK, USA) Used in endemic areas (India now switched to bOPV + IPV) ─── SN 6. Classify Herpesviridae / Subfamilies / Virus / Infection Subfamily Characteristics Members One infection Alphaherpesvirinae Fast growth; short cycle; latency in neurons HSV-1, HSV-2, VZV HSV-1 → oral herpes (cold sores); VZV → chickenpox Betaherpesvirinae Slow growth; large cells (cytomegaly); latency in glands, monocytes CMV, HHV-6, HHV-7 CMV → congenital CMV, retinitis in AIDS Gammaherpesvirinae Lymphotropic; latency in lymphocytes; oncogenic EBV (HHV-4), KSHV/HHV-8 EBV → Infectious mononucleosis; KSHV → Kaposi's sarcoma ─── SN 7. HIV - Pathogenesis / Opportunistic Infections / Lab Diagnosis / HIV Testing Strategies HIV Pathogenesis: 1. HIV gp120 binds CD4 on Th cells + CCR5 (macrophage-tropic, early) or CXCR4 (T-tropic, late) co-receptor 2. gp41 mediates membrane fusion → viral RNA enters cell 3. Reverse transcriptase (error-prone) → proviral DNA → integrase → integrated into host chromosome (provirus) - permanent 4. Provirus latent in resting CD4 cells; activated by NF-κB → viral replication → cell death 5. CD4 count progressively declines (normal 500-1500/µL) → immune deficiency 6. CD4 < 500: Symptomatic HIV; CD4 < 200: AIDS (opportunistic infections) 7. CD8 T cells initially control viremia; exhausted over time Two Opportunistic Infections in HIV/AIDS: 1. Pneumocystis jirovecii Pneumonia (PCP) - CD4 <200; bilateral interstitial pneumonia; "ground-glass" X-ray 2. Cryptococcal meningitis - CD4 <100; Cryptococcus neoformans; diagnosed by India ink + serum/CSF cryptococcal antigen 3. Others: CMV retinitis, MAC infection, Toxoplasma encephalitis, esophageal candidiasis, Kaposi's sarcoma Laboratory Diagnosis: 1. Screening (ELISA/CLIA): Detects anti-HIV antibodies ± p24 antigen (4th generation tests = combo test) 2. Confirmatory: Western blot (bands at gp41, gp120, p24 confirm) or Line Immunoassay (LIA) 3. CD4 count (flow cytometry) - staging, ART initiation, monitoring 4. HIV RNA viral load (RT-PCR/NASBA) - treatment monitoring, diagnosis in neonates 5. HIV p24 antigen - detected in window period before antibodies HIV Testing Strategies in India (NACO): Strategy When Method Strategy I Blood safety Single ELISA; if +ve, blood discarded Strategy II Surveillance (low prevalence)m 2 ELISAs; if discordant, 3rd test Strategy III Diagnosis in symptomatic individuals 3 sequential ELISAs with different antigens; 3 positives = HIV positive Window period: 2-8 weeks after infection; antibodies not yet detectable; diagnosed by p24 antigen or viral RNA PCR (NAT) ─── SN 8. Four Oncogenic Viruses 1. HPV (Human Papillomavirus) - types 16, 18 → Cervical cancer, oropharyngeal cancer; E6 (degrades p53), E7 (inactivates Rb) 2. HBV + HCV → Hepatocellular carcinoma (HCC) 3. EBV (Epstein-Barr Virus) → Burkitt's lymphoma (c-myc translocation), Nasopharyngeal carcinoma, Hodgkin's lymphoma, post-transplant lymphoma 4. KSHV/HHV-8 (Kaposi's Sarcoma Herpesvirus) → Kaposi's sarcoma (AIDS-defining) 5. HTLV-1 (Human T-cell Leukemia Virus type 1) → Adult T-cell leukemia/lymphoma (ATL) ─── SN 9. Epstein-Barr Virus (EBV) • Family: Herpesviridae, Gammaherpesvirinae (HHV-4) • Tropism: B lymphocytes (via CD21/CR2 receptor) and epithelial cells • Latency: Persists in memory B cells for life Diseases: 1. Infectious Mononucleosis (glandular fever, "kissing disease"): ◦ Fever, pharyngitis (exudative), lymphadenopathy (posterior cervical), splenomegaly, atypical lymphocytes (Downey cells - activated CD8 T cells) ◦ Monospot test (heterophile antibody test): Paul-Bunnell test; EBV-infected B cells produce heterophile antibodies that agglutinate sheep/horse RBCs ◦ Complications: Splenic rupture, airway obstruction, hemolytic anemia, Guillain-Barré 2. Burkitt's Lymphoma (endemic African type - jaw tumor in children; associated with malaria co-infection; t(8;14) c-myc tr

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