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BACTERIAL MORPHOLOGY - PROKARYOTES PORTIONSDETAILSTHE NUCLEOIDHave no true nucleus, nuclear membrane, & mitotic apparatus.Have nucleoid: Package of DNA in e cytoplasm.Consists of a single continuous circular molecule ranging in size from 0.58 to almost 10 million base pairs.E no. of nucleoid & chromosomes depend on e growth condition.Rapidly growing bacteria have > nucleoid per cell than slowly growing one.It is Feulgen-positive: indicates e presence of DNA (negatively charged).Nuclear region is filled with DNA fibrils.EXCEPTIONSPlanctomycetes: have nucleoid surrounded by a nuclear envelope with 2 membranes.Vibrio cholerae & Brucella melintesis: have 2 dissimilar chromosomes.Borrelia burgdorferi & Streptomyces coelicolor: have linear chromosome. CYTOPLASMIC STRUCTURESLack of autonomous plastids such as mitochondria & chloroplasts.E electron transport enzymes r localized in e cytoplasmic membrane.In photosynthetic bacteria: e photosynthetic pigments r located in intracytoplasmic membrane systems such as lamellae, chromatophores, or chlorosomes.In cynobacteria: have thylakoids & e major accessory pigments used for light harvesting r found on e surface of e thylakoid membranes. Storage of reserve material:Is in the form of insoluble granules which appear as refractile bodies in the cytoplasmFunction in e storage of energy or as a reservoir of structural building block.Most r bounded by a thin nonunit mebrane consisting lipids.Types of inclusion bodies of storage materials: Poly-β-hydroxybutyric acid (PHB): A lipid-like compund consisting of chains of β-hydroxybutyric acid units connected through ester linkages.Produced when e source of nitrogen, sulfur, or phosporus is limited while carbon is excess.Gylogen:Formed when carbon is excess.Used s carbon source when protein & nucleic aci synthes r resumed – same as PHB.Sulfur:Prokaryotes oxidize reduced sulfur compounds such as hydrogen sulfite & thiosulfate to produce intracellular granules of elemental sulfur.When e reduced sulfur is limiting, e granuled sulfur is oxidized to sulfate & e granule will slowly disappear.Phosphate:Inorganic phosphate is accumulated in e form of granules of polyphosphate.E granules can be used as sources of phosphate for nucleic acid & phospholipid synthesis to suport growth.E granules r also termed as volutin granules or metachromatic granules because they stain red with a blue dye. CO2 fixation:Autotrophic bacteria contain polyhedral bodies surrounded by a potein shell (carboxysomes).E bodies contain e key enzyme of CO2 fixation, ribulosebisphosphate carboxylase.Magnetosomes:Intracellular crystal particles of iron magnetite that allow certain aquatic bacteria to exhibit magnetotaxis.Surrounded by a nonunit membrane containing phospholipids, proteins, & glycoproteins. Gas vesicles:Provide buoyancy for aquatic bacteria.E mebrane is a 2 nm thick layer of protein, impermeable to water & solutes but permeable to gas.Cytoskeletal protein:Play cytoskeletal roles.Actin homologs (MreB & Mbl) help to determine ell shape, segregate chromosomes, & localize protein with e cell.Nonactin homologs (FtsZ) & bacterial cytoskeletal proteins (SecY & MinD) help to determine cell shape, regulate cell division, & segregate chromosomes. THE CELL ENVELOPEConsists of capsule, cell wall, cell membrane, flagella, & pili.Function to protect bacteria from hostile environments such as extreme osmolarity, harsh chemical, & antibiotic.THE CELL MEMBRANESTRUCTUREAlso called cytoplasmic membrane which is visible in electrom micrograph as thin posed of phospholipids & 200 kinds of proteins which make up 70% of the membrane mass.Have no sterols except mycoplasma which incorprate sterols into their membrane when growing in the sterol-containing media.At least 50% of e cytoplasmic membrane must be in semifluid state for cell growth to occur. At low temperature, this is done by incorpration of unsatured fatty acid into e phospholipids of e membrane.FUNCTIONSPermeability & Transport:Forma a hydrophobic barrier impermeable to most hydrophilic molecules which can only be transported through:Passive transport: relies on diffusion, uses no energy, & operates only when e solute is at higher concentration outside than inside e cell. The examples are simple diffusion, facilitated diffusion, & channel protein.Active transport: 2 types depending on e source of enegy employed:Ion-coupled transport: moves a molecule across e cell membrane across e cell membrane at e expense of a previously established ion gradient such as proton-motive or sodium-motive force. There r 3 basic types i.e. uniport, symport, & antiport.ABC transport: employs ATP directly to transport solutes into e cell. It is facilitated by specific binding proteins.Group translocation: e substrate is phosphorylated during e process, no concentration gradient is involved & it allows bacteria to utilize their energy sources by couling transport with metabolism. Special transport process: some bacteria secrete siderophores (compound that chelate Fe & promotes its transport as a soluble complex) such as hydroxamic acid.Electron Transport & Oxidative Phosphorylation:E cell membrane has a functional analog with e mitochondrial membrane.It contains cytochromes & other enzymes & e component of respiratory chain for oxidative phosphorylation.Excretion of Hydrolytic Exoenzymes & Pathogenicity Protein:E cell membrane excretes hydrolytic enzymes that degrade e polymers to subunits small enough to enetrate e cell membrane.Bacteria secrete them directly into e external medium or into e periplasmic space b/w e peptidoglycan layer & eouter membrane of e cell wall (gram –ve).Biosynthetic Function:E cell membrane is e site of e carrier lipid on which e subunits of e cell wall r assembled. E enzymes for cell wall biosynthesis & phospholipid synthesis are located here too.Chemotactic Systems:Attractants & repellents bind to specific receptors in e bacterial membrane.THE CELL WALLE functions:Gives osmotic protection.Helps in cell division.Serves as a primer for its own biosynthesis.Site of antigenic determinants.Exception to mycoplasma which r cell wall-lacking bacteria containing no peptidoglycan. PEPTIDOGLYCAN LAYERA complex polymer consists of:A backbone of alternating N –acetylglucosamine & N –acetylmuramic acid.Same in all species.A set of identical tetrapeptide side chain attached to e N –acetylmuramic acid.Vary from species to species.In all specis, there r features in common. Most hae L-alanine at position 1, D-glutamate at position 2, & D-alanine at position 4. Position 3 is e most variable one – Gram –ve bacteria have diamnopimelic acid (DAP) while Gram +ve bacteria have L-lysine. A set of identical peptide cross-bridges.Vary from species to species.In many Gram –ve cell wall, e cross bridge consists of a direct peptide linkage b/w DAP amino group of 1 side chain & e carboxyl group of e terminal D-alanine on e 2nd side chain. In Gram +ve bacteria, there r 40 sheets of peptidoglycan & on 1 or 2 sheets in Gram –ve bacteria.GRAM-POSITIVE BACTERIATechoic & Teichuronic Acids:Consists of glycerophosphate or ribitol phospate residues which r connected by phosphodiester linkages & usually have other sugars & D-alanine attached (to position 2 or 3 of glycerol or position 3 or 4 of ribitol).R –vely charged & partially responsible for e –ve charge of e cell surface.2 types i.e. wall techoic acid (WTA) which is covalently linked to peptidoglycan & membrane techoic acid or lipotechoic acid (LTA) whish is associated with lipids.Lie b/w e cytoplasmic membrane & e peptidoglycan layer, extending through pores in e peptidglycan layer.E repeat unit may be glycerols (joined by 1,3 or 1,2-linkages), ribitol (joined by 1,5-linkages), or complex consists of both joined to glucose, galactose, orN-acetylglucosamine.Functions:Make up a polyanionic network or matrix with peptidoglycan which provides functions relating to elsticity, porosity, tensile, strenghth, & electrostatic properties of e envelope.Constitue e major surface Ag of those Gram +ve species.Associated (LTA) with e M protein molecule forming microfibril that facilitate e attachment of S pyogenes to animal cells.Teichuronic acids: r similar polymer but e repeat units include sugar acids & r synthesized in place of techoic acids when phosphate is limiting. Polysaccharides:Consists of sugars such as mannose, arabinose, rhamnose, & glucosamines as well as acidic sugars such as glucuronic acid & mannuronic acid.Exist as subunits of polysaccharides in e cell wall.GRAM-NEGATIVE BACTERIAOuter Membrane:Bilayered in structure with inner leaflet resembles cell membrane while e outer leaflet contains LPS.Functions:Protection from deleterious substances such as bile salts by excluding hydrophbic molecules.Has channel protein called porins that permit passive diffusion of low MW hydrophilic compounds.Has a relatively high antibiotic resistance of Gram –ve bacteria.Participates in anchoring to peptidoglycan layer through OmpA protein.Acts as sex pilus receptor in F-mediated bacterial conjugation through OmpA protein.Contains proteins involved in e transport of specific molecules such as Vit. B12. Lipopolysaccharide (LPS):Consists of a complex glycolipid called Lipid A, attached to a polysaccharide & a terminal series of repeat units.Lipid A consists of phosphorylated glucosamine disaccharide units to which r attached long chains fatty acids (β-Hydroxymyristic is unique to e lipid).E polysaccharide core has LPS & includes ketodeoxyoctanoic acid (KDO) & a heptose.E repeat unit composed of linear trisaccharides & others & known as O Ag which functions to cover e bacterial surface & exclude hydrophobic compounds through its hydrophilic carbohydrate chain.Functions:E –vely charged LPS molecules r noncovalently cross-bridged by divalent cations to stabilize e membrane & provide a barrier to hydrophobic molecules.Has an important virulance factor (lipooligosaccharides- e sialylation of its N-acetyllactosamine causes molecular mimicry in e host).Lipoprotein:Cross-link e outer membrane & peptidoglycan layers.Contains 57 aa, representing repeats of a 15-aa-sequence.E lipid component consists of diglyceride thiother linked to a terminal cysteine which s noncovalently inserted in e outer membrane.Function: to stabilize e outer membrane & anchor it to peptidoglycan layers.Perisplasmic Space:Space b/w e inner & outer membrane which contains peptidoglycan layers & a gel-like solution of proteins.~20-40% of cell volume.E periplasmic proteins:Contains a high concentration of D-glucose which r variously substituted with glycerol phosphate & phosphatidylethanolamine residues or O-succinyl esters.These r called membrane-derived oligosaccharides which control osmoregulation.ACID-FAST CELL WALLComposed of:Peptidoglycan.An external asymmetrical lipid bilayer.E inner leaflet which contains mycolic acids (contain waxes) lnked to an arabinoglycan.E outer leaflet which contains other extractable lipids.Advantages:E highly ordered lipid embeded with proteins allows slow passage of nutrients & drugs by forming water-filled pores.E hydrophobic structure renders e bacteria resistant to many harsh chemicals like strong acid.Its permeability to hydrophilic molecules slows e bacterial grothw rate.Known as Acid-Fast because when introduced to a dye by heating or treatment with detergent, e dye can’t be removed by dilute HCL.E.g.: mycobacteria.CRYSTALLINE SURFACE LAYERMany both Gram +ve & -ve bacteria posses 2D crystalline subunit-type lyer lattice of protein or glycoprotein molecules (S-layer) as outermost component.S-layer:Composed of a single kind protein molecule which capable of self-assembly.Resistant to proteolytic enzymes & protein-denaturing agents.Protects e cell from wall-degrading enzymes & from e invasion of predatory bacerium. Maintains e cell shape.May involve in cell adhesion to host epidermal surface.CAPSULE & GLYCOCALYXGlycocalyx:Definition: e polysaccharide-containing material lyingoutside e cell.Formed from sucrose.Functions: resistant to desiccation & correlates dental carries with sucrose consumption in human.Capsule:Definition: a condensed, well-defined layer closely surrounding e cell that excludes particles.Functions in e:Invasiveness of pathogeinc bacteria.Protection from phagocytosis.Adherence to bacteria to surface of their environment. Slime layer: glycocalyx which is loosely associated with e cell & does not exclude particles.FLAGELLASTRUCTUREThread-like appendages composed of protein, 12 – 30 nm in an of locomotion, 3 types of arrangement:Peritrichous: flagella all around e body of e bacteria, e.g. Salmonella typhi.Amphitrichous: a single flagellum at each pole.Lophotrichous: bunch of flagella at one or both ends, e.g. Spirilium minus.Monotrichous: 1 flagellum at 1 end, e.g. V. cholerae.Building blocks: thousands molecules of a protein subunit called flagellin.E flagellum is formed by e aggregration of subunits to form a helical structureR highly antigenic: H Ag.Structure for attachment: a complex composed of a hook (short curved structure acts as universal joint b/w motor & flagellum) & basal body (bears a set of rings, i.e. one pair in Gram +ve & two pairs in Gram –ve). MOTILITYR semirigid helical rotors to which e cell imparts a spining movement.Flagellar motor & its components r located in e cell envelope.PILI (FIMBRIAE)R rigid surfce appendages.Shorter & finer than posed of structural protein subunits termed pilins.Minor protein called adhesins r located at e tips of e pili & r responsible for attachment properties.2 types:Ordinary pili: adherence of symbiotic & pathognic bacteria to host cells.Sex pili: attachment of donor & recipients cell in bacterial conjugation.Pilin molecules r arranged helically to form a straight cylinder that does not rotate & lacks a complete basal body.Pili grow from inside of e cell outward.ENDOSPOREOVERVIEWSPORULATIONPROPERTIESGERMINATIONTHE STAPHYLOCOCCIPORTIONDETAILSSPECIESS. aureus: ubiquitous, ant. nares (50% – 70% of healthy person), skin, mucous membrane of URT & GIT, hospital.S. epidermis: skin, gut, & URT.S. saprophyticus: decaying plant & animal, urinary bladder, & lower urinary tract of young female.PROPERTIESGram-positive spherical cells.Arranged in grape-like irregular clusters.Grow readily on many types of media.Active metabolically: fermenting carbohydrates & producting pigments (white to deep yellow) & lactic acids.Coagulase test: S. aureus – positive, others – negative.E pathogenic staphylococci:Hemolyze blood.Coagulate plasma.Produce various extracellular enzymes & toxin.Rapidly develop resistance to antimicrobial agents, drying, & heat.MORPHOLOGYSpherical cells about 1 ?m in diameter arranged in irregular cluster.Liquid cultures: single, pairs, tetrads, & chains cocci.Young cocci stain strongly Gram positive, on aging: many become Gram negative.Nonmotile & do not form spores.Lysis under influence of drugs like penicillin. CULTURE & COLONYGrow readily on aerobic or microaerophilic condition & most rapidly at 37oC.Colonies:On solid media: round, smooth, raised, & glistening.S. aureus: gray to deep golden yellow colonies, creamy growth, causes discoloration, & hemolyzes blood. S. epidermidis: gray to white colonies.S. saprophyticus: grey or white colonies.Pigment production:Form pigment best at room temperature (20 – 25 oC).Develop pigment only prolonged incubation.No pigment is produced anaerobically or in broth.GROWTHResistance to antimicrobial drugs:Resistance to penicillin:Due to e production of β-lactamase production.Under control of plasmids which r transmited by trasduction & perhaps also by conjugation. Resistance to nafcillin:Independent of β-lactamase roduction.Resistance is due to mecA gene which encodes a low affinity penicillin binding proteins.Susceptibility to vancomyocin:Susceptible if MIC is < 2 ?g/mL.Intermediate susceptibility if MIC is 4 – 8 ?g/mL.Resistant if MIC is > 16 ?g/mL. Plasmid-mediated resistance to tetracyclines, erythomycins, aminoglycosides, & etc.Tolerance: Implies that staphylococci r inhibited by a drug but not killed by it.Can be attributed to lack of activation of autolytic enzymes in e cell wall. ANTIGENIC STRUCTURESPeptidoglycan:A polysaccharide polymer containing linked subunits providing e rigid exoskeleton of e cell wall.Destroyed by strong acid or exposure y lysoenzyme.Elicits e production of IL-1 & opsonic Abs by monocytes.A chemoattractant for PMN leukocytes.Have endotoxin like activity & activates complement.Teichoic acids: polymers of glycerol or ribitol phosphate which linked to peptidoglycan & can be antigenic.Protein A: a cell wall component that binds to e Fc portion of IgG molecules except IgG3. This makes it as self.Capsules:Inhibits phagocytosis by PMN leukocytes.Have coagulase or clumping factors that bind to fibrinogen yielding aggregration of e bacteria.ENZYMES & TOXINCatalase: converts H2O2 into water & oxygen & differentiates staphylococci from streptococci.Coagulase: An enzyme-like protein that clots oxalated or citrated plasma.Binds to prothrombin & e complex initiates fibrin polymerization.Deposits fibrin on e bacterial surface & alters their ingestion by phagocytic cells.Other enzymes:Clumping factor: responsible for adherence of e organisms to fibrinogen & fibrin. When mixed with plasma, it forms clumps.Exotoxins:α-toxin: heterogenous protein that acts on eukoryotic cell membrane. Also acts as a potent hemolysinβ-toxin: degrades sphingomyelin which toxic to many cells.δ-toxin: heterogenous & dissociates into subunits in nonionic detergents. It disrupts biologic membranes & causes diarrheal disease.γ-hemolysin: efficiently lyse WBC by causing pore formation in e cellular membranes that increase cation permeability. Leukocidin: consists of 2 components which act synergistically to kill WBCs.Excofoliate toxins: consists of 2 distinct proteins of same molecular weight:Epidermolytic toxin A: chromosomal gene product & heat-stable.Epidermolytic toxin B: plasma mediated & heat-labile.Yield e generalized desquamation of e staphylococcal scalded skin syndrome.R superantigen.Toxic shock snydrome toxin: a superantigen which promotes e manifestations associated with fever, shock, & multisystem involvement includng desquamative skin rash.Enterotoxin: R superantigen, heat-stable, & resistant to e action of gut enzymes.E emetic effect is e result of CNS stimulation after e toxin acts on neural receptors in e gut.R on a chromosomal element called a pathogenecity island which interacts with bacteriophages to produce toxin.PATHOLOGYPrototype of staphylococcal lesion is furuncle or localized abscess.E mechanism: production of coagulase which coagulates fibrin around e lesion & within e lymphatic → formation of a wall that limit e process → accumulation of inflammatory cells & fibrous tissues at e centre → liquefaction of necrotic tissue occurs → drainage of e liquid centre → granulation tissue formation → healing. Organisms may spread via e lymphatics & bloodstream results in suppuration within veins associated with thrombosis.E complications:Osteomyelitis: in a terminal blood vessel of e mataphysis of a long bone leading to necrosis & chronic suppuration.Pneumonia, meningitis, emphyma, endocarditis or sepsis with suppuration in any organs.Skin infection: impetigo, pyoderma, or acne.Bullous exfoliation: e scalded skin syndrome caused by e production of exfoliative toxin.Toxic shock syndrome caused by TSST-1.CLINICAL FINDINGSLocalized staphylococcal infection:Appears as pimple, hair follicleinfection, or abscess.There is an intense, localized, painful inflammatory reaction that undergoes central suppuration & heals quickly when e pus is drained.E wall of fibrin & cells prevent e organism from spreading.Direct contamination of a wound: e.g. in postoperative staphylococcal wound infection or infection following trauma.Bacteremia: due to bacterial dissemination results in endocarditis, meningitis, organ dysfunction, & intense focal suppuration.Food poisoning: characterized by a short incubation perid (1 – 8 hours) with violent nausea, vomiting, diarrhea, & rapid convalescence.Toxic shock syndrome: manifested by an abrupt onset high fever, vomiting, diarrhea, myalgias, scarlatiniform rash, & hypotension with cardiac & renal failure.DIAGNOSTIC TEpiSTSGram staining: purple clustering cocci.Pigmentation.Coagulation test.Protein A test.Phosphate test.Deoxyribonuclease test.Mannitol fermentation.Catalase positive.TREATMENTTreat with penicillinase – resistant penicillins:CloxacillinNafcillinDicloxacillin If e organism is methicillin – resistant treat with:VancomycinClindamycinGentamcinCycloserineFusidic acidRifampicin THE STREPTOCOCCIPORTIONDETAILSCLASSSIFICATIONClassification into many categories has been based on:Colony morphology & hemolytic reactions on blood agar.Serologic specificity of e cell wall group-specific substance or capsular Ags.Biochemical reactions & resistance to Ags.Ecologic features.Categories of streptococci:Hemolysis:β-hemolysis: complete disruption of RBCs with clearing of e blood around e bacterial growth.α-hemolysis: incomplete reduction of hemoglobin & e formation of green pigment.γ-hemolysis: non-hemolytic steeptococci. Lancefield classification (group-specific substance):Based on e carbohydrate contained in e cell wall of many streptococci.E basic serologic grouping is Lancefield A-H & K-U. Group A: rhamnose-N –acetylglucosamine, Streptococcus pyogenes.Group B: rhamnose-glucosamine polysaccaharide, Streptococcus agalactiae.Group C: rhamnose-N –acetylgalactosamine, Streptococcus equislimilis.Group D: glycerol teichoic acid containing D-alanine & glucose, Streptococcus faecalis.Group F: glucopyranosyl-N –acetylgalactosamine.Capsular polysaccharide: based on e antigenicity of e capsular polysaccharides.Biochemical reactions:Used for species that typically do not react with commonly used Ab preparations for e group-specific substances.Based on biochemical tests such as sugar fermentation, presence of enzyme, test for susceptibility, & etc.Many streptococci r classified according to e combination of e above features.SPECIESStreptococcus pyogenesMORPHOLOGYSpherical & ovoid cocci arranged in chains which divide in a plane perpendicular to e long axis of e chain (one plane only).Streptococci r gram-positive, however as a culture ages & e bacteria die, they can appear to be gram-negative.Most group A strains produce capsules composed of hyaluronic acid which impede phagocytosis.E cell wall contains proteins, carbohydrates, peptidoglycans, & hair-like pilli (M-protein) which r covered with lipoteichoic acids (for attachment to epithelial cells). CULTURE & COLONYMost grow in solid media as discoid colonies of 1-2 nm in diameter.S. pyogenes r β-hemolytic which produces large zones of β-hemolysis around colonies greater than 0.5 mm in diameter.2 forms of colonies:Matte colonies: strains that produce much M protein & generally r > virulent.Glossy colonies: strains that produce little M protein & often r not virulent. They r PYR +ve but bacitracin sensitive.GROWTHEnergy is obtained from e utilization of glucose with e production of lactic acid.Growth tends to be poor on solid media or broth unless enriched with blood or tissue fluid.Most pathogenic strains grow best at 37oC.Most streptococci r facultative anerobes & grow under aerobic & anaerobic conditions while e peptostreptococci e obligate anaerobes.ANTIGENIC STRUCTURESM protein:A major virulent factor of group A S. pyogenes.Appears as hair-like projections on e cell wall.E presence indicates virulence & can resist phagocytosis, while its absence indicates no virulence.Immunity to infection is related to e presence of type-specific Abs to M protein.E M protein molecule has a rod-like coiled structure that separates functional domains. E structure allows for a large no. of sequence changes while maintaining function.Has 2 classes, I (may be a virulent determinant for rheumatic fever) & II. T substance:No r/ship to e virulence of e streptococci.T substance is acid & heat labile.Obtained from streptococci by proteolytic digestion.It permits differentiation of certain types of streptococci by agglutination of specific antisera.Nucleoproteins: also known as P substances which make up most of e streptococcal cell body.ENZYMES & TOXINStreptokinase (fibrinolysisn): transforms e plasminogen into plasmin that will digest fibrin & other protein.Streptodornase: depolymerizes DNA. Hyaluronidase: Splits hyaluronic acid which aids in spreading infecting microorganisms.R antigenic & specific for each bacterial or tissue source.Pyrogenic exotoxins (erythrogenic toxin): Exist in 3 antigenically distinct streptococcal pyrogenic exotoxins of A, B, & C.Exotoxin A (M protein types 1 & 3) has been associated with streptococcal shock syndrome & scarlet fever.Acts as superantigens which activate polyclonal T cells.Diphosphorydine nucleotidase: ability to kill leukocytes.Hemolysins:Streptolysin O: protein which is hemolytically active in e reduced state & responsible for hemolysis seen when growth is in cuts deep into e medium of blood agar plate.Streptolysin S: responsible for e hemolytic zones around streptococcal colonies on blood agar plate eventhough it is not antigenic.CLINICAL FINDINGSDiseases attributable to invasion by S. pyogenes , β-hemolytic group A streptococci:Erysipelas: massive brawny edema & a rapidly advancing margin of infection.Cellulitis: acute, rapidly spreading infection of e skin & subcutaneous tissues which is associated with pain, tenderness, swelling, & erythema. E lesion is not raised with indistinct boundries.Necrotizing fasciitis (streptococcal gangrene): extensive & very rapidly spreading necrosis of e skin & subcutaneous tissue (flesh-eating bacteria).Puerperal fever: a septicemia originating in e infected woud (endometritis).Bacteremia/sepsis: follows e infection of traumatic or surgical wounds which rapidly can be fatal.Diseases attributable to local invasion with S. pyogenes & their by-products:Streptococcal sore throat:D/t e adherence of S. pyogenes to pharyngeal epithelium via lipoteichoic acids.In infant & small children: e sore throat occurs as subacute nasopharyngitis with thin serous discharge, little fever & cervical lymph node enlargement. In older children & adults: > acute & characterized by intense nasopharyngitis, tonsiltis, & intense redness & edema of mucus membrane with purulent exudates, high fever, & tender cervical lymph nodes.Streptococcal pyoderma:Local infection of superficial layers of skin called impetigo.Consists of superficial vesicles that break down & eroded areas whose denuded surface is covered with pus & encrusted.It spreads by continuity & higly communicable especially in hot & humid climates.Cwidespread infection occurs in eczematous or wounded skin or in burn which progress to cellulitis.Invasive group A streptococcal infections:Streptococcal toxic shock syndrome:Characterized by shock, bacteremia, respiratory failure, & multiorgan failure.Death occurs in ~30% of patients.Infection tends to follow minor trauma such as necrotizings fasciitis, myositis, & etc.Erythema & desquamation may occur.Scarlet fever:Pyrogenic exotoxins A-C also cause scarlet fever in association with S. pyogenes pharyngitis or with soft tissue infection.E rash appears on e trunk after 24 hours of illness & involve e extremities.Poststreptococcal diseases:Acute glomerulonephritis:Develops 3 weeks after S. pyogenes skin infection.May be initiated by AgAb complexes on gromerular basement membrane.Clinical features: blood & protein in urine, edema, high blood pressure, & urea nitrogen retention with low level of serum complement.Some die, some develop chronic form & kidney failure, & majority recovers completely.Rheumatic fever:Results in damage to heart muscle & valve d/t cross reaction b/w cell membrane Ags & human heart tissue Ags.E onset is preceded by S. pyogenes infection 1-4 weeks earlier.Patients with > severe streptococcal sore throat have a greater chance of developing rheumatic fever.Symptoms: fever, malaise, a migratory non-suppurative polyarthritis, & inflammation of all parts of heart.E carditis leads to thickened & deformed valves & to small perivascular granuloma in myocardium which r replaced by fibrous tissues.Rheumatic fever has a marked tendency to be reactivated by recurrent streptococcal infection whereas nephritis does not. DIAGNOSTIC TESTSDirect detection - the antigen is extracted from a throat swab. The antigen extract will then bind with antibody specific to the group A streptococcal carbohydrate. This has classically involved agglutination of antibody coated beads. However, simpler tests have been recently introduced. Results are available within minutes.Lancefield grouping of isolated beta hemolytic colonies (see above).Colonies are beta hemolytic (figure 5) and their growth is inhibited by bacitracin (presumptive diagnosis).Patient serum shows antibodies to streptolysin O or other streptococcal antigens. This is important if delayed clinical sequelae occur.Beta hemolysis is caused by two hemolysins O and S; the former is inactive in the presence of oxygen. Thus, stabbing of the plate increases the intensity of the hemolysis reaction.TREATMENTAll r susceptible to Penicillin G & most r susceptible to erythromycin.Some r resistant to tetracyclines.Streptococcus pneumoniaMORPHOLOGYGram positive bacteria which r lancet-shaped diplococcic or arranged in chains.Readily lysed by surface active agents which remove or inactivate e inhibitor of cell wall autolysins.Pneumococci is inhibited by Optochin.CULTURE & GROWTHPneumococci forms small round colonies which r at 1st dome-shaped but later develop a central plateau with an elevated rim.It is α-hemolytic on blood agar form smooth colonies.Most energy r obtained from fermentation of glucose leading to e production of lactic acids (limits growth).Pneumococci which produce large amounts of capsules produce large mucoid colonies.ANYIGENIC STRUCTUREComponent structures: pneumococci have cell wall which contains peptidoglycan & teichoic acid which r immunogically distinct for each > 90 types.Quelling reaction: i.e. “capsule swelling test” for rapid identification & typing of e organisms.PATHOGENESISProduction of disease:Diseases r produced through e ability to multiply in tissues.E capsules resist phagocytosis from phagocytosis.No toxins r produced, but IgA protease is produced to help in colonization.Loss of natural resistance which predispose to pneumococcal infection:Viral & other respiratory tract infection that damage cell surface which leads to mucus accumulation, bronchial obstruction, & RT injury.Alcohol & drugs that depress phagocytic activity, cough reflex, & facilitates aspiration of foreign material.Abnormal circulatory dynamic.Malnutrition & general debility. Pathology of pneumococcal infection:Pneumococcal causes an outpouring of fibrinous edema fluid, RBCs, & WBCs into e alveoli which leads to consolidation of e lungs. However, e alveolar wall remains intact.They reach e bloodstream through lymphatic drainage & many reside in sinuses & middle ear.Later, mononuclear active cells phagocytose e debris & e lquid phase gradually reabsorbed.CLINICAL FINDINGSE onset is usually sudden, with fever, chills, & sharp pleural pain.E sputum is similar to alveolar exudates which is bloody or rusty in color.E complications: meningitis, endocarditis, & septic arthritis.LABORATORY DIANOSISStained smear: gram stain shows many PMNs & many RBCs.Capsule swelling test: positive.Culture: blood culture reveals α-hemolytic.TREATMENT & PREVENTIONDOC: Penicillin G with high doses.Pneumococci remain susceptible to vancomycin.Pneumococci r resistant to tetracycline & erythromycin. Corneybacterium diphtheriaePORTIONDETAILSMORPHOLOGYThey r 0.5-1.0 ?m in diameter & several micrometers long.They posses irregular swelling at one end that gives e club-shaped appearance.Granules (staining deeply with aniline dye) r irregularly distributed within e rod which gives a beaded appearance.Individual corneybacteria tends to lie parallel or at acute angles to one another.E colonies: Small, granular, gray, with irregular edges, & have small zone of hemolysis.In potassium tellurite, e colonies r brown to black with a brown-black halo because tellurite is reduced intracellularly.4 biotypes of corneybacteria: gravis, mitis, intermedius, & belfanti.They grow aerobically & tend to pleomorphism in microscopic & colonial morphology.In Loeffler’s serum medium, thay grow > readily & also called as Klebs-Loeflerr Bacillus.PATHOGENESISThey r spread by droplets or by contact to susceptible persons, & then they grow on mucous membranes & skin abrasion.Diphtheria toxin:Characteristics: Heat-labile polypeptides & bipartite molecule which is composed of fragment A & B.Production:E toxin is produced by toxigenic bacteria.Toxigenic & lysogenic bacteria r produced when nontoxigenic bacteria r infected by bacteriophage from toxigenic diphtheria bacilli. E trait is subsequently heredirity.E actual production of toxin occurs when e prophage of e lysogenic diphtheria become induced & lyses e cells.E toxigenicty is under control of e phage gene & e invasiveness is under control of bacterial gene. Mechanism of action:Fragment A: inhibits polypeptide chain elongation by inactivating e elongation factor EF-2 with e presence of NAD.Fragment B: no independent activity but required for e transport of fragment A into e cell.Toxin fragment A inactivates EF-2 by catalyzing a reaction that yields free nicotinamide and inactive adenosine diphosphate-ribose-EF-2 complex.Result: abrupt arrest of protein synthesis which leads to necrotizing & neurotoxic effects.PATHOLOGYEffects of bacilli themselves:Formation of a false membrane which adhere to underlying tissue.Diphteria produce toxin which is absorbed into e mucus membranes → destruction of epithelium → superficial inflammatory response → e necrotic epithelium becomes embedded in exuding fibrin & red & white cells → grayish pseudomembrane is formed (over tonsil, pharynx, or larynx).It is a grayish white, wrinkle, & tough which is difficult to strip off. If removed, it will leave behind minute bleeding spot. E regional lymph nodes in e neck enlarged & there might be marked edema of e entire neck.In fauceal & laryngeal diphtheria e false membrane may cause mechanical obstruction of e passage.Wounded or skin diphtheria occurs chiefly in tropics: e membrane may form an infected wound which fails to heal.However, diphtheria does not actively invade deep tissues & practically never enters bloodstream.Effects of exotoxin:In e heart, muscle, liver, kidneys, adrenal, & sometimes nerve damage (results in paralysis of soft palate, eye muscles, & extremities.E diphtheria within e membrane produce toxin actively.Toxin is absorbed & causes distant toxic damage:Parenchymatous degeneration.Fatty infiltration.Necrosis with gross hemorrhage. CLINICAL FINDINGSInitial features: sore throat & fever.Later development d/t to membrane formation & e toxin:Prostration & dyspnea which leads to suffocation.Irregularities in cardiac rhythm d/t toxic myocarditis.Paralysis effects on vasomotor control of e blood vessels.Toxic & interstitial nephritis in kidneys.Fatty changes & hemorrhage in adrenal glands.Nerves: neuritis.LABORATORY DIGANOSISGram stain: By using e Dcaron swabs from nose, throat, or other suspected lesion.Must be done before e registration of antibiotic.Culture: by using a Loeflerr slant (12-18 hours) or a tellurite plate (36-48 hours).Toxigenicity test:Elek test: immersed a filter paper in diphtheria toxin → set it on serum agar → streak e strain of C. diphtheria on e agar at e right angle to e strip paper → incubate at 37oC precipitation (toxin-antitoxin interaction).An immunochromographic strip assay: a highly sensitive assay.PCR & EIA.TREATMENT & PREVENTIONTreatment of diphtheria antitoxin is mandatory for strong clinical suspicion of diphtheria.Antimicrobial drugs such as penicillin & erythromycin.Active immunization with diphtheria toxoid (vaccine) to yield adequate antitoxin level. Usually in combination with tetanus toxoid or pertussis vaccine.CLOSTRIDIUM SPECIESPORTIONDETAILSMORPHOLOGYTypical organisms:Spores r wider than e diameter of e rods. Spores r located centrally, subterminally, or terminally.Moat species r motile & possess peritrichous bacteria. Culture & colony:Clostridia r anaerobes which grow under anaerobic condition.Some r aerotolerant & will also grow in ambient air.They grow well in blood-enriched media used to grow other bacteria.E colony: Multiple sizes (large-raised & small) colonies which can be spread on agar surface.Many produce zones of hemolysis on blood agar.Growth: clostridia can ferment a variety of sugars, digest protein, turn milk into acids & undergo stormy fermentation.Clostridium tetaniOVERVIEWCauses tetanus & is worldwide in distribution in e soil & in e feces of horses.Can be distinguished by specific flagellar Ags.All share a common O Ags which may be masked & produce neurotoxin, tetanospasmin.E spores r centrally located.TOXINE toxin is tetanospasmin which is cleaved by a bacterial protease into 2 peptides linked by disulphide bond.Mechanism: toxin binds to receptors on e presynaptic membranes of motor neurons → it migrates by e retrograde axonal transport system to e cell bodies to spinal cord & brain stem → it diffuses to terminals of inhibitory cells → it degrades synaptobrevin (protein for docking of neurotransmitter vesicles) → release of inhibitory glycine & GABA is blocked → hyperreflexia, muscle spasm, & spastic paralysis results.Extremely small amount of toxin can be lethal to human. PATHOLOGYC. tetani is not an invasive organism. Thus, e infection remains localized in e area of devitalized tissue into which e spores have been introduced.Germination of e spores & development of vegetative organisms r aided by:Aid e establishment of low redox potential.Necrotic tissue.Calcium salts.Associated pyogenic infection.CLINICAL FINDINGSE incubation period: 4-5 days to as many weeks.E disease is characterized by tonic contraction of voluntary muscles:Muscular spasm of e area of injury & infection → muscle of e jaw (trismus & lock jaw) which contract so that mouth can’t be opened → spasm of other voluntary muscle → tonic spasm → by e presence of external stimuli, generalized muscle spasm can occur.In newborn infant, risus sardonicus occur d/t a peculiar grin caused by acute spasm of facial muscles. E patient is fully conscious & pain may be intense.Death usually results from interference with e mechanics of respiration.Mortality rate in generalized tetanus is very high.LABORATORY DIAGNOSISE clinical pictures & history of injury is of diagnostic test.Proof of isolation of C. tetani must rest on e production of toxin & its neutralization by specific antitoxin.TREATMENT & PREVENTIONActive immunization with toxoids (vaccine).Produced by detoxifying w toxin with formalin & concentrating it.3 injections should be given for e initial course with a booster injection given upon entry to school. Proper care of wound contaminated with soils.Prophylactic use of antitoxin.IM administration which neutralizes e toxin that has not been fixed to nervous tissues.Administration of penicillin.Strongly inhibits e growth & stops further toxin production.May also control associated pyogenic infection.Surgical debridement for removing e necrotic tissues.Patients who develop symptoms of tetanus should be given muscle relaxant, sedation, & IV assisted ventilatoxin to neutralize e toxin. Clostridium perfringensTOXINE toxin produced causes a spreading infection which has lethal, necrotizing, & hemolytic properties.E α-toxin of C. perfringens type A is a lecithinase & its lethal action is proportionate to e rate at which it splits lecithin to phosphorylcholine & diglyceride.E theta toxin has similar hemolytic & necrotizing effects. It is not a lecithinase but a DNAase & hyaluronidase, a collagenase that digests collagen is subcutaneous tissue & muscle.They also produce enterotoxin when grown in meat dishes which induces intense diarrhea in 6-18 hours. E spores r terminally located.PATHOGENESISInvasive clostridial infection:Spores reach tissue by contamination or traumatized area or from intestinal tract → spores germinate, multiply, & ferment carbohydrate in tissue → form gases → tissue distention & blood supply interference → secretion of necrotizing toxin & hyaluronidase → spread of infection → extension of tissue necrosis → opportunity for increased bacteria growth, hemolytic anemia, & severe toxemia death. Gas gangrene or clostridial myonecrosis:Involves mixed infection of toxigenic clostridia, proteolytic clostridia, & various cocci & gram-negative anism from fecal flora may contaminate a wound & may grow & multiply in poorly perfused tissue.ifection spread rapidly & gas can be felt in tissue.CLINICAL FINDINGSFrom a contaminated wound, e infection spreads in 1-3 days to produce precipitation in e subcutaneous tissue & muscle, results in foul-smelling discharge, rapidly progressing necrosis, fever, hemolysis, toxemia, shock, & death.E disease: anaerobic fasciitis or cellulitis.Enterotoxin: Toxin forms when e organisms sporulate in e gut.E onset of diarrhea (w/o vomiting & fever) is 6-18 hours, but e illness lasts only 1-2 days.LABORATORY DIAGNOSISGram stain of specimens obtained from wound, pus, & tissue with e presence of large gram positive rods suggestion for gas gangrene clostridia.Culture in chopped meat-glucose medium & thioglycolate medium & onto blood agar plates incubated anaerobically.Final identification rests on toxin production & neutralization by specific antitoxin.TREATMENT & PREVENTIONTreatment:Prompt & extensive surgical debridement of e involved area & excision of all devitalized tissues.Administration of antimicrobial drugs such as penicillin at e same time.Antitoxin in e form of concentrated Igs & polyvalent antitoxin. Prevention: early & adequate cleansing of contaminated wounds & surgical debridement together with e administration of antimicrobial drugs.Clostridium botulinumOVERVIEWCauses botulism which is worldwide in distribution & found in soil & occasionally in animal feces.Spores of e organism r highly resistant to heat, withstanding 100oC for several hours.However, heat resistance is diminished at acid pH or high salt concentration.TOXINToxin is liberated during e growth & during e autolysis of e bacteria.Seven antigenic varieties r known from A-G, but only Type A, B, & E cause human illnesses.Type A & B r associated with a variety of foods & type E with fish products.Botulinum toxin is absorbed from e gut & binds to receptors of presynaptic membranes of motor neurons of e peripheral nervous system & cranial nerve.Proteolysis of e target protein in e neuron inhibits e release of Ach at e synapse, resulting in lack of muscle contraction & paralysis.E toxin is destroyed by heating for 20 minutes at 100oC.PATHOGENESISE illness is d/t intoxication resulting from e ingestion of food with e bacteria.E most common food: spiced, smoked, vacuum-packed, or canned alkaline foods that r eaten w/o cooking.In e food, e spores germinate & under anaerobic condition, vegetative forms grow & produce toxin.E toxin blocks Ach at NMJ results in faccid paralysis.CLINICAL FINDINGSSymptoms begin 18-24 hours after ingestion with visual disturbances, inability to swallow, & speech difficulties, signs of bulbar paralysis r progressive & death d/t respiratory paralysis or cardiac arrest.No fever is associated, e patient remains fully conscious shortly before death.E mortality rate is high.Recovered persons do not develop antitoxin in e blood.Newborn infants: poor feeding, weakness, & sign of paralysis which leads to sudden infant deah syndrome.LABORATORY DIAGNOSISE toxin can be demonstrated in e serum & leftover of e food by using RIA or passive hemagglutination.E antigenic type of toxin is identified by neutralization of specific antitoxin in mice.TREATMENT & PREVENTIONPrompt IV administration of antitoxin with customary precautions.Adequate ventilation must be maintained by mechanical respirator.E risk from home-canned food can be reduced by boiling e food > 20 minutes before consumption.Toxoid for active immunization.Bacillus anthracisPORTIONDETAILSMORPHOLOGYPrevalent in soil, water, air, & on vegetation.E typical cells:Large aerobic gram positive rods occurring in chains.Measure 1x3-4 ?m, have square ends, non-motile, & spore r located at e center of e rods.Use simple sources of nitrogen & carbon for energy & growth.Its capsule contains poly-D-glutamic acid which is antiphagocytic where its gene in plasmid.B. anthracis which is non-capsulated is not virulent & does not induce anthrax in test animal. E colonies:R round & have a cut glass appearance in transmitted light.Hemolysis is uncommon but gelatin is liquefied & growth in gelatin stab resembles as inverted fir tree.E spores:R resistant to environmental changes, dry heat, & certain chemical disinfectant for moderate periods.Persist for years in dry earth.Can be sterilized by autoclaving.PATHOGENESISA zoonosis where human is accidently infected by contact with infected animals or their products.E infection is acquired by e entry of e spores through:Injured skin (cutaneous anthrax): 95%.Rarely e mucus membrane (GI anthrax): very rare.By inhalation of spores into e lungs (inhalation anthrax): 5%.E mechanism: e spores germinate in e tissue at e site of entry → vegetative organisms grow → formation of a gelatinous edema & congestion → spread via lymphatic to e bloodstream → multiply freely in e blood & tissues → death.Anthrax toxin:Made up of 3 proteins: Protective Ag (PA): binds to specific cell receptors & following proteolytic activation, it forms a membrane channel that mediates entry of EF & LF into e cell.Edema factor (EF): an adenylyl cyclase, with PA it forms a toxin known as edema toxin. Lethal factor (LF): with PA, it forms lethal toxin which is e major virulence factor & cause death.E anthrax toxin genes r on another plasmid.Inhalation anthrax or woolsorter’s disease: spores r inhaled & phagocytosed in e lungs → transported by lymphatic drainage to e mediastinal lymph nodes → germination occurs → toxin production → development of hemorrhagic mediastinitis & sepsis → death.PATHOLOGYIn susceptible animal: E organisms proliferate at e site of entry.E capsules remain intact & e organisms r surrounded by a large amount of proteinaceous fluid containing few leukocytes.E leukocytes help them in rapid dissemination & in reaching e bloodstream.In resistant animal:E organisms proliferate for a few hours in e present of massive accumulation of leukocytes.Capsules gradually disintegrate & disappear.E organisms remain localized.CLINICAL FINDINGSCutaneous anthrax:Occurs on e exposed surface of e arms or hands, followed by face & neck.A prurutic papule develops 1-7 days after e entry of spores or e organisms through scratch.E papule rapidly changes into a vesicle or small ring vesicles that coalesce & a necrotic ulcer develops.E lesion is 1-3 cm in diameter & has a characteristic of central black eschar.After 7-10 days, eschar is fully developed & it dries, loosens, separates, & heals by granulation & leaves scar.Marked edema occurs with lymphangitis & lymphadenopathy.Systemic signs & symptoms: fever, malaise, & headache. Inhalation anthrax:E incubation period: as long as 6 weeks.Early clinical manifestations: marked hemorrhagic necrosis & edema of e mediastinum.Other complications: substernal pain, pronounced mediastinal widening, hemorrhagic pleural effusion, cough, sepsis, & hematogenous spread to GIT causing bowel ulceration, or to meninges causing hemorrhagic meningitis.E mortality rate is high. GI anthrax:E GI anthrax is acquired through ingestion of spores or spread of organisms from e intestinal tract.Clinical signs: abdominal pain, vomiting, & bloody diarrhea.LABORATORY DIAGNOSISSpecimens examination:Specimens r obtained from fluid or pus from a local lesion, blood, & sputum.Can be examined using:IF staining techniques.Gram stain: large gram positive rods. Culture:Blood agar plate: Non-hemolytic gray to white colonies with a rough texture & a ground-glass ma-shaped outgrowths (Medusa Head) may project from e colonies.Semisolid medium: non-motile organisms r present.Mice or guinea pigs: killed upon intraperitoneal injection.Serological test:ELISA is used to measure Abs against edema & lethal toxins.A positive results is a fourfold change on a single titer of greater than 1:32.TREATMENT & PREVENTIONAntibiotics r effective & must be started early: ciprofloxacin & penicillin G with gentamycin or streptomycin.Propghylaxis with ciprofloxacin & doxycycline for potential exposure.Control measures:Disposal of animal carcasses by burning or by deep burial in lime pits.Decontaminating by autoclaving of animal products.Protective clothing & gloves for handling potentially infected materials.Active immunization of domestic animals with live attenuated vaccines.Immunization of people with high occupational risk.MYCOBACTERIUMPORTIONDETAILSOVERVIEWAlso known as fungus bacterium.Class 1: Actinobacteria which consists of:Actinomyces.Nocardia.Srynebacteria.Rhodococcus.Properties:Acid (alcohol) fastness: resist decolorization by acidified alcohol after stained with basis fuchsin.Presence of mycolic acids in e cell wall.A G + C content of DNA are 61-71%.MYCOBACTERIAAt least 100 species.Causative agents of tuberculosis, leprosy, & opportunistic infection (saprophytes).Some r slow grower (> 7 days) & some r rapid grower (< 7 days).Morphology:Rod-shaped, aerobic, & do not form spores.Some r coccobacillary, filamentous, or branched.Staining:Ziehl-Neelsen stain: hot staining (result: red in color).Kinyoun: cold staining.Auramine or fluorescence: yellow-orange fluorescence.Culture:M. leprae can’t be grown in culture, only in mouse foot pad & 9 banded armadillos.Others: Lowenstein-Jensen (LJ) medium (inspissed egg media), Middlebrook 7H9 (liquid), Middlebrook 7H10 & 7H11 which r agar based. (Culture’s color is green).Pigment production (yellow to orange):Scotochromogens: form pigment in e dark.Photochromogens: form pigment after exposure to light.Non-chromogens: no pigmentation at all.Mycobacterium tuberculosisMORPHOLOGYThin straight uncapsulated rods measuring about 0.4 x 0.3 ?m.AFB, obligate aerobe which r non-motile & non-spore forming.Survive in milk, organic matters, pasture land, phenolic, & hypochlorides.Sensitive to alcohol, formaldehyde, glutaraldehyde, heat, & UV light.Resistant to acids, alkali, & quaternary ammonium compounds.Derive energy from e oxidation of many simple carbon compounds.TUBERCLE BACILLIFound mainly in cell wall which can induce hypersensitivity type 4 in previously infected animal.E constituents of tubercle bacilli:Lipids:Include mycolic acids, waxes, & phosphatides which r largely bound to protein & polysaccharide.Muramyl peptide complexed with lipid can cause granuloma formation & caseous necrosis.Proteins: elicit e tuberculin reaction & formation of a variety of Abs.Polysaccharides: can induce immediate type of hypersensitivity & can serve as Ags in reaction with sera of infected persons.PATHOGENESISVirulence is d/t bacterial invasiveness & ability to survive in macrophages.No toxin r produced.Immune response is mediated by cellular immunity which leads to hypersensitivity type 4.2 types of tuberculosis:Primary tuberculosis:Site of infection: Usually lungs:Port of entry is through respiratory tract by inhalation of bacilli.E primary complex or Ghon focus: enlargement of hilar lymph nodes.Can be spread to other parts of body through hematogenous or lymphatic spread.Occasionally GIT: primary complex consists of tonsil & cervical lymph nodes enlargement (which results in scrofula or purplish in e region) as well as enlargement of ileocaecal & messentric lymph nodes enlargement.Skin: primary complex consists of regional lymph nodes enlargement (prosector’s wart).E mechanism (1-2 weeks time): T cell clones (Ag specific) → cytokines → macrophage activation → granuloma formation → central caseation (usually limit e 1st infection) → quiescent → fibroblast scar & calcification → recovery or incomplete destruction of mycobacterium → dormant → immunocompromised condition leads to reactivation → post-primary tuberculosis. 2 principal lesions:Exudative type:Consists of an acute inflammation reaction with edema fluid, PMNs, & monocytes around e tubercle bacilli.Seen in lungs that may heal by resolution & leads to massive necrosis of tissue.During this phase, tuberculin test is positive.Productive type or chronic granuloma:Consists of 3 zones:A central area of large, multinucleated giant cells containing tubercle bacilli. Later, e area will undergo casaetion.A mid zone of pale epitheliod cells arranged radially.A peripheral zone of fibroblasts, lymphocytes, & monocytes.A caseous tubercle may break into a bronchus, empty its contenst & form a cavity which then heals by fibrosis or calcification.Post-primary tuberculosis:Usually caused by tubercle bacilli that have survived in primary lesions. Characterized by longer local lesions, minimal lymphatic involvement, frequent cavitation, & usually involved e apical region of lungs which is e highest oxygen concentration.Reactivation can be spontaneously when e IR is low.Process of granuloma formation is e same but > extensive with large area of caseation, a condition known as tuberculomata.Protease & cytokines levels r high.E mechanism: tubercle bacilli → erode wall of bronchus → liquefied content → expectorated → aerobic → bacilli growth increases.In immunocompromised patients, e cavitation is rare; however, lymphatic & hematogenous spread is common: cryptic disseminated tuberculosis.LABORATORY DIAGNOSISTuberculin test: old tuberculin & purified protein derivative (PPD).Mantoux test: intracutaneously.Heaf test: guns with 6 prongs.Tine test:Endemic areas: > 10 mm.Non-endemic areas: < 5 mm.Children & immunocompromised.Laboaratory specimen: sputum, bronchial washing, biopsy, gastric aspirates, CSF, & pleural fluids.Culture: LJ medium or middlebrook media.Drug susceptibility testing: in LJ medium containing doubling dilutions of drugs.X-ray.TREATMENT & PREVENTIONSterilizing: rifampicin & pyrazinamide.Bacteriocidal: isoniazid, streptomycin, & ethambutol.Bacteriostatic: ethionamide, prothionamide, cycloserine, thiacetazone, & D-aminosalicyclic acids.Mycobacterium lepraeOVERVIEWLeprosy: a chronic mycobacterial disease primarily affecting e peripheral nervous system & secondarily involving skin & other tissues.Etiology: mycobacterium leprae:unculturable in e lab, but can be grown in mouse footpad & 9 banded armadillos.Man is e only significant reservoir.Transmission is via nasal secretion by droplets & by skin.Acid-fast bacilli are singly & arranged in parallel bundles or in globular masses.Often found in scraping from skin or mucous membrane & within endothelial cells of blood vessels or in e mononuclear cells.PATHOGENESISPrinciple target cells r Schwann cells which leads to nerve damage & results in anesthesia & muscle paralysis. Skin:Non-specific lesions with pigmentary changes (hypopigmented): anesthesia or hypoaesthesia.Often heals spontaneously.Clinical menifestations depends on immune status of patients, ranging from tuberculoid to lepromatous.Borderline tuberculoid < mid-borderline < borderline lepromatous.Hyperactive IRAnergy Few localized skin lesions with few bacilli (paucibacillary).Intense granulomatous response: damage in major nerve (hardening).Numerous skin lesions: often confluent.Bacilli are many (multibacillary) with clustering.IR lowered: anergy.Ear lobes: bacillary +++++Leads to blindness.Other signs & symptoms:Eyes:Uveitis.Corneal infection to eyelid paralysis.Bone resorbtion:Collapse of nasal bone.Shortening of fingers & toes (autoamputation).Neuritis, orchitis, & immune-complex nephritis.LABORATORY DIAGNOSISHistory & physical examination.Split skin smear & purich biopsy from ear lobes, nasal secretion, & nasal mucosa.Histology: granuloma & caseation.AFB: Viable if stained strongly & evenly.Dead when stained unevenly & weakly.Morphological index (MI): High MI: active disease.Low MI: with chemotherapy.Skin test:Lepronin test (only guide).Fernandez (early): reaction in 1-2 days.Mitsude (late): reaction in 7 days (usually –ve in LL).TREATMENT & PREVENTIONDapsone: diaminophenyl suphone-DDS.DDS: resistant, thus MDT with rifampicin & clofazimine.Other drugs: prothionamide, ofloxacin, & minocycline.WHO:Paucibacillary: rifampicin & dapsone for 6 days.Multibacillary: rifampicin, dapsone, & clofazimine for 2 years or more.Stages of 1o tuberculosis in childhoodTime from onsetCharacteristics 3-6 weeks1o complex developsTuberculin conversion is +ve2-6 monthsProgressive healing of 1o complexPossibility of pleural effusion6-12 monthsPossibility of military or meningeal tuberculosis1-3 yearsPossibility of bone or joint tuberculosis3-5 yearsPossibility of gastrourinary or chronic tuberculosisSplit-skin smear: Acid Fast StainingFor diagnosis of leprosyBacillary index (record bacteria in high power)1+1-10 per 100 fields2+1-10 per 10 fields3+1-10 per field4+10-100 per field5+100-1000 per field6+> 1000 per field THE SPIROCHETESPORTIONDETAILSOVERVIEWTreponema pellidum ssp. pallidumMORPHOLOGYPATHOGENESISDIAGNOSISTREATMENTGENERAL VIROLOGYPORTIONDETAILSOVRIGINRegressive theory: Viruses may be degenerate form of intracellular parasites.Progressive theory: Viruses r derived from cellular RNA & DNA components.Normal cellular nucleic acids that gained e ability to replicate autonomously & therefore evolve.DNA viruses came from mitochondrial DNA, plasmid, or transposable elements. They then evolve coat proteins & transimissibility.RNA viruses from bination theory: Viruses evolved through a combination of e above mechanisms.GENERAL CHARACTERISTICSObligate intracellular parasites.Small (20 – 300 nm in diameter) – filterable through bacterial-tight filters.Contain:A single type of nucleic acid, either DNA or RNA.A protein coat (capsid) consisting of individual protein units (capsomeres).A host derived lipid membrane (envelope) through which may be inserted viral protein (soikes or peplomers).Multiply inside living cells by using e biosynthetic machinery of e host cell.CLASSIFICATIONDisease: encephalitis & hepatitis.Modes of transmission: arbovirus.Host cells: animal, plant, & bacteria.Tissue or organ: adenoviruses & enteroviruses.GENOMIC SEQUENCINGVirion morphology: size, shape, types of symmetry, peplomers, or membranes.Virus genome properties: types of nucleic acid, size of genome, strandedness, & etc.Physicochemical properties of virion: molecular mass, buoyant density, pH stability, & etc.Genome organization & replication: gene order, number, & position of open reading frames.Antigenic properties.Biologic properties: natural host range, mode of transmission, vector relationship, & etc.BALTIMORE’s- According to e strategy of mRNA transcription Double stranded DNA: Adenoviruses, Herpesviruses, Poxviruses, & etc.Single stranded +ve sense DNA: Parvoviruses.Double stranded RNA: Reoviruses.Single stranded +ve strand RNA: Picornaviruses, Togaviruses, & ETC.Single stranded –ve sense RNA: Orthomyxoviruses, Rhabdoviruses, & etc.Segmented: Orthomyxoviruses.Non-segmented: Rhabdoviruses.VIRUS MORPHOLOGYTypes of symmetry:Cubic symmetry:Icosahedral pattern: e most efficient arrangement for subunits in a closed shell.Have 20 equilateral triangle faces, 12 vertices, & 5 or 3 or 2 fold axes of rotational symmetry.E vertex units have 5 neighbors (pentavalent) & all others have 6 (hexavalent).Have 60 identical subunits on e surface of icosahedrons.E viral nucleic acid is condensed within e isometric particles (virus-encoded core proteins) or cellular histones.E.g.: Poliovirus.Helical symmetry:Protein subunits r bound in periodic way to e viral nucleic acid, winding it into a helix.E viral nucleic acid protein complex (nucleocapsid) is coiled inside lipid-containing envelope.All known examples of animal viruses with helical symmetry contain RNA genome (except rhabdoviruses), have flexible nucleocapsids that r wound into a ball inside plex structures: > complicated in structure such as poxviruses (brick shaped with ridges in external surf.).Envelopes:Enveloped: All animal viruses with helical nucleocapsids r enveloped & have RNA genome.Polyhedral capsid: Herpes simplex virus.Helical capsid: Influenza virus.Non-enveloped:Polyhedral capsid: Poliovirus.Helical capsid: Bacteriophage virus.CHEMICAL COMPOSITIONPROTEINFunctions of structural proteins:Facilitate transfer of e viral nucleic acid from one cell to another.Protect e viral genome against inactivation by nucleases.Participate in e attachment of e virus particle to a susceptible cell.Provide e structural symmetry to e virus particles.Determine e antigenic characteristics & activities of e virus.Enzymes:Present in very small amounts.Essential for e initiation of e viral replicative cycle when e virion enters a host cell.E.g.: RNA polymerase & reverse transcriptase.NUCLEIC ACIDEncodes e genetic information necessary for replication of e virus.E size:Viral DNA genome: 3.2 kbp to 375 kbp.Viral RNA genome: 7 kbp to 30 kbp.Viral RNA genome:May be single linear molecule.May be loosely associated with e virion.E isolated RNA of viruses with +ve sense genomes is infectious & functions as mRNA within e infected cell.Virions carry an RNA polymerase that in e cell transcribes e genomic RNA molecules into several complementary RNA molecules which serve as mRNA.Many viral genomes have been sequenced which reveal e genetic r/ship among isolates.May be characterizaed by its G + C content.LIPID ENVELOPESE lipid is acquired when e viral nucleocapsid buds through a cellular membrane in e course of maturation.Budding occurs only at sites where virs-specific proteins have been inserted into e host cell membranes.E specific phospholipid composition of e virion envelope is determined by e specific type of cell membrane involved in e budding process.There r viral glycosated proteins protruding from e envelope & exposed on e external surf. of e virus particle.There r unglycosated proteins of viral origin underneath e envelope that anchor e particle.Lipid-containing viruses r sensitive to ether or other organic solvents which leads to loss of infectivity.GLYCOPROTEINViral envelopes contain glycoprotein which r virus-encoded (e sugar often reflects host cell)Functions:Attaches e virus particle to a target cell by interacting with cellular receptor.Often involved in e membrane fusion step of infection.Important viral antigen.Frequently involved in e interaction of e virus particle with neutralization Ab.REACTION TO STIMULIHeat & cold:Icosahedral virus: stable, losing little infectivity after several hours at 37oC.Enveloped virus: heat-labile, rapidly dropping in titre at 37oC, lose infectivity after prolonged storage at -90oC, & sensitive to repeated freezing & thawing.Viral infectivity generally destroyed by heating at 50 – 60 oC for 30 minutes.Virus can be preserved by storage at subfreezing temperatures.Some may withstand lyophilization & be preserved at room temperature (> resistant when heated in dry state).Salt:Many viruses can be stabilized by salts in concentration of 1 mol/L, e.g.: MgCl2 & MgSO4 (important in vaccine)Salt stabilization can maintain e viral potency for weeks at ambient temperature.pH: Usually stable b/w pH of 5.0 to 9.0.Some r resistant to acidic condition but all r destroyed in alkaline condition.Radiation: UV light, X-ray, & high energy particles inactivate viruses & lose its infectivity.Photodynamic inactivation: viruses become susceptible to inactivation by visible light when stained with vital dyes because the dyes bind to e nucleic acid. Thus may inactivate progeny virus & cease plaque development.Ether susceptibility: enveloped viruses r e most susceptible.Detergent: Non-ionic detergents solubilize lipid constituents of viral membranes.Anionic detergents solubilize viral envelopes & disrupt capsids into several polypeptides.Formaldehyde: destrots virus infectivity by reacting with nucleic acids (single stranded genomes r > affected).VIRAL ASSAYPORTIONDETAILSANALYSIS OF VIRAL STRUCTUREElectron microscopy (EM):Transmission EM: -ve staining of whole virus particles, staining of ultrathin section, & metal shadowing.Scanning EM.X-ray diffraction pattern of crystallized viruses: Has a resolution power down to e atom level.NMR nuclear magnetic resonance.MEASURAMENT OF VIRUS SIZEDirect observation in e electron microscope: Viruses can be visualized in preparations from tissue extracts & in ultrathin sections of infected cells.Sedimentation in e ultracentrifuge: If particles r suspended in a liquid, they will settle to e bottom at e rate that is proportionate to their size.E r/ship b/w e size & shape of a particle & its sedimentation rate permits determination of particle parative measurement: B/w viruses or b/w bacteria & viruses.CULTIVATION OF VIRUSESDetection of virus-infected cells (multiplication of viruses):Development of CPE.Appearance of a virus-encoded protein such as hemagglutinin.Adsorption of RBC to infected cells: hemadsorption (d/t virus-encoded hemagluitinin).Detection of virus-specific nucleic acid.Viral growth in an embryonated chick egg: may result in e death of e embryo.Inclusion body formation:E site of development of e virions.May be considered as diagnostic aid.QUANTITATION OF VIRUSESPhysical method:Quantitative assays such as PCR can determine e no. of viral genome copies in a sample.Virus sequence variation may reduce virus detection & quantification by this method.Physical Counting under EM: by comparison with a standard suspension of small latex particles.Hemagglutination Assay.Biological method:Depend on measurement of animal death, animal infection, or CPE in tissue culture.Multiple cycles of replication & cell killing produce a small action of infection or plaque.Plaque Assay: A single plaque can arise from a single infectious virus particle called plaque forming unit (PFU).End Point Dilution Assays: to estimate e 50% infectious dose (semi-quantitative).PURIFICATION OF VIRUS PARTICLEE starting material: large volumes of tissue culture medium, body fluids, or infected cells.It is a multistep procedures:Concentration of e virus particles by:Precipitation with ammonium sulphate, ethanol, or polyethylene glycol, or by ultrafiltration.Hemagglutination & elution for orthomyxoviruses.Once concentrated, virus then can be separated from host materials by:A preliminary purification: Differential centrifugation which removes most nonviral material.E final purification method: Density gradient centrifugation: In rate zonal centrifugation, e concentrated virus is layered onto a preformed linear density gradient of sucrose or glycerol. During e centrifugation, e viruses sediment as a band at a rate determined 1o by e size & weight of e virus particle.High speed centrifugation: Involves e density gradient of cesium chloride, potassium citrate, or sucrose (one which is least toxic to e virus is to be chosen). Virus particles migrate to an equilibrium position where e density of e solution = buoyant density & form a visible band. Other methods:Column chromatography: virus is bound to a substance such as diethylaminoethyl or phosphocellulose & eluted by changes in pH or salt concentration.Zone electrophoresis: permits e separation of virus particles from contaminants n e basis of charge.Specific antisera: remove virus particles from host.Icosahedral viruses r easier to purify than envelope viruses b/cause e latter contain variable amounts of envelope per particle, thus e virus particles e heterogenous in both size & density.IDENTIFICATION OF VIRUS PARTICLESE particle can be obtained only from infected cells or tissues.Particles obtained from various sources r identical.E degree of infective activity of e preparation varies directly with e no. of particles present.Destruction of e physical particle is associated with a loss of viral activity.Certain properties of e particles & infectivity must be shown to be identical.E absorption spectrum of e purified particles in e UV range should coincide with e UV inactivation spectrum of e virus.Antisera prepared should react with e infective particles & vice versa.E particles should be able to induce characteristic disease in vivo.Passage of e virus in tissue culture should result in e production of e progeny. METHOD OF VIRAL INACTIVATIONReasons to inactivation:To sterilize laboratory supplies & equipment.To disinfect surfaces or skin.To make drinking water safe.To produce inactivated virus vaccines.Methods of inactivation:Sterilization accompanied by steam under pressure, dry heat, ethylene oxide, & gamma irradication.Surface disinfectants include sodium hypochlorite, glutaraldehyde, formaldehyde, & peracetic acid.Skin disinfectants include chlorhexidine, 70% ethanol, & iodophores.Vaccine production may involve UV or detergents to inactivate e viruses. PRESERVATION OF VIRAL INFECTIVITYGenerally, viruses r more sensitive than bacteria & fungi to inactivation by physical & chemical agents.Enveloped viruses r more sensitive than naked ones.Half life of viruses at certain temperatures:60oC: measured in seconds.37 oC: measured in minutes.20 oC: measured in hours.4 oC: measured in days.-70 oC or lower: measured in years.LONG TERM STORAGE OF VIRUSESFreezing of small aliquots of virus suspended in medium containing protective protein such as albumin followed by storage at:Temperature of CO2 ice: -70 oC.Temperature of liquid nitrogen: -196oC.Freeze-Drying (lyophilization): dehydration of a frozen viral suspension under vacuum followed by storage at 4 oC or -20 oC.Stabilization in one molar concentration of certain salts.“Once in a lifetime, u find a friend, who touches not only ur heart but also ur soul.Once in a lifetime, u discover someone, who stand beside u, not even u.Once in a lifetime, if u r lucky, u find someone, as I’ve found u.Very special people, we can be ourselves with, talk with, laugh with, hope with, & believe with.”“Ukhwah bukan terletak pada kerapnya pertemuan atau manisnya bicara, tapi terletak pada ingatan terhadap sahabat dalam doanya.”- Imam Ghazali - VIRAL REPLICATIONPORTIONDETAILSOVERVIEWFor virus to multiply it must obviously infect a cell.All must make proteins with 3 sets of functions:Ensure e replication of e genome.Package e genome into virus particles.Alter e metabolism of e infected cell so that viruses r produced.STEPS OF VIRAL REPLICATIONATTACHMENT, PENETRATION, & UNCOATINGAttachment:I.e. interaction of a virion (viral attachment protein) with a specific receptor (mostly glycoprotein) site on e surface of a cell.Receptor binding is believed to reflect fortuitous configurational homologies b/w a virion & a cell.E absence or presence of receptors determines e cell tropism & pathogenesis of a virus.Penetration:I.e. virus particle is taken up or engulfed inside e cell.May involve 3 mechanisms:Receptor-mediated endocytosis by using endosome & also called as viropexis.Translocation or direct penetration oe e entire virion across e plasma membrane.Fusion of e viral envelope with e plasma membrane of e cell: involve e interaction of a viral fusion protein with a 2nd cellular receptor or coreceptor.Uncoating:I.e. e physical separation of e viral nucleic acid from e outer structural components of e virion so that it can function.E genome may be released as a free nucleic acid or as a nucleocapsid (usually contain polymerases).Occurs concomitantly with or shortly after penetration. May require acidic pH in e endosome & e infectivity of e parental virus is lost at uncoating stage.EXPRESSION OF VIRAL GENOMES & SYNTHESIS OF VIRAL COMPONENTSE specific mRNAs must be transcribed from e viral nucleic acid for successful expression & duplication of genetic information.Then, viruses use cell component to translate e mRNA. However, virus-specific proteins may regulate e extent of transcription of e genome or e translation of viral mRNA.Viral protein is synthesized in e cytoplasm on polyribosome composed of virus specific mRNA & host cell ribosome. E larger e virus, e > independent of cellular function & is > susceptible to antiviral chemotherapy (> virus-specific processes r available for drug action).Replication strategy:RNA containing viruses:-ve sense viruses: carry RNA polymerase to synthesize mRNA.Most genetic information is expressed at e same time.Widest variation in strategies of gene expression:Some virions carry polymerases.Some systems utilize subgenomic messages which sometimes generated by splicing.Some viruses synthesize large polyprotein precursors that r processed & cleaved to generate final gene products.Viral genomic RNA is generally duplicated in cell cytoplasm.DNA containing viruses:Early viral protein r synthesized soon after infection & late protein r made only late in infection, i.e. after viral DNA synthesis.DNA viruses that replicate in e nucleus use host cell DNA & RNA polymerases & processing enzymes.MORPHOGENESIS & RELEASE.Morphogenesis or assembly:Involve e assembly of all e components necessary for e formation of e mature virion at a particular site of e cell.During this stage, e basic structure of virus is formed & newly synthesized viral genomes & capsid polypeptide assemble together to form progeny viruses.Icosahedral capsid can condense in e absence of nucleic acid, whereas nucleocapsids of viruses with helical symmetry cannot form w/o viral RNA.Non-enveloped viruses accumulate in infected cells & e cell eventually lyse & release e virus particles. E site of assembly varies for different viruses.Maturation:I.e. e stage of e life-cycle at which e virus becomes infectious.Usually involves structural changes in e particle, often resulting from specific cleavage of polyproteins to form e mature individual protein products.Enveloped viruses may mature by a budding process. So that they r not infectious until they have acquired e envelopes. Therefore, infectious progeny do not accumulate in e cell.However, excess amounts of viral components may accumulate & lead e formation of inclusion body as CPE which cause cell death.Release:Lytic viruses (most non-enveloped): e cell breaks open & release e virus.Enveloped viruses: acquire e lipid membrane as e virus buds out through e cell membranes. Virion envelope proteins r picked up during this process as e virus is extruded.Budding may or may not kill e cell (controlled by e virus): e physical interaction of e capsid proteins on e inner surf. of e cell membrane forces e particle out though e membrane. REPLICATION STRATEGIESMammalian cells can only make DNA & RNA in e nuclear compartment i.e. viruses replicating in e cytoplasm must make their own polymerase.There is no mammalian cell enzyme able to use RNA as a template for RNA or DNA synthesis. Viruses with an RNA genome have to make their own polymerase.Many RNA viruses with a +ve sense strand do not need to contain a virion-associated polymerase. E genomic RNA can serve as mRNA, to make e various viral proteins including polymerase enzymes, upon cell entry.RNA viruses with a –ve sense strand must contain a virion associated polymerase to make a +ve sense strand RNA i.e. mRNA upon cell entry.VIRUS-HOST INTERACTIONPORTIONDETAILSTYPES OF INFECTIONTypes of viral infection:Productive infection: occurs in permissive cells & results in e production of infectious virus (few to thousand).Abortive infection: fails to produce infectious progeny, either because e cells r non-permissive or unable to support e expression of all viral genes or because e infecting virus may be defective.Latent infection: may ensue with e persistence of viral genomes, e expression of no or a few viral genes, & e survival of infected cells.PRODUCTIVE INFECTIONCYTOCIDAL OR LYTICWhen host cells caused to be lysed when virus progeny virions released.Can be seen in cell culture which is known as cytophatic effect (CPE).CPE: virus-induced damage to e cells that alters its microscopic appearance.E effect: cells r killed.E.g.:Poliovirus: cell lysis.Smallpox & influenza viruses: cell rounding.Adenovirus: cell clumping.PRODUCTIVE TRANSFORMATIONHost cells r not killed (lysed) but undergo neoplastic transformation.Entire genome integrates into that of host cells & expresses oncogenes.Host cells r transformed to a state of uncontrolled cell division.Continuous release of infectious viral progeny is usually by budding.E.g.: RNA tumor (oncogenic) virus which r:Human T cell leukemia virus.Sarcoma virus.Mammary tumor virus.PERSISTENCEInfected cells & virus coexist over a long period of not.E exact mechanism is not known.Defective interfering particles temperature sensitive mutants & infection (all implicated).E.g.: lymphocytis chonemeningitis.NON-PRODUCTIVE INFECTIONE initial stage: same as for productive viral infection.Then, non-productive infection can have different courses: apoptosis, non-productive transformation, & latency.May be due to:Thus, replication can’t be completedA block on one stage of replication cycleViral genome may be defective.Viral genome mat be too small: parvoviridae (need 2nd helper virus like adenovirus).Mutation.APOPTOSISEarly viral replication steps: trigger host cell apoptosis response.Host & virus both have no propagation.E.g.: vaceinra virus in Chinese hamster ovary cells results in cell rounding, condensation of chromatin, & fragmentation of DNA.NON-PRODUCTIVE TRANSFORMATIONVirus infects e cell. Then, virus oncogenes integrate with cell DNA leading to cell transformation.Transformed cells do not yield infectious progeny virus: non-productive.E.g.: papilloma virus (ware-viruses) of 77 types, some r causally involed in cancer of servix & cancer of anogenital organs.LATENCYIn 1oinfection, viral infection produces progeny (productive).Then, 2o latent infection affects neural cells (for life).Viral genome remains intact during latency in nucleus of host cell.Can reemerge t/out life: especially during immunosuppresed.E.g.:HSV & H2V: cause chickenpox & shingles (appear in accordance to dermatosome).Epstein-Barr virus (EBV): cause nasopharyngeal carcinoma, Burkitt’s lymphoma, & infectious mononucleosis. TRANSFORMATIONA type of virus-cell interaction.Properties of all change dramatically.Transform cells have similar properties to tumor cells.E.g.: Herpes virus, adenovirus, hepadna virus, papovavirus, & poxvirus.Characteristics of transformed cells:Loss of contact inhibition of growth.Fewer requirements for serum factors.Indefinite no. of cell divisions.Viral Ag exposed.Fibroretinas absent.Fetal Ag present.VIRAL ENTRYSkin:Breached through transcutaneous infection & IV route.Infection via vectors, usually insects. E.g.: togavirus, flavivirus, bunyavirus, reovirus, & rhabdovirus (bite).Few r from skin lesion: HSV, varicella virus, mollusoum contagiosum, & ebola virus.Iatrogenic infection: Hepatitis B, C, & D while STD: papilloma virus, HSV, Hepatitis A virus, & HIV. Mucosa: oropharyngeal, GIT, respiratory tract, & eye.VIRAL SPREADOnce infection is established, virus spread:At e site of entry:Cell to cell: viral particles r released at apical end of epithelium.Disseminated: viral particles r released at basal end of epithelium via hematogenous (viremia) & lymphatic spread.Neural spread:Mechanism is similar to entry into other cells.Uncoated nucleocapsid is carried passively along axons & dendrites by axoplasmic particles.E.g.: rabbit virus, poliovirus, reovirus, & herpes virus. GENETIC & PHENOTYPIC CHANGES IN VIRUSESPORTIONSDETAILSMUTATIONMutation is a heritable change in e genotype & virus which subjected to mutation causes minor or subtle genetic changes.Mechanisms of mutation:By e effects of chemical or physical mutagens such as UV light & x-ray on nucleic acids.By e natural behavior of e bases that make up nucleic acids (resonance from keto to same in all enol & from amino to imino form). viruses.Through e fallibility (lack of fedility) of e enzymes that replicate e viral nucleic acids (viruses with high-fidelity transcriptases have relatively low mutation rates & vice versa).Mutation rates:DNA viruses: similar to those of eukaryotic cells because their replicatory enzymes have proofreading functions. 10-8 to 10-11 errors per incorporated nucleotide.RNA viruses: lack of proofreading function in their replicatory enzymes. 10-3 to 10-4 errors per incorporated nucleotide.Types of mutations:Lethal mutation: mutations that interfere with e essential functions of attachment, penetration, uncoating replication, assembly, & release. Rapidly lost from e population.Neutral mutation: d/t redundancy of e genetic code which results in either no change in e viral protein or in replacement of an amino acid by a functionally similar amino acid.Favorable mutation: mutations that do not impair essential viral functions which persist or become fixed in a virus population.Phenotypic Variations caused by mutation (e mutants):Conditional-lethal mutants: mutants that r lethal under non-permissive condition but that yields normal infectious progeny in permissive condition.Temperature-sensitive mutants: grow at low temperatures (cold-adaptation mutants) but not at high temperature.Host-range mutants: able to grow in permissive cells but r abortive in non-permissive cell.Antigenic drift virus (minor antigenic variation): antigenically altered viruses which r able to cause disease in previously resistant or immune hosts. E.g.: antigenic drift in e epitope of e hemagglutinin spike of influenza A virus.Attenuated Vaccine Strains from mutation: occur in e mutations result in (1) - (3) mutants but with intact antigenicity & reduced pathogenicity. E.g.: e Sabin vaccine strain of polioviruse.Back mutations (reversion): vaccine strain which has fewer mutations & thus less stable & is subjected to restore e neural virulence. This vaccine strain causes disease in vaccinated individuals.Others: drug resistant mutants & suppressor mutation (intragenic & extragenic).GENETIC INTERACTION B/W VIRUSESRECOMBINATION & REASSORTMENTRecombination results in e production of progeny virus that carries trait not found together in either of e parents.Generally occurs b/w members of e same virus type & yields genetically stable progeny.2 mechanisms of recombination:Independent reassortment:Occurs when related viruses that have segmented genomes exchange segments during replication.These genes r unliked & assorted at random.Only RNA viruses have segmented genomes such as orthomyxoviridae, reoviridae, arenaviridae, & etc.E.g.: Antigenic shift (immediate & major antigenic change) in Influenza A virus (b/w animal & human strain) which gives rise to pandemic infection.Intramolecular recombination:Known to occurs in viruses with non-segmented genomes either by:DNA viruses: occur by annealing, breakage, crossing over, & reunion.RNA viruses: occurs by strand strand switching or strand jumping where no breakage in viral RNA molecules. Results in e production of:New viral serotypes (antigenic types).Viruses with altered virulence.Reactivation of genetically inactivated viruses.Genetic Reactivation:Marker rescue:Occurs b/w e genome of active virion & e genome of a virus particle that has been inactivated in some way.Results in e rescue of certain markers (active gene) of inactivated parents which r present in viable progeny.None of e progeny produced r identical to e inactivated parent, but r genetically stable.Multiplicity reactivation:E production of infectious virus by a cell infected with 2 or > virus particles of e same strain, each of which has suffered a lethal mutation in a different gene.Occurs b/w e damaged nucleic acids of e parents, producing viable genome that can replicate.E greater e damage to e parental genomes, e larger e no. of inactive particles required per cell to ensure e formation of viable genome. INTERFERENCEI.e. e inhibition of multiplication of one of 2 viruses infecting e same cell cultures or e whole animals.Does not occur with all viral combinations, 2 viruses may infect & multiply within e same cell as efficiently as in single infection.Causes of interference:One virus may inhibit e ability of e second to adsorb to e cell, by blocking or destroying its receptors.One virus may compete with e second for components of e replication apparatus.E first virus may cause e infected cell to produce an inhibitor that prevents replication of e second virus.PHENOTYPIC INTERACTIONS B/W VIRAL GENE PRODUCTSPOLYPLOIDYSeen in enveloped viruses. 2 types:Polyploidy: virus particles having multiple copies (nucleocapsid) of same genome.Heteroploidy: virus particles having multiple copies of different genomes.PHENOTYPIC MIXING3 types:Transcapsidation.Mixed capsidation.Mixed PLEMENTATIONE interaction of viral gene products in cell infected with 2 viruses, one or both of which may be defective.Results in e replication of one or both under condition in which replication will not ordinarily occur.One virus provides a gene product in which e second is defective, allowing e second to grow.E genotypes of e 2 viruses remain unchanged.However, if both mutants r defective in e same gene product, they will not able to complement each other’s growth.GENE THERAPYGene transfer to mammalian cells through transformation, transfection, & microinjection.Recombinant viruses r developed to carry normal human gene that could be useful as vectors for gene therapy.Target diseases: diabetes, cystic fibrosis, SCID, & etc.VIRAL VACCINESPORTIONSDETAILSOVERVIEWE purpose: to utilize e immune response of e host to prevent viral disease.Immunity to viral infection is based on e development of an immune response to specific Ags located on e surface of virus particles (for enveloped virus r e surface glycoprotein) or virus-infected cells.TYPES OF VACCINEKILLED-VIRUS VACCINESInactivated vaccines r made by purifying viral preparations to a certain extent & then inactivating viral infectivity in a way that does minimal damage to e viral structural proteins.Generally stimulate e development of circulating Ab against e coat proteins of e virus, conferring some degree of resistance but e replicative function should be destroyed.Inactivation may be mediated by heat or chemicals such as formaldehyde or β-propiolactone.Advantages: No reversion to virulence by e vaccine virus.Gives sufficient humoral immunity if boosters r given.Can be used in immunodeficient patients.Sometimes better withstands adverse environmental conditions in e tropics.Disadvantages:Extreme care is required in their manufacture to make certain that no residual live virulent virus is present in e vaccines.E immunity conferred is often brief & must be boosted which involved e concern about e possible effects (HS reactions) of repeated administration of foreign proteins.Parenteral administration of this vaccine sometimes gives limited protection because local resistance (IgA) is not induced adequately at e natural portal of entry.E cell-mediated immune response to inactivated vaccine is generally poor.Some killed-virus vaccines have induced HS to subsequent infection.ATTENUATED LIVE-VIRUS VACCINESPrepared from attenuated strains that r almost or completely devoid of pathogenicity but r capable of inducing a protective immune response.Methods of attenuation:Administration of pathogenic or partially attenuated virus by an unnatural route: used in e immunization of military recruits against ARDS using enterically coated live adenovirus types 4, 7, & 21.Passage of e virus in an unnatural host or host cells to induce host-range mutation: Used in e most vaccines for men & animals.Examples of human vaccines attenuated by inducing host-range mutation:17D vaccine strain of yellow fever passed in mice & chick embryos.Live-attenuated poliovaccine were passaged in monkey kidney cells.Measles passaged in e chick embryo fibroblast.Rubella & mumps.Development of temperature-sensitive mutants: used in e conjunction with e above method.Advantages:Act like e natural infection with regard to their effect on immunity.They multiply in e host & tend to stimulate longer-lasting Ab production, to induce a good cellular immunity, & to induce Ab production & resistance at e portal of entry.Disadvantages:E risk of reversion to greater virulence during multiplication within e vaccine & underattenuation.Unrecognized adventitious agents latently infecting e culture substrate may enter e vaccine stocks.E storage & limited shelf life of attenuated vaccines present problems, but can be overcome by e use of viral stabilizers.Interference by coinfection with a naturally occurring, wild-type virus may inhibit e replication of e vaccine virus & decrease its effectiveness. Preparation instability & heat lability.Cannot be given to immunocompromised or pregnant patients.Spread to contacts of vaccine who have not consented to be vaccinated.SUBUNIT VACCINESVaccines with purifying viruses & viral Ag.Examples:HA vaccines for influenza A & B.HBs Ag vaccine: in e past derived from e plasma of chronic carriers.Problem: increasing purification may lead to loss of immunogenicity & this may necessitate coupling to an immunogenic carrier protein or adjuvants such as aluminium salt.RECOMBINANT VACCINESProduced by recombinant DNA technology.2 types:Recombinant viral proteins:Virus proteins expressed in bacteria, yeasts, & mammalian cells.Examples:E. coli r 1st to be used, but e expressed proteins r not glycosylated & caused drawback.Recombinant Hep B vaccine is e only recombinant vaccine licensed at present.Hybrid virus vaccine:An alternative application of recombinant DNA technology.Example: polyvalent live hybrid virus vaccine – e gene of several viruses or even other organism can be inserted into virus (e.g.: Vaccinia virus) to generate a hybrid virus vaccine that can express several exogenous immunizing Ags.SYNTHETIC PEPTIDESE vaccination depends on e identification of immunogenic sites. E.g.: food & mouth disease.Advantages of defined viral Ags or peptides:Production & quality control simpler.No NA or other viral or external protein, therefore < toxic.Safer in cases where viruses r oncogenic or establish a persistent infection.Feasible even if virus cannot be cultivated.Disadvantages of defined viral Ags or peptides:May be less immunogenic than conventional inactivated whole-virus vaccines.Requires adjuvant or carrier protein.Requires 1o curse of injection followed by boosters.Fails to elicit CMI.ANTI-IDIOTYPE ANTIBODIESHas been developed d/t e ability of anti-idiotype Abs to mimic foreign Ags & induce IR against e viruses, bacteria, & protozoa.Advantages of anti-idiotype antibodies:Have many potential uses as viral vaccines, particularly when e Ag is difficult to grow or hazardous.Have been used to induce immunity against a wide range of viruses, including HBV, rabies, Newcastle, disease virus & FeLV, reoviruses, & polioviruses.DNA VACCINESDNA coding for e foreign Ag is directly injected into a relatively no-dividing tissue such as muscle of an animal so that e foreign Ag is directly produced by e host cells.Advantages:DNA is relatively inexpensive & easier to produce than conventional vaccines.E time for development is relatively short which may enable timely immunization against emerging infectious diseases.Precautions:Delivery of e DNA to cells is still not optimal.E possibility that e vaccine’s DNA will be integrated into host chromosomes & turn on e oncogene or turn off e tumor suppressor gene.Extended immunostimulation by e foreign Ag may provoke chronic inflammation or autoAB production.Examples:1st clinical trial: HIV in 1995 & subsequent trial in 1996. Technique used: direct injection of plasmids which is Vaccines for influenza virus, HSV, & T cell lymphoma. taken up by host cells for expression.REVERSE VACCINOLOGYAdvantages:Fast access to virtually every Ag.No-cultivable can be approached.Non-abundant Ags can be identified.Ags not expressed in vitro can be identified.No-structural proteins can be used.Disadvantages: non-proteinous Ags like polysaccharides & glycolipid cannot be used.PRESENTATION OF IMMUNOGENIC PROTEINS & PEPTIDESProteins separated from virus particles r generally much less immunogenic than e intact particles.This different is d/t e change in configuration of a protein when it is released from e structural requirements of e virus particle.Attempts to enhance e immunogenic activity of separated proteins by:Adjuvants:Potentiate e IR with modes of action:Functions to localize & slowly release Ag at or near e site of administration.Functions to activate APCs to achieve effective Ag processing or presentation.Materials used as adjuvants:Aluminium salt: e only used for human vaccines.Mineral oils: vaccines administrated as a water-in-oil emulsion which is slow in breakdown & gradually release e Ag. It can be potentially carcinogenic.Bacterial cell wall: mycobacterial product (Freund’s adjuvants).Liposomes:I.e. artificial lipid membrane spheres into which immunizing proteins (& adjuvants) r incorporated. Can stimulate CMI.E immunostimulating complexes have activities equivalent to those of e virus particles from which e proteins r derived.PROBLEMS IN VACCINATIONDifferent types of virus may cause similar disease.Antigenic drift & shift, especially true of RNA viruses & those with segmented genomes.Animal reservoirs which can lead to reinfection after elimination from e human population.Integration of viral DNA which can hide from e IR.Transmission of viruses from cell to cell via syncytia for potential AIDS vaccine.Recombination & mutation of e virulent strain or of e vaccine virus. VACCINATION PROGRAMMEMalaysia vaccination programme:ImmunizationAge (months)Age (years)012356121861215BCG1If no scarHep B123DPT1234DTTOPV12345Hib123Measles Sabah MMR12Other available vaccines in KKM:Pneumococcal polysaccharide vaccine: single dose, booster 3-5 years.Meningococcal vaccine: single dose, immunity up to 3 years.Cholera: 2 doses 4 weeks apart (min 1 week), booster every 6 months.Japanese B encephalitis vaccine: 3 doses, booster at 4 years of age.Rabies: pre-exposure immunization (3 doses, booster every 2-3 years) & post exposure vaccination (treatment).Thypoid: Vi polysaccharide vaccine (single dose), oral thypoid vaccine (3 doses 2 days apart), & whole cell thypoid vaccines.Vaccines in Malaysia but not yet in KKM program:Varicella zoster: 12 months to 12 years (single dose) & >12 years (2 doses, 28 days apart).Hepatitis A whole inactivated vaccine: children (3 doses) & adult (2 doses, 6 to 12 moths apart).Influenza: yearly revaccination with e latest composition by WHO.Yellow fever vaccine: for travel purpose only.SUCCESSFUL VACCINATION Small pox:Variolation:1st used by Chinese & widespread in England in 1700s.Procedure: material was obtained from e pustules of an infected person who had minor smallpox (milder form). This material was used to infect another person who also got e milder disease. If e person does not die, he will develop lifelong immunity.Vaccination:In 1796, Jenner discovered vaccination using vaccinia virus, e agent of cowpox.Those who had cowpox, will gain protective immunity against > virulent smallpox.Reasons for successful smallpox vaccination:No animal reservoir.Lifelong immunity.Subclinical cases r rare.Infectivity does not precede overt symptoms.One variola serotype.Availability of effective vaccine.Poliomyelitis.Live versus Killed VaccinesLive vaccinesFeatures Killed vaccinesLow DoseHigh Single (few)No. of dosesMultiple Many yearsDuration of immunityLess IgG & IgA Ab responseIgGGood (d/t replication)Cellular immunityPoor Possible Reversion to virulenceNot possibleNo Need to adjuvantsYes [47.7] Wahai orang-orang yang beriman, kalau kamu membela (ugama) Allah nescaya Allah membela kamu (untuk mencapai kemenangan) dan meneguhkan tapak pendirian kamu.98171023495ARBOVIRUS & DENGUE FEVERPORTIONDETAILSOVERVIEWArbovirus: arthropod borne virus (those infecting human r zoonotic).Multiply in tissue of arthropods & r passed into new vertebrates when arthropods take a subsequent blood meal after a period of extrinsic incubation.TAXONOMYNo taxonomic significance with 535 heterogenous group of virus.Under 7 taxonomic families & many r zoonotic which do not require arthropod vectors.Family Genus Shape Nucleic acidEnvelope Togaviridae Alpha virusungroupCube +ve ssRNA+Flaviviridae FlaviviparousCubic +ve ssRNA+Bunyaviridae Bunyavirus Phlebovirus Nairovirus Uukuvirus Hantavirus Helical -ve ssRNA+Reoviridae Orbivirus Cotivirus Unassigned Cubic dsRNA-Rhabdoviridae Vesicuvirus Lyssavirus Unassigned Bullet-shape-ve ssRNA+DISEASES3 clinical syndromes:Fever of an undifferentiated type with or w/o a moculopapular rash & usually benign.Encephalitis often with high fatality rate.Hemorrhagic fever which is frequently severe & fatal. E degree of viral multiplication & its predominant site of localization in tissue determine e clinical syndromes.DENGUE FEVEREPIDEMIOLOGYDengue (breakbone fever) is a mosquito-borne infection caused by a flavivirus that is characterized by fever, severe headache, muscle & joint pain, nausea & vomiting, eye pain, & rash.4 serotypes of dengue virus transmitted in tropical area b/w 35oN + 35oS.Vectors: A. aegypti (principle), A. albofictus, A. polynesiesi, A. cochi, & A. scuttelaris bebrideus. Definitive & reservoir host: only human, thus not a zoonosis.VECTORSPrinciple vector which is A. aegypti:Bread around human, but aviposits in stagnant water.Adults take shelter indoor.Bite during 1-3 hours, in e morning & late afternoon.Fly as long as 0.8 to 2.0 km.In endemic area: 1/20 mosquitoes is infected.Transmission in tropical area is t/out e year, high during raining season, high humidity, long lifespan, high temperature, & low extrinsic incubation period.PATHOGENESISDengue fever:Usually is a self-limited infection.Clinical disease begins 4-7 days after infection. E onset of fever may be sudden with malaise (d/t cytokine response), chills, & headache.Pain develops especially in e back, joints, muscles, & eyeball: d/t HDE perivascular mononuclear infiltrate & lipid accumulation (high creatinine phosphokinase & hepatic transaminase).Viremia at e onset of fever & may persist for 3-5 days but e temperature returns to normal after 5-6 days & subside on e 3rd day & rise again about 5-8 days after onset (saddleback pattern). Rash (lymphatic dermal vasculitis) may appear on e 3rd or 4th day & lasts for 1-5 days.Virus infects bone marrow & causes peripheral cytopenia & hemophagocytosis.Dengue hemorrhagic fever (DHF):May occur in individuals with passively acquired or preexisting nonneutralizing heterologous dengue Ab d/t previous infection with a different serotype of virus.Initial symptoms stimulate normal dengue, but e patient’s condition is abruptly worsen. Dengue shock syndrome (DSS):Characterized by shock & hemoconcentration.Fatal cases d/t kuppfer cells hyperthropy & focal ballooning.Shock is d/t sudden extravasation of plasma into extravascular compartment including pleura & abdominal cavity usually when fever has declined.DSS is also d/t high TNF, ILs, IFN-γ, & complement activation.Results: loss of fluid, hypoproteinemia, & albuminemia.Reversal usually within 48 hours of DHF.Pathogenesis: 2ndary infection → heterologous or enhancing, non-neutralizing Abs increase uptake of e virus → increase replication in Fc-bearing cells (Abs mediated immune enhanced) → high TNF-α, CD8, & IL2 → cross reactivity → CD4 & CD8 increase → high IFN-γ & IL-2 → DSS.VIROLOGYFamily: flaviviridae.Virions:Consists of M (membrane) glycoprotein & E (envelope) glycoprotein.Dimmers packed horizontally head to tail on virion surface.Unstable in heat, UV light, disinfectant (alcohol & iodine), acid, & ether.CLINICAL PICTURESDengue fever: acute fibrile disease, headache, musculoskeletal pain, rash, fever, malaise, rash, irritability, & URTI.DHF & DSS:Effusion of pleura (80%), ascites, gallbladder, pararenal, liver, spleen, & pericardial.Myocardial dysfunction, metabolic acidosis, respiratory distress, encephalitis, liver & renal failure.Fatality rate: up to 50%.Adults:> severe & acute.Chills, severe frontal headache, retroorbital pain, severe musculoskeletal & lumbar back pain.Skin flushed & rashes.LAB DIAGNOSISPCR: for rapid identification & serotyping of dengue virus in acute phase serum during e period of fever.Viral isolation: done 1 week after onset by inoculation of a mosquito cell line with patient’s serum, coupled with NA assays.Culture: specimen obtained from biopsy or autopsy & cultured in suckling mice.Serological test:By cross-reactivity of IgG to heterologous flavivirus Ags.Specimen obtained from blood & CSF.E.g.: ELISA & hemagglutinin inhibition test.TREATMENTTreatment is symptomatic & supportive (fluid replacement therapy).PREVENTIONMainstay: Public health: insectidal fogging & vaccine (under development).Personal hygiene at home.HERPESVIRUSESPORTIONSDETAILSOVERVIEWUnique property: ability to establish lifelong persistent infections in their hosts & to undergo periodic reactivation. However, e reactivated infection may be clinically quite different from e disease caused by 1o infection, but is > serious in immunocompromised patients.Structure & composition:Virion: large & spherical virus with diameter of 150 – 200 nm (enveloped) & of 125 nm (naked).Genome: double-stranded, linear, 125-240 kbp DNA in e form of toroid & reiterated sequences, surrounded by a protein coat that exhibits icosahedral symmetry & has 162 capsomeres. Nucleocapsid: surrounded by an envelope that is derived from host’s nuclear membrane & contains viral glycoprotein spikes about 8 nm long & Fc receptors.Proteins: > than 35 proteins in e virion.Tegument: an amorphous, asymmetric structure b/w capsid & envelope.CLASSIFICATIONα-herpesviruses:Fast-growing, cytolytic viruses that tend to establish latent infection in neurons.Members: HSV 1&2 & Varicella-zoster virus. β-herpesviruses:Slow-growing & cytomegalic viruses which become latent in secretory glands & kidneys.Members: CMV, HHV-6, &HHV-7. γ-herpesviruses:Infect & become latent in lymphoid cells.Members: EBV & HHV-8.REPLICATIONVirus binds to receptor on envelope glycoprotein (glycosaminoglycan) → enters e cell by fusion with e cell membrane → e capsid is transported trough e cytoplasm to a nuclear pore → capsid uncoating → DNA associated with e nucleus → viral DNA becomes circle immediately upon release from e capsid → VP16, a tegument protein, complexes with several cellular protein & activate initial viral gene expression → immediate-early genes r expressed → α-proteins r produced → e proteins permit expression of e early set of genes → translated into β-proteins → viral DNA replication begins → late transcripts r produced & produce γ-proteins → maturation occurs by budding of nucleocapsids through e altered inner nuclear membrane → enveloped viral particles r transported by vesicular movement to e cell surface.Length of replication is 18 hours (HSV) – 70 hours (CMV).HERPES SIMPLEX VIRUSPROPERTIES2 serotypes of HSV-1 & HSV-2 which grow rapidly & r highly cytolytic.E genomes:Large (~ 150 kbp) & can encode at least 70 polypeptides.Similar in organization & exhibit substantial sequence homology.Can be distinguished by sequence analysis or by restriction enzyme analysis of viral DNA.They share several cross-reactive epitopes with each other & also with VZV.Man is e only natural host.Mode of transmission:HSV-1: spread by contact usually involving infected saliva or tears.HSV-2: transmitted sexually or from a maternal genital infection to a newborn.Types of viral glycoproteins:One (gD): e most important inducer of neutralizing Abs.Glycoprotein C: C3b-binding protein.gE: an Fc receptor.Glycoprotein G: type-specific & allow for antigenic discrimination b/w HSV-1 & HSV-2.EPIDEMIOLOGYHSV-1:1o infection occurs early in life (6 months to 3 years) & usually asymptomatic with development of Abs, but e virus is not eliminated.A carrier state is established t/out life & punctuated by transient recurrent attacks of HSV.Risk factors: lower socio-economic population with crowded living condition & poorer hygiene.Generally causes infection above e belt.E frequency of recurrences varies widely among individuals.HSV-2:Seroprevalence is higher among women than men & higher among blacks than white.Abs r seldom found before puberty d/t STD.Recurrent genital infections may be symptomatic or asymptomatic & tend to recur > often than HSV-1.Maternal genital HSV infection poses risks both to mother & fetus.Genital HSV infection increases in HIV type 1 patients.PATHOGENESISPathology:Cytolytic infection d/t necrosis of infected cells with inflammatory response.Lesion in e skin & mucous membrane is e same in HSV-1 & HSV-2.Cytophatic effects:Cell ballooning.Productions of Cowdry type A intranuclear inclusion bodies.Margination of chromatin.Formation of multinucleated giant cells.Cell fusion: provides method for cell to cell spread.1o infection: Virus enters through mucosal surface or broken skin → replication at e site of infection → invasion of local nerve ending → transported to dorsal ganglia by retrograde axonal flow → further replication → latency.Latent infection:Establish latency in: trigeminal ganglia (HSV-1) & sacral ganglia (HSV-2).Latency: non-replicating state which uses microRNA to prevent cell death & maintaining e latency. Latency lasts for e lifetime & no virus can be recovered b/w recurrences.Provocative stimuli can reactivate e virus such as axonal injury, stress, or exposure to UV light.Reactivation: virus follows axons back to e peripheral site & replication proceeds at e skin or mucous membrane.CLINICAL FINDINGSOropharyngeal disease (HSV-1):Most frequently in small children (1-5 years) & involves e buccal & gingival mucosa of e mouth.Incubation period: 3- 5 days with clinical illness lasts 2-3 weeks.Symptoms: fever, sore throat, vesicular & ulcerative lesions, gingivostomatitis, pharyngitis, tosillitis & malaise.Recurrent disease: cluster of vesicles localized at e border of e lip with intense pain which fades over 4-5 days.Vesicles → pustules → crusting stage → heal w/o scarring after 8-10 days.Keratoconjunctivitis (HSV-1):Recurrent lesion: dendritic keratitis, corneal ulcers, or vesicles on e eyelids.There may be progressive involvement of corneal stroma with permanent opacification & blindness.Genital herpes (HSV-2):10 infections: Lasts about 3 weeks & characterized by vesiculoulcerative lesion of e penis or cervix, vulva, vagina, & perineum which is painful & present with fever, malaise, dysuria, & inguinal lymphadenopathy. Complications: extragenital lesions, aseptic meningitis, & viral excretion (~ 3 weeks).Recurrences: common & mild with appearance of vesicle about 10 days.Skin infections:Occur in abrasion that becomes contaminated with e virus.Lesions r seen on e fingers of dentists & hospital personnel & e body of wrestlers.Become severe & life threatening in individuals with skin disorders that permits extensive local viral replication & spread:Eczema herpeticum: a potentially serious disease that occurs in patients with eczema.Herpetic withlow which arise from implantation of e virus into e skin & typically affect e fingers.Encephalitis: A necrotizing encephalitis which is localized & usually at hippocampus.Has a high mortality rate & often survive with neurological defects.Neonatal herpes:Acquired in utero, during birth (through birth canal with herpetic lesion: avoid by using cesarean) or after birth (from family members or hospital personnel). Viral pneumonitis or intravascular coagulopathy.3 categories of disease:Lesions localized to e skin, eyes, & mouth.Encephalitis with or w/o localized skin involvement.Disseminated disease involving multiple organs such as CNS.Mortality rate of untreated disease is 50% & e prognosis can be worst.Infections in immunocompromised patients: Involved patients who r HIV patients, organ transplantation, malignancies, malnutrition, & immunosuppressed d/t diseases or therapy.IMMUNITYMany newborns acquire passively transferred maternal Abs which lost after 1st 6 months of life which is e period of greatest susceptibility until 2 years old.HSV-1 Abs begins to appear at e early childhood, & by adolescence, they appear in most persons.HSV-2 Abs rise during e age of adolescence & sexual activity. In 1o infections, IgM appears 1st, then followed by IgG & IgA Abs that persist for long periods.E Abs do not prevent reinfection or reactivation of latent virus but may be modify subsequent disease. LAB DIAGNOSISCytopathology: specimen obtained from e base of e vesicles & e presence of multinucleated giant cells indicates HSV is present.Isolation & identification: virus may be isolated from herpetic lesions, throat washing, CSF, & stool both during 1o & asymptomatic periods, but is not sufficient evidence for indication of causative agent. Serology: Abs appear in 4-7 days after infection & reach a peak in 2-4 weeks which is useful to document to recent infection. Direct detection:Electron microscopy of vesicle fluid: ra[I but can’t distinguish b/w HSV & VZV. PCR: now is used routinely for e diagnosis of herpes simplex encephalitis (from CSF).Immunofluoresence of skin wrappings: can distinguish b/w HSV & VZV. TREATMENT & PREVENTIONDrug therapy:DOC: acyclovir, valacyclovir, & vidarabine which r e inhibitors of viral DNA synthesis.E drugs may suppress clinical manifestation, shorten time to healing, & reduce recurrences of genital herpes.Vaccination: purified glycoprotein Ags found in e viral envelope.Prevention: susceptible patients should be protected from e virus.VARICELLA-ZOSTER VIRUSPROPERTIESProperties of e virus:It has no animal reservoir, but virus propagates in e cultures of human embryonic tissue & produce typical intranuclear inclusion bodies.CPE r > focal & spread much > slowly than HSV.Infectious virus remains strongly cell-associated & serial propagation is > easily accomplished by passage of infected cells than of tissue culture fluids.Same virus produces both chickenpox & shingle, & children who have recovered from zoster virus-induced infection r resistant to varicella.Overview of e diseases:Varicella (chickenpox): mild, highly contagious disease (children) characterized by a generalized vesicular eruption of e skin & mucous membranes. E disease is severe in adults & immunocompromised patients.Zoster (shingle): a sporadic, incapacitating disease of adults & immunocompromised individuals that is characterized by a rash on e skin innervated by a single sensory ganglion.EPIDEMIOLOGYVaricella & zoster occur worldwide.Varicella is highly communicable & is a common epidemic disease of childhood, but rare in adulthood (common in winter & spring).A live attenuated vaccine is available.Varicella spreads readily by airborne droplets & by direct contact but r less common in zoster because e virus is absent from e upper respiratory tract.A varicella patient is infectious shortly before e appearance of rash to e 1st few days of rash.PATHOGENESISVaricella:Route of infection: e mucosa of e upper respiratory tract or e conjunctiva.Initial replication in regional lymph nodes → 1o viremia spreads virus → replication in liver & spleen → 2o viremia involving infected mononuclear cells transports virus to skin → rash develops → swelling of epithelial cells, ballooning degeneration, & accumulation of tissue fluid → vesicle formation.Virus replication & spread r limited by host humoral & cellular IR.Zoster:Skin lesions r histopathologically identical to varicella.Sensory neurons & ganglia: acute inflammation & only single ganglion may be involved.E distribution of lesions in skin corresponds closely to areas on innervation from an individual dorsal root ganglion.Waning immunity allows viral replication to occur in a ganglion, causing intense inflammation & pain. Virus travels down e nerve & induces vesicle formation.Cellular immunity is e most important host defense & reactivations r sporadic & recurs infrequently.CLINICAL FINDINGSVaricella:Incubation period of typical disease is 10-21 days.Symptoms: malaise & fever → rash on e trunk → rash on e face, limbs, buccal & pharyngeal mucosa in e mouth → successive fresh vesicles appear in crops → all stages of macules, papules, vesicles, & crusts may be seen at one time (e rash lasts about 5 days).Complications: rare in normal children but encephalitis can occur & varicella pneumonia is most common complication & mortality rate is very low.Neonatal varicella: E infection is contracted from e mother just before or after but w/o sufficient IR to modify e disease.Virus is often widely disseminated & may prove fatal.Varicella according to e week of gestation:1st 20 weeks of pregnancy: scarring of skin & hypoplasia of limbs (3% chance of transmission).After 20 week gestation: VZV can cross e placenta in e late stages of pregnancy to infect e fetus congenitally & e disease very fom mild to a fatal disseminated infection. Immunocompromised patients: increased risk of complications & DIC may occur & rapidly fatal.Zoster:Starts with severe pain in e area of skin & mucosa supplied by one or > group of sensory nerves & ganglia.Few days after onset, a crop of vesicles appears along e dermatotomes & e trunk, head, & neck r most commonly affected.E complication in elderly is postherpetic neuralgia which is protracted pain that may continue for months.IMMUNITYPrevious infection with varicella is believed to confer lifelong immunity to varicella.Abs induced by varicella vaccine persist for at least 20 years.Zoster occurs in e presence of neutralizing Ab to varicella.Increases in varicella Ab titer may occur in persons with HSV infection.Cellular immunity is important host defense for recovery.LAB DIAGNOSISDirect detection: electron microscopy may be used for vesicle fluids but can’t distinguish b/w HSV & VZV, but IF on skin scrapping can distinguish b/w e two.Virus isolation: rarely carried out as it requires 2-3 weeks for result.Serology: e presence of VZV IgG is indicative for past infection & immunity, while e presence IgM is indicative of recent 1o infection.Tzank smear: multinucleated giant cells r seen which r absent in non-herpetic lesions.TREATMENT & PREVENTIONTreatment:Varicella in normal children is mild & self-limiting.Acyclovir is shown to accelerate e resolution of e disease & should be given promptly immunocompromised individuals with varicella infection & normal individuals with serious complications such as pneumonia & encephalitis.Prevention:A live attenuated varicella vaccine is generally used & is highly effective at inducing protection from varicella in children, but less in adults & 95% effective in preventing severe disease.Shingle vaccine is effective in older adults at reducing both e frequency of outbreaks of zoster & e severity of disease that occur.CYTOMEGALOVIRUSPROPERTIESUbiquitous herpesviruses belong to β-herpesvirus subfamily.Has e largest genetic content of human herpesviruses with DNA genome of 240 kbp.Nucleocapsid is 105 nm in diameter with 162 capsomers.E cell surface glycoprotein acts as an Fc receptor that can non-specifically bind e Fc region of Abs which may help in immune system invasion.CMV is very species-specific & cell-type specific.CMV replicates very slowly in cultured cells & infection is spread 1o cell to cell.CPE: perinuclear cytoplasmic inclusion, intranuclear inclusions typical of herpesvirus, multinucleated giant cells, enlargement of many cells, & inclusion-bearing cytomegalic cells.Infection sources: breast milk, urine, saliva, semen, & cervical secretion.EPIDEMIOLOGYCMV is endemic in all over e world causingcytomegalic inclusion disease.Prevalence of infection varies with socioeconomic status, living conditions, & hygienic practices.Abs prevalence may be moderate in adults of high economic status.Human r e only one known host for CMV & once infected, e person carries e virus for life which may be activated from time to time.Transmission requires close person to person contact & virus may be shed in urine, saliva, semen, breast milk, & white blood cells.E dominant routes r oral & respiratory spread but can also be spread transplancentally, by blood transfusion, by organ transplantation, & by sexual contact.Reactivation of latent maternal infections can lead to vertical transmission.CMV infections r markedly increased in immunosuppressed populations such as HIV patients & organ transplant recipients.PATHOLOGYNormal host:May be transmitted person to person requiring close contact with virus-bearing material.Incubation period: 4-8 weeks in normal older children & adult.Virus causes systemic infection & r isolated from lung, liver, esophagus, colon, kidneys, monocytes, T & B cells.E disease is known as infectious mononucleosis-like syndrome.CMV establishes lifelong latent infections & can be shed intermittently from e pharynx & urine.CMI is depressed during 1o infection which contributes to e persistence of viral infection & may take several months to recover.Immunosuppressed host:1o infection is > severe in immunosuppressed persons such as organ transplantation, malignancy, chemotherapy, & AIDS patients.Viral excretion is increased & prolonged & e infection is > apt to become disseminated & pneumonia is e most common complication.In seropositive individuals, e host immune response presumably maintains CMV in a latent state. Congenital & perinatal infection:CMV can be transmitted in utero with both 1o & reactivated maternal infection can also be acquired from e exposure to virus in mother’s genital tract during delivery & from maternal breast milk.Fetal & newborn infections with CMV may be severe.Generalized cytomegalic inclusion disease results from 1o maternal infection.No evidence supports that gestational age at e time of maternal infection affects expression of disease in fetus CLINICAL FINDINGSNormal host:Usually asymptomatic but occasionally causes a spontaneous mononucleosis syndrome (heterophil-negative) which is mild & rarely results in complications.Characteristics: malaise, myalgia, protracted fever, liver function abnormalities, & lymphocytosis.Subclinical hepatitis is common, but hepatosplenomegaly in younger children is frequently observed.Immunocompromised host:Complications: pneumonia, interstitial pneumonitis (BM transplantation), virus-associated leucopenia (solid organ transplantation), obliterative boncholitis in lung transplants, graft artherosclerosis (heart transplantation), & CMV-related rejection of renal allograft.In AIDS patients: disseminated disease, gastroenteritis, chorioretinitis, & progressive blindness.Congenital & perinatal infection:Characterized by involvement of e CNS & RED system: may result in death (rate: 20%).Clinical features: intrauterine growth retardation, jaundice, hepatosplenomegaly, thrombocytopenia, microcephaly, & retinitis.Survivors will develop CNS defect, severe hearing loss (10%: deaf), ocular abnormalities, & mental retardation.Acquired infection: virus is shed in e saliva & urine of infected individuals for weeks & months. IMMUNITYAbs to CMV occurs in most human sera.E presence of Abs in breast milk does not prevent transmission of infection to breast-feeding infants.Maternal Abs protect > against development of serious disease in e infant than viral transmission.LAB DIAGNOSISDirect detection:Biopsy specimens may be examined histologically for CMV inclusion Abs or for e presence of CMV Ags. However, sensitivity is low.E pp65 CMV antigenaemia test is now routinely used for e rapid diagnosis of CMV infection in immunocompromised patients.PCR for CMV-DNA is used in some centers but there may be problems with interpretation.Virus isolation: Conventional cell culture regarded as gold standard but requires up to 4 weeks for result.> useful & rapid culture method as DEAFF test which can provide result in 24-48 hours.Serology: e presence of IgM indicates 1o infection while e presence IgG indicates 2o infection. TREATMENT & PREVENTIONTreatment:Congenital infection: perhaps an abortion.Perinatal & postnatal infection: no treatment.Immunocompromised patients: make e diagnosis early & give prompt antiviral therapy.DOC: ganciclovir which reduces e symptoms.Prevention:No licensed vaccine is available but Towne strain (live attenuated) has been developed w/o used yet concerns to its latency & reactivation.Measures taken to avoid CMV infection in organ transplantation:Screening & matching e CMV status of e donor & recipients.Use of CMV –ve blood for transfusion.Administration of CMV Ig to seronegative recipients prior to transplantation.Gadministration of antiviral prophylaxis such as acyclovir & ganciclovir. EPSTEIN-BARR VIRUSPROPERTIESA ubiquitous γ-herpesvirus that causes acute infectious mononucleosis (a polyclonal stimulation lymphocytes).E genome:172 kbp, has a G + C content of 59% & encodes about 100 genes.Does not normally integrate into e cellular DNA but forms circular episomes which reside in e nucleus & amplified during S phase in a cell cycle. Target cell is B-lymphocytes:Infection leads to e establishment of continuous cell lines indicating e immortalized B-cells.Mechanism: EBV binds to B cell by binding to C3d receptor → EBV directly enters latent state in e cells w/o undergoing complete viral replication → EBV enters cell cycle → a limited repertoire of EBV genes r expressed → indefinite cell proliferation → release virus particles.EBV-immortalized B cells express different function such as secretion of Igs, B cell activation products expression, & expression of at least 10 viral gene products. Latency: viral persistence, restricted virus expression, & e potential of reactivation & lytic replication.Replication: replicates in vivo in epithelial cells of e oropharynx, parotid gland, uterine cervix, & nasopharyngeal carcinoma.Viral Ags:Latent phase Ags: synthesized by latently infected cells which include EBNAs & LMPs.Early Ags: nonstructural proteins whose synthesis is not dependent on viral DNA replication.Late Ags: structural component of e viral capsid.EPIDEMIOLOGYTransmission is by contact with saliva, particularly by kissing.2 epidemiological patterns:In developed countries: 2 peaks of infection i.e. b/w age 1-6 years old & b/w age 14-20 years old. Eventually, 80-90% adults r infected.In developing countries: infection occurs at 2 years old, & 90% of children r seropositive.PATHOLOGYPrimary infection:Infection initiates in e oropharynx where viral replication occurs in epithelial cells of e pharynx & salivary gland as well as B lymphocytes → infected B cell spread e infection t/out e body.Normal individuals: most virus-infected cells r eliminated, but small no. of latently infected lymphocytes persist for e life time in e host.Autoantibodies r typical of e disease with heterophil Abs that react with Ags on sheep RBC.Reactivation from latency:Occurs as evidence by increased levels of virus in saliva & of DNA in blood cells.Usually clinically silent or subclinical. Cancer:EBV is a cause of Burkitt’s lymphoma, nasopharyngeal carcinoma, Hodgkin’s disease, & others.E patients will have elevated level of Abs to virus specific Ags & e tumor tissues contain EBV DNA.CLINICAL FINDINGSInfectious mononucleosis:Incubation period: 30-50 days.Symptoms: headache, fever & malaise (persists for months after acute illness), fatigue, sore throat, enlarged lymph nodes & spleen.Typical illness is self limited & lasts about 2-4 weeks.Clinical features: increase in e no. of circulating WBC, mainly large & atypical T lymphocytes.Burkitt’s lymphoma:Occurs endemically in parts of Africa & Papua New Guinea usually in children aged 3-14 years.It restricted to areas with holoendemic malaria which becomes e cofactor of e disease.It responds favorably to chemotherapy.Multiple copies of EBV genome & Ags can be found in BL with high titres of Abs against EBV Ags.BL shows reciprocal translocation b/w e long arm of chromosome 8 & chromosomes 14, 2, or 22 which results in e deregulation of e c-myc gene.Sporadic cases occur especially in AIDS patients which may or may not be associated with EBV.In theory, it can be controlled by e eradication of malaria or vaccination against EBV.Nasopharyngeal carcinoma:I.e. a malignant tumour of e squamous epithelium of nasopharynx which is very prevalence in S. China but rare in other parts of e world.Multiple copies of EBV genome & EBNA-1 Ags can be found in cells of undifferentiated NPC.High titres of Abs against EBV Ags r present & usually e prognosis is poor.Disease in immunodeficient hosts: After infection, EBV maintains a steady low-grade infection in e body, & if in immunocompromised patients, e virus will be reactivated.In transplant recipient: lymphoproliperative disease may be developed & r fatal with e lesions tend to be extranodal & in unusual sites such as at GIT or CNS.In AIDS patients: oral leucopenia & non-Hodgkin’s lymphoma.Ducan X-linked lymphoproliperative syndrome which occurs exclusively in males with a defective gene in e X-chromosomes which counts for half of e fatal cases of immunocompromised patient. IMMUNITYCellular immunity & cytotoxic T cells r important in limiting primary infections & controlling chronic infections.LAB DIAGNOSISAcute EBV infection is usually made by e heterophil Ab test or detection of anti-EBV VCA IgM.Burkitt’s lymphoma: diagnosed by histology or e tumor can be stained with Abs to lambda light chain which will reveal a monoclonal tumor of B cell origin.NPC: diagnosed histologically or screening of early lesion of NPC (also for treatment) by determination of e titre of anti-EBV VCA IgA.TREATMENT & PREVENTIONThere is no EBV vaccine available.Acyclovir reduces EBV shedding from e oropharynx during period of drug administration but it does not affect e no. of EBV-immortarlized B cells.VIRAL HEPATITIS A TO EPORTIONDETAILSPATHOLOGYHepatitis: inflammation of e liver.Microscopic characteristics:Spotty parenchymal cell degeneration.Necrosis of hepatocytes.A diffuse inflammatory reaction.Disruption of liver cell cords.Kupffer cell hyperplasia.Periportal infiltration by mononuclear cells.Accumulation of macrophages near degenerating hepatocytes.However, preservation of e reticulum framework allows hepatocytes regeneration & liver architecture can be maintained. This takes place in 8-12 weeks.Acute viral hepatitis r characterized by fulminant & massive hepatocellular necrosis with jaundice.Chronic hepatitis:Chronic carriers:Sporadically abnormal aminotranseferase values & hepatomegaly.Portal inflammation & swollen & pale hepatocytes.Chronic active hepatitis: inflammation, necrosis, & collapse of e normal reticulum framework with bridging b/w e portal triads or terminal hepatic veins.None of e viruses r cytophatogenic & is belived that e cellular damages seen r immune-mediated.CLINICAL FEATURESVirusIncubation periodJaundice(years)Acute case-fatality rateComplication/ chronicityMortality from chronicityAAverage: 30 days.(15 - 50 days)< 6: < 10%6-14: 40 - 50%> 14: 70 - 80%NoneNo chronicityFulminant, cholestatic, & relapsing hepatitis.NoneBAverage: 60-90 days(45 - 180 days)< 5: < 10%> 5: 30 - 50%– 1.0%< 5: 30 - 90%> 5: 2 – 10%15 – 25%CAverage: 6-7 weeks(2 - 26 weeks)< 20%Low 60 - 85%Cirrhosis: 5 - 20%3%EAverage: 40 days(15 - 60 days)-Overall: 1-3%Pregnancy: 15-25%Increased with age, but no chronicityNone Asymptomatic > Symptomatic > Fulminant liver failure > Death.General symptoms: nausea, vomiting, upper abdominal pain, loss of appetite, fever, diarrhea, light colored stool, dark urine, jaundice, & extrahepatic manifestations.TREATMENT Viral hepatitis: supportive & directed treatment at allowing e hepatocellular damage to resolve % repair itself.Chronic liver disease:HBV: Lamivudine, a reverse transcriptase inhibitor reduces HBV DNA level, but once stopped, viral replication resumes & resistant virus mutants r selected.HCV: combination therapy of IFN-α & ribavirin with response rate of up to 50%.Both HBV & HCV:Therapy: recombinant interferon-alfa therapy (HBV: 35% remission & HCV: 25% of sustained response).With end stage liver damage: orthotopic liver transplantation (problem: risk of reinfection of e graft). HEPATITIS A VIRUSPROPERTIESFrom family of picornaviridae & genus hepatovirus.E size is b/w 27 – 32 nm spherical particle with cubic symmetry.E genome: a linear ssRNA genome of 7.5 kb.Stability:Stable to treatment with 20% ether, acid (pH 1 for 2 hours), & heat (60oC for 1 hour).UV radiation (1 minute at 1.1 watts).Treatment with formalin (for 3 days at 37 oC).Treatment with chlorine (10-15 ppm for 30 minutes).Infectivity can be preserved for at least 1 month after being dried & stored at 25oC & 42% relative humidity at -20oC.R destroyed by:Autoclaving (121oC for 20 minutes).Boiling water (for 5 minutes).Dry heat (180 oC for 1 hour).EPIDEMIOLOGYOutbreaks:Common in families, institutions, day care centres, or military cops.D/t fecal-oral route, close contacts with others, & blood exposure.Recurrent explosive epidemic usually result from fecal contamination of food or water.Users of injection & noninjection drugs.Persons with clotting disorders & chronic liver disease.Persons working with nonhuman primates.Risk factors:Living in crowded & poor sanitation.Travelers to developing countries.Men having sex with men. LAB DIAGNOSISHAV can be detected in e liver, stool, bile, & blood by IA, PCR, & NA hybridization assays.HAV is detected in e stool about 2 week prior to onset of jaundice up to 2 weeks after.Anti-HAV in IgG fraction: Appears soon after e onset of disease.Persists for decades.Anti-HAV IgM confirms e diagnosis by ELISA.Anti-HAV in IgM fraction:Appears during acute phase.Peaks about 2 weeks after elevation of liver enzymes.Declines to non-detectable levels within 3-6 months.PREVENTIONVaccination strategy (licensed for age 1 & above):Target groups: persons with increased risks of infection & community with historically high risk of HAV. Side effects: sore at injection site, headache, & malaise with no severe reactions & safe for pregnant ladies.Usage for infants: safe & immunogenic for infants w/o maternal Abs because passively-acquired maternal Abs blunts e IR towards e vaccine.Immunoglobulin therapy:Pre-exposure: for travelers to intermediate & high HAV-endemic regions.Post-exposure (within 14 days): routine (household & other intimate contacts) & selected situations (institutions & food prepared by infected food handlers). Good hygiene practices.Clean water & sewage system to avoid food & water contamination.HEPATITIS B VIRUSPROPERTIES3 forms:Spherical particles of 22 nm in diameter made up of HBsAg.Tubular or filamentous form with same diameter but over 200 nm long results from HBsAg overproductionLarger 42 nm spherical virions.E outer surface or envelope contains HBsAg & surrounds a 27 nm inner nucleocapsid core that contains HBcAg.E viral genome consists of partially double-stranded circular DNA, 3200 bp in length.HBV is sensitive to:Higher temperature (100 oC for 1 minute).Longer incubation period (60 oC for 10 hours).Sodium hypochlorite, 0.5% (for 3 minutes).4 phenotypes: adw, ayw, adr, & ayr.HBV is stable:At 20 oC for over 20 years.To repeated freezing & thawing.At 37 oC for 60 minutes. Remains viable after being dried & stored at 25oC.At pH 2.4 for 6 hours. To UV radiation of plasma & blood.EPIDEMIOLOGYHBV is a worldwide distribution with no seasonal trend or predilection for any age of group.Those who r infected at infant develop chronic disease & at high risk of developing hepatocellular carcinoma.Modes of transmissions:HBsAg is found in saliva, nasopharyngeal washing, semen, menstrual period, & vaginal secretion.By close contact & sexual intercourse.By improperly sterilized syringes, needles, scalpels, or by tattooing & ear pricing. Risk factors:Parenteral drug abusers.Institutionalized persons.Health care personnel.Multiply transfused, organ transplantation, & hemodialysis patients.Highly promiscuous persons.Newborns born to mothers with HBV.Global patterns of chronic HBV infection:High (> 8%), 45% of global population: lifetime risk of infection is > 60% & early childhood infections r common.Intermediate (2-7%), 43% of global population: lifetime risk of infection is 20-60% & infections occur in all ages.Low (< 2%), 12% of global population: lifetime risk of infection is < 20% & most infections occur in adult risk goups.LAB DIAGNOSISDNA polymerase activity, HBV DNA, & HBeAg present early in e incubation period, after e 1st appearance of HBsAg.During initial phase of infection, high level of HBV particles is detected in e blood.HBsAg is detectable 2-6 weeks in advance of clinical evidence & persists t/out e clinical course of e disease but disappears after 6 months of exposure.Ab to HBsAg is detected after e of HBsAg in low concentration. Detectable by ELISA for HBV Ag, butAnti-HBc IgM is detected in acute disease but is low in chronic HBsAg carriers. Abs & PCR for viral DNA.PREVENTIONPrevention of perinatal HBV transmission.Vaccination strategies:For all infants.For children in high-risk group.For adolescents (<18 years old).For adults in high-risk groups.Side effects of vaccination r rare, but anaphylaxis appears in 1:600000 doses given.HEPATITIS C VIRUSPROPERTIESFrom family of Flaviviridae & genus Hepacivirus with 6 clades & > 100 subtypes.E genome is +ve ssRNA virus of 9.4 kbp in size which encodes a core protein, 2 envelope glycoproteins, & several nonstructural proteins.E virus undergoes sequence variation during chronic infection which is called quasi-species (not related to difference in clinical disease).Genotypes 1 & 4 r > resistant & require > prolonged treatment, but genotype 2 & 3 show a better response to treatment.In Malaysia, 3a > 1 > 2 > 4.EPIDEMIOLOGYHCV infections r extensive t/out e world.HCV is transmitted by:Through direct percutaneous exposures to blood.High-risk sexual practices.Health care workers.Mother to child through vertical transmission.By commercial IV Ig preparation.Risk factors:Illegal injection drug users.Transfusion or transplant from infected donor.Occupational exposure to blood (needle stick).Iatrogenic: unsafe injection.Multiple sex partners.Sexual/household exposure to anti-HCV +ve contacts.LAB DIAGNOSISMost 1o infections r mild & asymptomatic.Serological assays such as EIA detect Abs toward HCV for diagnosis but cant’ distinguish e stage of infection.NA-based assays is used for:Detection of circulating HCV RNA.Monitoring patients on antiviral therapy.To genotype HCV isolates.PREVENTIONScreening & testing donors of blood, organs, & tissues.Virus inactivation of plasma-derived products.Risk-reduction counseling & services.Infection control practices: avoiding direct exposure to infected blood:Anti-HCV +ve persons should not donate any parts of theirs.Do not share items that might have blood on them (personal care & home therapy).Cover cuts & sores on e body.HEPATITIS D VIRUSPROPERTIESE virus contains HDAg surrounded by HBsAg envelope.It has particles size of 35-37 nm & a buoyant density of 1.24-1.25 g/mL.E genome is circular, -ve ssRNA which is 1.7 kb in size.HDV is a defective virus which acquires an HBsAg coat for transmission, however, no homology exists b/w e 2. HDV can be coinfected with HBV (acute infection) or superinfected on top of chronic HBV (severe chronic liver disease).EPIDEMIOLOGYHDV is found t/out e world but with a non-uniform distribution & infects all groups of age.Highest prevalence in Italy, e Middle East, central Asia, West Africa, & South America. Risk factors: Persons who receive multiple transfusions. Persons who r in close contact with them.IV drug abusers.Modes of transmission r similar with HBV but no STD & it can also be transmitted perinatally.2 epidemiological patterns:In Mediterranean countries: HDV is endemic among persons with HBV & most r transmitted by intimate contact.In non-endemic area: HDV is confined to persons exposed frequently to blood & blood products such as drug addict.LAB DIAGNOSIS2 patterns:Coinfection pattern: Abs to HDAg develop late in e acute phase of infection & may be of low titre.Superinfection pattern: usually results in HDV persistence with high level of IgM, IgG, HDV RNA, & HDAg.All markers for HDV replication disappear during convalescene, even HDV Abs may disappear within months to years.PREVENTIONHBV-HDV coinfection: pre or postexposure prophylaxis to prevent HBV infection (HBIG &/or Hepatitis B vaccine).HBV-HDV superinfection: education to reduce risk behaviors among persons with chronic HBV infection.HEPATITIS E VIRUSPROPERTIESE genus is Hepevirus.E virus is transmitted enterically & occurs in epidemic form in developing countries with fecal contamination.E viral genome is a +ve sense ssRNA 7.6 kb in size.There is possibility of animal to hman transmission.EPIDEMIOLOGYMost outbreaks occur associated with focally contaminated drinking water.Minimal person to person contact transmission.Cases in non-endemic areas usually have history of travelling to endemic areas.PREVENTIONMostly precautions r for travelers to endemic regions: avoid drinking water of unknown purity, uncooked shellfish, & uncooked fruits or vegetables not peeled by e travelers themselves.RETROVIRUSESPORTIONDETAILSPROPERTIESVirion: spherical, 80-110 nm in diameter, helical nucleocapsid within icosahedral position: RNA (2%), protein (~60%), lipid (~35%), & carbohydrate (3%).Genome: linear & +ve ssRNA, 7-11 kb, diploid, & may be defective & carrying oncogene.Protein: reverse transcriptase contained in side virion.Envelope: contains glycoprotein & lipid.MORPHOLOGICAL PATTERNSType A: Occur only intracellularly & non-infectious. Intracytoplasmic type A particles r 75 nm in diameter & precursors of extracellular type B viruses.Intrecisternal type A r 60-90 nm in diameter & unknown entities. Type B: 100-130 nm in diameter & contain an eccentric nucleoid.Type C: e largest group of retroviruses, r 90-110 in diameter, & contain centrally located nucleoid.Type D: r 100-120 in diameter, contain an eccentric nucleoid, & exhibit surface spikes shorter than type B’s.CLASSIFICATIONα-retrovirus: avian leukosis & sarcoma viruses.β-retrovirus: mouse mammary tumor.γ-retrovirus: mammalian leukemia & sarcoma virus.δ-retrovirus: HTLV & bovine leukemia virus.ε-retrovirus: fish virus.Spumavirus: foamy degeneration of inoculated cells.Lentivirus: HIV & neurologic impairment disease. HOST RANGEEcotropic viruses: infect & replicate oly in cells from animal of e original host species.Amphotropic viruses: exhibit a broad host range (infect natural host & heterologous species as well).Xenotropic viruses: replicate in some heterologous (foreign cells) but not in cells of e natural hosts.GENETIC CONTENTSGenes required for viral replication:gag gene: encodes e core proteins (group specific Ag).pro gene: encodes a protease enzyme.pol gene: encodes e reverse transcriptase enzyme. 5’-gag-pro-pol-env-3’ (gene order in env gene: encodes e glycoproteins that form projections retrovirus).on e envelope of e particles.Additional genes downstream from e env gene: tax or tat gene (transactivating regulatory gene) which encodes for a non-structural protein which alters e transcription or translational efficiency of other viral genes. Onc gene: carried by directly transforming retroviruses which r highly oncogenic in appropriate host animal & can transform cells in culture.REPLICATIONAttachment of e virion to a specific cell surface receptors → penetration of e virion core into e cell → reverse transcription within e core structure to copy e genome RNA into DNA → transit of e DNA to e nucleus → integration of e viral DNA into e random sites in cellular DNA to form e provirus → synthesis of viral RNA by cellular RNA polymerase II using e integrated provirus as a template → processing of e transcripts to genome & mRNAs → synthesis of virion proteins → assembly & budding of virions → proteolytic processing of capsid proteins → mature infectious virion.RETROVIRAL ONCOGENESISTRANSFORMATIONDefined as e introduction of inheritable changes in a cell causing changes in e growth phenotype & immortalization.It is a multistep process involving initiation, promotion, & progression.It involved gene mutation, amplication of cells containing these mutations & further changes leading to e fully transformed phenotypes.TYPESAcutely transforming viruses:Cause tumors in 1 to few weeks.High efficiency of disease induction.Carry oncogenes & polyclonal in nature.Transform cells in vitro.Nearly all r defective: need a helper virus for replication.E.g.: avian, murine, & feline sarcoma.Slowly transforming viruses:Cause tumor in 6-12 months.Low efficiency.Do not carry oncogenes & monoclonal or oligoclonal in nature.No visible effects on cells in culture.Most r replication competent.Some r endogenous.E.g.: HTLV-1.HTLV-1 VIRUSE causative agent of adult T cell lymphomas (ATL) & nervous system disorder called tropical spastic paraparesis.It does not carry oncogenes (i.e. slowly transforming) & completely exogenous to infected animal & human.HTLV-1 & HTLV-2 share about 65% homology & display significant serologic cross-reactivity.Have a high affinity for mature T cells (cell tropism), but r expressed at very low level in infected individuals.HTLV-1 ia a complex retrovirus, encoding a transactivator, tax:Its product alters e expression of other viral genes which is necessary for viral replication in vivo. May be contributed to oncogenesis.Stimulates e expression of genes involved in cellular gene regulation including IL-2 & IL-2R genes which r known to affect T-cell growth regulation.Modes of transmission:Involved cell associated virus.Mother to child transmission via breast feeding.Blood transfusion by sharing blood-contaminated needles.Sexual intercourse.Clinical features: Linked to HTLV-1 associated myelopathy or tropical spastic paraparesis.Development of progressive weakness of e legs & lower body.Leukemia: 1% of infected persons after a latent period of 20-30 years.Epidemiology:Worldwide distribution with estimated of 10-20 millions infected.HTLV associated disease clusters found in Southern Japan, Melanesia, e Caribbean, Central & South America, & parts of Africa.< 1% people have HTLV-1 Abs.> 10% people in endemic areas r seropositive, Abs may be found.Mechanism of oncogenesis:Viral-promoter enhancer sequences in e long terminal repeat r responsible to e signals that cause e activation & proliferation of T cells.Replication of virus linked to host cell replication to ensure efficient virus propagation.Carry tax gene & transactivating regulatory genes.106426020320LENTIVIRUSES, HIV, & AIDSPORTIONSDETAILSPROPERTIESAre a retrovirus & a member of Lentivirus genus.Have a cylindrical nucleoid in e mature virion. Contains e 4 genes required for a replicating retrovirus: gag-pro-pol-env.Other additional genes responsible for regulating viral expression & important in disease pathogenesis in vivo:Tat gene: functions in transactivation & transcriptional activation of other viral genes.Rev gene: expression of viral structural proteins & facilitation of e export of unsliced viral transcripts from e nucleus. Nef gene: increases viral infectivity, facilitate activation of resting T cells, & downregulates e expression of CD4 & MHC class 1.Vpr gene: increases transport of e viral preintegration complex into e nucleus & arrest G2 phase of e cell cycle.Vpu gene: promotes CD4 degradation.Vif gene: promotes viral infectivity by suppressing e effects of an inhibitory cellular protein present in some human cells.E env gene:E regions of greatest divergence among isolates r localized.Codes for e viral envelope proteins.E SU product:Contains binding domain responsible for viral attachment to CD4 molecule & coreceptors.Determine lymphocytes & macrophages tropism.Carries e major antigenic determinant that elicit neutralizing Abs.E Tm product:Contains both a transmembrane domain that anchors e glycoprotein in e viral envelope.A fusion domain that facilitates viral penetration into target cells.Host factors important for pathogenesis: age, stress, genetics, & concurrent infection.DISINFECTION & INACTIVATIONE virus is inactivated:Completely by treatment for 10 minutes at room temperature with:By extreme pH (pH 1.0-3.0).By exposure to undiluted bleach for at least 30 seconds (for viruses in clotted or unclotted blood in e needle).By paraformaldehyde (for virus in free solution).In liquids or 10% serum by heating at 56oC for 10 minutes.By heating at 68oC for 72 hours (for lyophilized blood products).COMMON FEATURESViruses r transmitted by exchange of body fluid.Virus persists indefinitely in infected hosts, though it may present in low amount.Viruses have high mutation rates.Virus infection progresses slowly through specific stages.Viruses can infect non-dividing terminally differentiated cells.’It may take years for disease to develop.VIRAL RECEPTORSReceptors necessary for HIV infection:Receptors: CD4 molecule on macrophages & T cells – to gain entry into e cells.Co-receptors: Chemokine receptors: CCR5 (macrophages-tropic strains) & CXCR4 (lymphocytes-tropic strains).E co-receptors used by HIV for cell entry r found on lymphocytes, macrophages, thymocytes, on neurons, & cells on colon & cervix.A dendritic cell-specific lectin, DC-SIGN:Appears to bind HIV-1 but not to mediate cell entry.May facilitate transport of HIV by dendritic cells to lymphoid organs.Enhance infection of T cells.HIV & AIDSCLASSIFICATION oh HIV-1E strain can be classified into 3 groups:E major group M: > 90% of HIV-1 infection belongs to M group.E outier group O: appears to be restricted to west central Africa.E new group N: discovered in 1998 in Cameroon & is extremely rare.These 3 groups may represent 3 separate introduction of simian immunodeficiency virus into human.PHASES OF HIV INFECTIONPhase 1:Acute infection: lasting from infection until seroconversion (onset of detectable virus-specific Abs).Usually lasts 3-8 weeks.Virus replicate to high titre (usually R5 type).Concluded when CTL develop, seroconversion occurs, & virus load is diminished in e blood.Phase 2:Asymptomatic phase which lasts for months to > 15 years.Continuation of viral replication.Active immune response & antigenic escape.Concluded by development of OI (CD4+ T cell < 200/mm3).Phase 3:Host immune response collapses.Clinical progression to AIDS.Syncytium inducing (X4 viruses) r detected in most if not all cases.CD4+ T CellsViral load 1 2 3PROGRESSION PATTERNS~20% of HIV infected people become fast progressors:They lose > 100 CD4 cells /mm3 / year.Twice this percentage in perinatally infected infants, transfusion recipients, & hemophiliacs.~35% become progressors:Lose 31 CD4 cells /mm3 / year.~25% become long term non-progressors:No decrease in CD4.Level of ~ 500 – 800 CD4 cells /mm3 / year which is still on e low side of normal.PATHOLOGYCD4 T lymphocytes & memory cells:E cardinal feature: CD4 T cell depletion. WhichDue to: viral replication in these cells & e death of uninfected cells by indirect mechanism.Early in infection, primary isolate is M tropic, but then later replaced by T tropic viruses.At any given time, only small fraction of T cell r productively infected & others r killed.E survivors r infected & revert to a resting memory state being a long term, stable reservoir for e virus.When exposed to Ag, they become activated & release infectious viruses.Monocytes & macrophages:E co-receptor of HIV on macrophage is CCR5 chemokine receptors.In e brain, monocytes & macrophages r highly infected which leads to e development of neuropsychiatric manifestation in HIV infection. M tropic strains of HIV are important for initial infection.Monocytes & macrophages r major reservoirs for HIV in e body because they r relative refractory to CPE of HIV so that e viruses survive in these cells & can be transported to e whole body.Infected macrophages may continue to produce viruses for a long time. Lymphoid organs:In untreated infection & latency state, HIV is actively replicating in lymphoid tissues because e microenvironment in e organ is ideal for e virus.Cytokines r released & activate a large pool of T cells for e virus.At late stage, e architecture of e lymph node becomes disrupted. Neural cells: Virus enter brain via infected monocytes & release cytokines which r toxic to neurons & chemotatctic factors that lead to infiltration of inflammatory cells into e brain. E diseases: HIV encephalopathy, peripheral neuropathies, & AIDS dementia complex.HALF-LIVES OF HIV-1Activated cells infected by HIV:Produce virus immediately & die within 1 to 2 days.Half-live: 1.6 days.E virus production accounts for e vast majority of virus present in plasma.Virus in e plasma:It is estimated that 10 billion HIV particles r produced & destroyed each day.E half-live: 6 hours.HIV life-cycle: complete life cycle requires ~ 2.5 days.Resting cells infected by HIV:Half-live of at least 5-6 months.Present as a major challenge for virus eradication & anti-retroviral therapy.HIV-1 RESISTANCEResistant variants: pre-existing in e host, can be transmitted & evolve.Selective pressure of therapy:Drug has limited potency.Non-adherence to potent anti-retroviral therapy.Poor absorption or rapid clearance of drugs.Drugs r not active in cells.TRANSMISSIONSexual contact: male to male, male to female, & female to female.Fluid exposure: Semen, vaginal secretion, breast milk, & blood. Routes of transmission:Injecting drug by using or sharing needles.Occupational exposure.Transfusion of blood products.Open cuts, skin abrasion, & mucous membrane.Perinatal infection:Transmission from mother to baby.Breastfeeding.CLINICAL FINDINGSGeneral symptoms: Common symptoms: fatigue, rash, headache, nausea, & night sweat.Serious symptoms: prodrome which includes fatigue, malaise, weight loss, fever, dyspnea, chronic diarrhea, white patches on e tongue, & lymphadenopathy.No treatment: death after ~2 years. Plasma viral load:Viral load is of significant prognosis value which reflects e total no. of productively infected cells & their average burst size.Plasma viral load appears to be e best predictor of long term clinical outcome while CD4 counts r e best predictor of short term risk of developing an opportunistic disease. Also important for measurement of antiretroviral drug therapy.Pediatric AIDS:Clinical findings: lymphoid interstitial pneumonitis, severe oral candidiasis, encelophaty, wasting, generalized lymphadenopathy, bacterial sepsis, hepatosplenomegaly, diarrhea, & growth retardation.Viral RNA load: generally low at birth, then rise rapidly within e 1st 2 months, & followed by a slow decline until e age of 2 years (suggesting that IS has difficulty containing e infection).Neurologic disease:E diseases: subacute encephalitis, vacuolar myelopathy, aseptic meningitis, & peripheral neuropathy.AIDS dementia complex: poor memory, inability to concentrate, apathy, psychomotor retardation, & behavioral changes.Psychology: high risks of psychiatric disorders & e most common is anxiety disorder.Pediatric neurology: seizure disorders, progressive loss of behavioral developmental milestones, encephalopathy, attention deficit disorder, meningitis, & developmental delays.Opportunistic infection: protozoa, fungi, bacteria, & viruses.Cancer:Tend to be those with a viral cofactor.Includes non-Hodgkin’s lymphoma, Kaposi’s sarcoma, cervical cancer, & anogenital cancer.Effective anti-retroviral drug therapy results in marked decrease in Kaposi’s sarcoma viruses. People infected with HIV:Can look healthy.Can be unaware of their infection.Can live lifelong productive lives when their HIV infection is managed.Can infect people when they engaged in high risk behavior.LABORATORY INVESTIGATIONSVirus isolation:HIV can be cultured from lymphocytes in peripheral blood.Higher titres of virus r found in e patients indicating e HIV infection.Viral growth is detected by testing culture supernatant fluids after 7-14 days for virus reverse transcriptase activity or for virus-specific Ags.However, they r time consuming, laborious, & r limited to research studies.Serology or screening HIV Ab testing:EIA/ELISA (reactive) → repeat EIA/ELISA (reactive) → IFA, others e.g. Western Blot (reactive) → positive for HIV (sensitivity & specificity exceeding 98%).Detects e body’s Abs response to HIV infection, not detecting e virus.Most will have detectable Abs within 6-12 weeks after infection, whereas virtually all will be positive within 6 months.Detection of viral NA or Ags:Amplification assays such as RT-PCR, DNA PCR, & bDNA tests r used to detect viral RNA.E tests can be quantitative, very sensitive, & form e basis for plasma viral load determination.E HIV RNA level r important predictive markers of disease progression & valuable tools with which to monitor e effectiveness of antiviral therapies.However, e presence of maternal Abs makes serological tests uninformative.TREATMENT &PREVENTIONTreatment: Antiviral drug: Combination therapy: Referred as highly active antiretroviral therapy (HAART). Can suppress viral replication to below limit of detection in plasma & decrease viral loads in lymphoid tissues which may prolong patient survival.However, it fails to cure HIV-1 infection because e virus persists in reservoirs of long-lived, latently infected cells, including memory CD4 cells.Monotherapy: rapid emergence of drug resistant mutants.Zidovudine: Reduce e transmission of HIV from mother to infant.Therapy during pregnancy & during e birth process & of e baby after birth to reduce transmission.Important of early diagnosis & testing:Allows for early treatment to maintain & stabilize e immune system response.Decreases risk of HIV transmission from mother to newborn baby.Allows for risk reduction education to reduce or eliminate high-risk behavior.Prevention:Vaccination: vaccine development is difficult because HIV mutates rapidly, is not expressed in all cells that r infected, & is not completely cleared by e host IR after primary infection.Gene therapy: intracellular immunization genetically alters target cells & makes them resistant to HIV. HIV risk reduction:Avoid drug & alcohol use to maintain good judgment.Don’t share needles used by others for drugs, tattoos, & body piercing.Avoid exposure to blood products.Health education:Any sexual intercourse should be protected by a condom.Do not share unsterile needles or syringe.All women who r potentially exposed to HIV should seek HIV Abs testing before becoming pregnant.HIV-infected mothers should avoid breastfeeding to reduce transmission of virus to their children.Control measures:Consistent & proper use of condom.Toothbrushes, razors, & other implements that could become contaminated with blood should not be shared with others.Seropositive women or women with seropositive sexual partners because if they pregnant, offspring is high risk of acquiring AIDS.Accidents that lead to bleeding should be cleaned with household bleach.Devices that have punctured e skin should be steam-sterilized by autoclaving before reuse or discarded.If seropositive, inform medical or dental officers.Testing for HIV Abs. INFLUENZA VIRUSESPORTIONSDETAILSPROPERTIESFrom e family orthomyxoviridae & causes respiratory tract infection.3 immunologic types divided by differences exhibited in e nucleocapsid (NP) & matrix (M) proteins: No cross reaction b/w e 3.A (man & animal): has subtypes determined by antigenic variationin e surface glycoprotein, HA & NA.B (man & animal).C (man only).Virion: spherical, pleomorphic, 80-120 nm in diameter with helical, 9 nm position: RNA (1%), proteins (73%), lipid (20%), & carbohydrate (6%).Genome: -ve sense segmented (A & B: 8, C: 7) ssRNA of 13.6 kb for e overall size. Proteins: contains 9 structural proteins & 1 non-structural protein:E nucleoprotein associated with viral DNA to form ribonucleoprotein (RNP) that assumes e helical configuration & forms e viral nucleocapsid.3 large proteins, PB1, PB2, & PA r bound to viral RNP for RNA transcription & replication.E matrix (M1) protein forms e shell underneath e viral lipid envelope & important for particle morphogenesis & is e major component of e virion.Envelope:Made of lipid derived from e cell surround e virus particle.2 virus encoded glycoprotein, hemagglutinin (HA) & neuraminidase (NA) r inserted into e envelope & exposed as spikes about 10 nm long on e surface of e particle.D/t HA & NA, envelope becomes e important Ag.M2 ion channel & NS2 proteins r also present in e envelope.D/t segmented nature of e RNA, e virus undergoes genetic reassortment which results in sudden changes in viral surface Ags which contribute to e epidemiological features of influenza & difficulty in developing vaccine.Infectivity is destroyed by lipid solvents, protein denaturants, formaldehyde, acidic environment, & irradiation. EPIDEMIOLOGYInfluenza virus infections occur worldwide & cause annual outbreaks which occur in waves & of variable intensity.Epidemiology according to immunogenic types:Type A:Undergoes both antigenic shift & drifts involving e genomic reassortment of HA & NA proteins. As a result, e population has no immunity against e new strain.Causes pandemics (massive epidemics) which can sweep across e continents & around e world. This occurred in 1918 (H1N1), 1957 (H2N2), & 1968 (H3N2).Epidemics arise through minor antigenic drifts as a result of mutation which tend to be 2-3 years.Type B: Sometimes causes epidemics which have a longer interepidemic period of 3-6 years d/t antigenic drift.Does not undergo antigenic shift.Type C: e least significant because it causes mild sporadic respiratory disease but not epidemic influenza.Surveillance for influenza outbreaks is necessary to identify e early appearance of e new strains. Isolation of a virus with an altered HA in e late spring during a mini epidemic signals a possible epidemic e following winter.Avian influenza:E influenza viruses do not appear to undergo antigenic change in e birds d/t birds life span.This means that genes that caused previous influenza pandemics in human still exist unchanged in e aquatic bird reservoir.Influenza virus of e ducks multiply in e living cells lining e intestinal tract & r shed highly in fecal matters.Evidence supports that pigs serve as mixing vessels for reassortants as their cells contain receptors recognized by both human & avian viruses. E school-age children r e predominant vectors of transmission.If given enough opportunities, e highly pathogenic H5NI avian strain could spread and sustain among humans either by reassortment or by adaptive mutation. This can cause a devastating influenza pandemic.Seroarcheology shows that e highest Ab levels in a particular age group reflect dominant Ags of e virus responsible for childhood infections of e group. ANTIGENIC STRUCTURESHEMAGGLUTININHA protein binds virus particles to susceptible cells & e major Ag against neutralizing Abs.Responsible for e continual evolution of new strains & subsequent influenza epidemics.Able to agglutinate RBCs under certain conditions.E mechanism: a short signal sequence at e aa terminal inserts e polypeptide into ER → e signal is removed → e HA protein is cleaved into 2 subunits, HA1 & HA2 → HA2 anchors e HA molecule in e membrane with a short hydrophilic tail extending into e cytoplasm → oligosaccharide residues r added at several sites → e HA molecule is folded into a complex structure → 5 antigenic sites (exposed on e surface of e structure) on HA molecule exhibit extensive mutation.E HA spike one virus particle is a trimer composed of 3 intertwined HA1 & HA2 dimers.E cleavage of HA protein is necessary for e virus particle to be infactious & is mediated by cellular protease.Influenza virus remain confined to e respiratory tract because e enzymes r common there.NEURAMIDASEE spike of NA is a tetramer composed of 4 identical monomers & contains 4 active (catalytic) sites.NA functions at e end of e viral replication:Acts as a sialidase enzyme which removes sialic acid from glycoconjugates.It facilitates release of virus particles from infected cell surface during e budding process.It helps prevent self aggregation of virions by removing sialic acid residues from viral glycoproteins.It helps e virus negotiate through e mucin layer in e RT to reach e target epithelial cells.ANTIGENIC DRIFT & SHIFTAntigenic drift:I.e. e minor antigenic changes.D/t e accumulation of point mutations in e gene resulting in aa changes in e protein.Sequences changes can alter antigenic sites on e molecule such that a virion can escape recognition by e host immune system.E immune system also acts as selection force that allows new antigenic variants to expand.A variant must sustains 2 or > mutations before a new strain emerges.Antigenic shift:I.e. major antigenic changes in HA & NA results in a new subtype.Reflects e drastic changes in e sequence of a viral surface protein which causes epidemics.E segmented genomes of influenza viruses reassert readily in doubly infected cells.E mechanism involves genetic reassortment b/w human & avian influenza which is possible only for influanze virus type A. REPLICATIONVirus attaches to cell surface sialic acid via e receptor site located on HA → virus particles r internalized within endosomes (receptor-mediated endocytosis) → fusion b/w viral envelope & cell membrane (uncoating) → viral RNPs r released d/t low pH in endosome → acid pH causes conformational change in HA & brings HA2 fusion peptide in contact with e membrane → M2 ion channel protein permits e entry of ions in e endosome into e virus particle → viral nucleocapsids r released into cytoplasm & nucleus → transcription & replication by virus encoded polymerase → nucleocapsids r assembled in e nucleus & moved out to cell surface → HA & NA r synthesized in ER, modified, & assembled into trimers & tetramers & r inserted into plasma membrane → maturation by budding → NA removes terminal sialic acids from cellular & virus surface glycoproteins → release of free virus particles. PATHOLOGYVirus spreads from person to person through air borne droplets or by contact with contaminated hands or surface.Infection is successful if e cough reflex, mucous secretion, & neutralizing IgA fail to prevent to remove e virus from respiratory epithelial cells.Viral NA lowers e viscosity of e mucous film in RT, laying bare e cellular surface receptors & promoting e spread of virus containing fluid to e lower portion of e tract.E incubation period is from 1-4 days depending upon e size of viral dose & e immune status of e host.Viral shedding starts e day preceding onset of symptoms, peaks within 24 hours, remains elevated for 1-2 days, & declines over e next 5 days.Infection causes cellular destruction & desquamation of superficial mucosa of e RT which results in edema & mononuclear infiltration, but does not infect e basal layer of e epithelium.Viral damage to RT lowers its resistant to 2o bacterial invaders such as staphylococci & many more. CLINICAL FINDINGSUncomplicated influenza:Appear abruptly & include chills, headache, & dry cough followed by high fever (3-5 days), generalized muscular aches, malaise, & anorexia.Respiratory symptoms last for another 3-4 days while e cough, coryza, & weakness may persist for 2-4 weeks after major symptoms subside.These symptoms r characterized of type A & B, while type C only causes common cold illness.In pediatrics, GI manifestations such as vomiting r common, while febrile convulsion & otitis media can also occur. Type A is an important cause of croup in children under 1 year old of age.Pneumonia:E lethal impact of influenza epidemic is death d/t pneumonia & cardiopulmonary diseases.Pneumonia can be result of viral, 2o bacterial, or combination of both.Increased mucous secretion helps carry agent to LRT & is attributed to loss of ciliary clearance, dysfunction of phagocytic cells, & provision of a rich bacterial growth medium by e alveolar exudates which leads to bacteria superinfection.Reye’s syndrome:Is an acute encephalopathy of children & adolescents b/w 2-16 years old.E cause is unknown but it is a complication of type A & B & varicella-zoster virus.IMMUNITYImmunity, i.e. Abs against HA & NA is long-lived & subtype-specific.Resistance to initiation of infection is related to Abs against HA, whereas decreased severity of disease & decreased ability to transmit virus to contacts r related to Abs against NA.Serum Abs persist for months or years, but IgA r for shorter duration.Original antigenic sin: subsequent infections or immunization reinforce e Abs to response to e 1st subtype of influenza experienced earlier.E CMI is responsible for e clearance of an established infection. Cytotoxic T cells response is cross-reactive & can be directed against both internal proteins & surface glycoprotein. LAB DIAGNOSISDetection of Ags: a rapid diagnosis can be made by e detection of influenza Ag from nasopharyngeal aspirates & throat washings by IFT & ELISA.Virus isolation: virus may be readily isolated from nasopharyngeal aspirates & throat swabs & cultured in embryonated eggs or MDCK cells.Serology: a retrospective diagnosis may be made by serology via CFT type-specific, HIA & NT subtype & strain specific, & EIA for type-specific diagnosis. Molecular amplification test such as PCR.TREATMENT & PREVENTIONTreatment:Amantidine: effective against influenza A if given early in e illness. However, resistance emerges rapidly.Rimantidine: similar to amantidine but fewer neurological side effects.Ribavirin: effective against both influenza A & B.Neuraminidase inhibitors: highly effective with fewer side effects. Resistance towards it emerges slowly.Prevention:Vaccination:Inactivated split/subunit vaccines r available against influenza A & B.E vaccine is normally trivalent, consisting of one A H3N2 strain, one A H1N1 strain, & one B strain.Prophylaxis: Amantidine can be used for those who r allergic to e vaccine (embryonated egg) r during period before e vaccine takes effect.PARAMYXOVIRUSESPORTIONDETAILSPROPERTIESUnder e paramyxoviridae family whose members r all initiate infection via respiratory tract.Virion: spherical, pleomorphic, > 150 nm in diameter with helical nucleocapsid of 13-18 position: RNA (1%), protein (73%), lipid (20%), & carbohydrate (6%).Genome:Linear, non-segmented, -ve sense, ssRNA which is non-infectious & of ~15 kb in size.No-segmented genome negates any opportunity for frequent genetic reassortment which causes e virus to be antigenically stable.Do not undergo antigenic drift as a result of mutation introduced during replication because nearly all aa involved in structural & functional roles.6 structural proteins:3 proteins r complexed with e viral RNA: e nucleoprotein (N or NP) that forms e helical nucleocapsid of 13-18 nm in diameter.This represents e major internal protein & 2 other larger proteins which involved in e viral polymerase activity functioning in RNA transcription & replication.3 proteins participate in viral envelope: matrix (M) protein underlies e viral envelope which has an affinity for both N & e viral surface glycoprotein & essential for virion assembly. Envelope:Made up of lipid which surrounds e nucleocapsid.Studded with 8-12 nm spikes of 2 different transmembrane glycoproteins.Glycoproteins:Help differentiate e various genera in e family.E larger tetramer glycoprotein (HN or H or G) is responsible for attachment to e host cells.E (F) glycoprotein mediates membrane fusion & hemolysisn activities.E pneumoviruses appear to contain 2 additional small envelope proteins (M2-1 & SH).CLASSIFICATIONDivided into 2 subfamilies & 7 genera whose most of e members r monotypic & antigenically stable.E genera:Respirovirus: 2 serotypes of human parainfluenza viruses.Rubulavirus: 2 other parainfluenza virus & mump virus.Avulavirus: avian parainfluenza virus.Morbillivirus: measles virus.Heipavirus: zoonotic paramyxoviruses.Pneumovirus: respiratory syncytial viruses & pneumonia virus.Metapneumovirus: human respiratory pathogen.REPLICATIONVirus attaches to host cell via hemagglutinin glycoprotein (CD46 or CD150 molecules) → virion envelope fuses with e cell membrane by e action of e fusion of glycoprotein F1 cleavage product → release of viral nucleocapsid directly into cell → mRNA transcripts r made in e cytoplasm by viral RNA polymerase → viral proteins r synthesized in e cytoplasm → viral glycoproteins r synthesized & glycosylated in e secretory pathway → progeny nucleocapsids form in e cytoplasm & migrate to cell surface → virus matures by budding.PARAINFLUENZA VIRUSESEPIDEMIOLOGYR major cause of LRT disease in young children & r widely distributed geographically.Type 3 is most prevalent during e 1st year of life while infection with type 1 & 2 occur at a lower rate.Type 3 is endemic with some increase during spring while type 1 & 2 tend to cause epidemics during fall or winter.Reinfections r common t/out childhood & in adults & results in mild URTI.Viruses r ransmitted by direct person-to-person contact, large droplet aerosols & through both nose & eyes which spread readily in a population.E incubation period: 5-6 days.Can also cause nosocomial infection in pediatric wards in hospitals.PATHOLOGYViral replication is limited to respiratory epithelia & viremia is rare.E infection involves only nose & throat resulting in harmless common cold syndrome.If involves e larynx & upper trachea, croup characterized by respiratory obstruction d/t swelling of e larynx & related structures presents.If spread deeper to lower trachea & bronchi, pneumonia or bronchiolitis occur.Immune status of e patients.Airway hyperreactivity.E production of virus-specific IgE during primary infection.Virus shedding is ~1 week after onset of illness & persistent shedding especially in children may facilitate e viral spread.Severity is determined by:Susceptibility of e protein to cleavage by proteases.Production of inappropriate protease by host cells.CLINICAL FINDINGSImmunocompetant patients:Type 1 & 2: rhinitis, pharyngitis, laryngotracheitis, croup, bronchiolitis, & pneumonia.Type 3: severe illness of croup & febrile illness.E most common complication: otitis media.Immunocompromised patients: susceptible to severe infection with mortality rates in BM transplantation of <20%.LABORATORY DIAGNOSISDetection of Ags: a rapid diagnosis can be made by e detection of parainfluenza Ag from nasopharyngeal aspirates & throat washing.Virus isolation: virus may be readily isolated from nasopharyngeal aspirates & throat swabs.Serology: a retrospective diagnosis made by serology, CFT is most widely used.TREATMENT & PREVENTIONContact isolation precautions to manage nosocomial outbreaks: restriction of visitors, isolation of infected patients, & growning & hand washing by medical personnels.E antiviral ribavirin for treatment of immunocompromised patients with LRTI.Both subunit vaccine & live attenuated type 3 virus vaccine for prevention.RESPIRATORY SYNCYTIAL VIRUSESEPIDEMIOLOGYDistribution is worldwide & is e major pediatric respiratory tract pathogen.Subgroup A infections appear to cause > severe illness than subgroup B infections.Viruses r spread by large droplets & direct contact with e eyes & e nose as e main portal of entry.Although virus is very labile, it can survive on environmental surfaces for up to 6 hours.In tropical areas, e virus epidemics may coincide with rainy seasons.E virus can also cause nosocomial infections in nurseries & on pediatric hospital wards. Transmission occurs primarily via hospital staff members.E virus can also cause symptomatic disease in healthy young adults in crowded conditions like in military camps.PATHOLOGYReplication occurs initially in epithelial cells of e nasopharynx.Virus may be spread into e lower respiratory tract & causes bronchiolitis & pneumonia.There is lymphocyte migration, resulting in peribronchiolar infiltration, submucosal tissues become edematous & plug consisting of mucus, cellular debris, & fibrin occlude e smaller bronchioles.E incubation period b/w exposure & onset of illness is 3-5 days.Viral shedding may persist for 1-3 weeks from infants & young children with e presence of high viral titre, whereas adults shed for only 1-2 days.Patients with impaired CMI may become persistently infected with e virus & shed virus for months.Infants at risk of severe infection:Infants with congenital heart disease.Infants with underlying pulmonary disease especially bronchopulmonary dysplasia.Immuocompromised infants d/t congenital immunodeficiency disease.CLINICAL FINDINGSClinical features range from inapparent infection or common cold through pneumonia in infants to bronchiolitis in very young babies with wheezing.Progression of symptoms may be very rapid, culminating in death.Reinfection is common in both children & adults, but usually is limited the upper respiratory tract.Can also cause otitis media.LABORATORY DIAGNOSISDetection of Ags: a rapid diagnosis can be made by e detection of RSV Ags from nasopharyngeal aspirates & throat washing.Virus isolation: virus may be readily isolated from nasopharyngeal aspirates & throat swabs.Serology: a retrospective diagnosis made by serology, CFT is most widely used.TREATMENT & PREVENTIONAerosolized ribavirin can be used for infants with severe infection & for those at risk of severe disease.No vaccine yet available.RSV Igs can be used to protect infants at risk of severe RSV disease.MUMPS VIRUSESEPIDEMIOLOGYMumps occur worldwide where e cases appear t/out e year in hot climates & peak in winter & spring.Outbreaks occur where crowding favors dissemination of e virus.Epidemics may occur in army camp.Mumps is primarily an infection of children where it reaches e highest incidence in children aged 5-9 years.Mumps is contagious & most susceptible persons can acquire infection from e close member.Modes of transmission: direct contact, air borne droplets, & fomites contaminated with saliva or urine.E overall mortality rate for mumps is low, mostly d/t encephalitis. PATHOLOGYHumans r e only natural hosts for mumps virus.1o replication occurs in nasal or upper respiratory tract epithelial cells.Viremia then disseminates e virus to e salivary glands & other major organ systems.E incubation period ranges from 2-4 weeks but is typically 14-18 days.Virus is shed in saliva from about 3 days before & 9 days after e onset of salivary gland swelling.Virus frequently infects e kidneys & viruria may persist for up to 14 days after e onset of clinical symptoms. CLINICAL FINDINGS> 1/3 of all mumps infections r subclinical including e majority of infections in children under 2 years old.Swelling of e gland: 50% of patients.A prodromal period of malaise & anorexia is followed by rapid enlargement of parotid glands with S involvement: aseptic meningitis & meningoencephalitis (bith resolve w/o sequelae).Pacreatitis is reported in ~4% of cases.Genital system: Testes & ovaries may be involved.Orchitis: 20-50% of men with mumps (lack of elasticity of tunica albuginea does not allow inflamed testis to swell, e complication is extremely painful). Atrophy of testis may occur as s result of pressure necrosis.Mumps oophoritis occurs in 5% of women.LABORATORY DIAGNOSISIsolation & identification of virus: Samples r from saliva, CSF, & urine.Monkey kidney cells r preferred for viral isolation.For rapid diagnosis, IF using mumps-specific antiserum can be used to detect mumps virus Ags.CPE of mumps: cell rounding, giant cell formation, & hemadsorption.Serology:Positive IgM Abs strongly suggests recent infection.Significant increase in IgG Abs b/w acute & convalescent specimens.Methods used: ELISA or HI.TREATMENT & PREVENTIONImmunization with attenuated live mumps virus vaccine to reduce mumps associated morbidity & mortality.Mumps vaccine is in combination with measles & rubella: MMR.2 doses of MMR r recommended for school entry.Infected students & health care worker should be excluded from work until 9 days after e onset of parotitis. MEASLES (RUBEOLA) VIRUSESEPIDEMIOLOGYMeasles is higly contagious, a single serotype, no animal reservoir, & infection confers lifelong immunity.Prevalence & age incidence r related to population density, economic, & environmental factors, & e use of MMR.Modes of transmission: respiratory routes & hematogenous transplacental transmission.A continuous supply of susceptible individuals is required for e virus to persist in e community.Measles is endemic t/out e world.Epidemics recur regularly every 2-3 years & tend to occur in late winter & early spring.PATHOLOGYHumans r e only natural hosts for measles virus.Pathogenesis: entry via RT → multiplies locally → infection spreads to e regional lymphoid tissue → further multiplication occurs → 1o viremia disseminates e virus → virus replicates in RED system → 2o vviremia seeds e epithelial surfaces of e body (skin, RT, & conjunctiva) → focal replication.CPE: multinucleated giant cells with intranuclear inclusion body in lymphoid tissue t/out e body.E incubation period: 8-12 days but may lasts up to 3 weeks in adults.CLINICAL FINDINGS2 phases of measles infection:Prodromal period of catarrhal stage: Lasts for 2-4 days.Characterized by fever, sneezing, coughing, running nose, eye redness, Koplik’s spots, & lymphopenia.E cough & coryza reflects an intense inflammatory reaction involving e mucosa of e RT.Koplik’s spots: pathognomonic for measles, small, bluish-white ulcerations on e buccal mucosa opposite e lower molars. The spots contain giant cells & viral Ags & appear about 2 days before rash.E fever & cough persist until e rash appears & then subside within 1-2 days.Exanthematous stage:Lasts for 5-8 days.E moculo-popular skin rash:Starts on e head & then spreads progressively to e chest, e trunk, & down e limbs.Appears as light pink, discrete macula-papules that coalesce to form blotches, becoming brownish in 5-10 days.E fading rash resolves with desquamation.Modified measles:Occurs in partially immune persons such as infants with residual maternal Abs.E incubation period is prolonged.Prodromal symptoms r diminished.Koplik’s spot r usually absent & rash is limited.E complications of measles:E most common complication is bacterial superinfcetion which causes otitis media (5-9% of cases), sinusitis, pneumonia, & sepsis.Giant cells pneumonia in patients with deficiencies in CMI which d/t unrestrained viral replication & has a high fatality rates.Acute postinfectious encephalitis: 1: 1000 which is very serious autoimmune demyelinating disease (no virus production in e CNS).Subacute sclerosing panencephalitis:1: 300000.E disease begins insidiously 5-15 years after a case of measles.Characterized by progressive mental deterioration, involuntary movements, muscular rigidity, & coma.Usually fatal within 1-3 years after onset.Exhibits high titers of measles Ab in CSF & serum & defective measles virus in brain cells.LABORATORY DIAGNOSISVirus detection: can be detected directly in epithelial cells in respiratory secretions & urine.Isolation & identification: Samples: nasopharyngeal & conjunctival swabs, blood samples, respiratory secretion, & urine.CPE: multinucleated giant cells containing both intranuclear & intracytoplasmic inclusion bodies.Shell using fluorescent Ab staining.Serology:Depends on a fourfold rise in Ab titer b/w acute-phase & convalescent-phase sera.ELISA, HI, & Nt tests. TREATMENT & PREVENTIONVitamin A treatment has decreased mortality & morbidity.Measles virus is susceptible in vitro to inhibition by ribavirin.Vaccination for measles:Monovalent form.In combination with live attenuated rubella vaccines (MR).Live attenuated rubella & mumps vaccine (MMR).Vaccine induced Abs persist for up to 33 years.Contraindications:Pregnancy.Allergy to eggs or neomycin.Immune compromise (except for AIDS patients).Recent administration of Igs.RUBELLA VIRUSPORTIONDETAILSOVERVIEWAn acute febrile illness characterized by a rash & lymphadenopathy that affect children & young adults.Rubella virus is a member of Togaviridae family & e sole member of genus Rubivirus.EPIDEMIOLOGYRubella is worldwide in distribution.Infection occurs t/out e year with a peak incidence in e springs.Epidemics occur every 6-10 years with explosive pandemics every 20-25 years.Infection is transmitted by e respiratory route, but rubella is not contagious as measles.POSTNATAL RUBELLAPATHOLOGYNeonatal, childhood, & adult infections occur t/out e mucosa of e upper respiratory tract.Initial viral replication in e respiratory tract → multiplication in cervical lymph nodes → viremia develops after 7-9 days & lasts until e appearance of Abs on about day 13-15 days. After e rash appears, e virus remains detectable only in e nasopharynx where it may persist for several weeks.One attack of e disease confers a lifelong immunity because rubella IgG persists for life.Immune mother transfer Abs to their offspring who r then protected for 4-6 months.CLINICAL FINDINGSBegins with malaise, low-grade fever, & a morbilliform rash appearing on e same day.E rash starts on e face, extends over e trunk & extremities, & rarely lasts > than 3 days.Transient arthralgia & arthritis r commonly seen in adults, especially women.Rare complications include thrombocytopenia & penic purpura & encephalitis.LABORATORY DIAGNOSISIsolation & identification of virus:Specimen r obtained from nasopharyngeal or throat swabs.Various lines of monkey or rabbit origin may be used for cell culture.Viral Ags can be detected by IF 3-4 days postinoculation.Serology:Detection of IgG is evidence of immunity.HI & ELISA tests are a standard serological test for rubella.TREATMENT & PREVENTIONRubella is a mild, self-limited illness, & no specific treatment is indicated.IGIV is injected into e mother cannot protect e fetus against rubella.MMR vaccine: to prevent congenital infection with lifelong immunity & less side effects.CONGENITAL RUBELLA SYNDROMEPATHOLOGYMaternal rubella associated with rubella infection during pregnancy may result in infection of e placenta & fetus.E infection may lead to deranged & hypoplastic organ development resulting in structural anomalies in e newborn.Tetratogenic effects depend on e age of pregnancy: 1st trimester: abnormalities in e infant in ~85% of cases.2nd trimester: detectable defects r found in ~16% of infants.After 20 weeks of gestation: birth defects r uncommon.E earlier in pregnancy infection occurs, e greater e damage to e fetus.At birth, virus is usually detected in pharyngeal secretion, multiple organs, CSF, urine, & rectal swabs.Viral excretion may last for 12-18 months after birth, but e level of shedding gradually decreases with age.CLINICAL FINDINGSCan be divided into 3 broad categories:Transient effects in infants.Permanent manifestations that may be apparent at birth or become recognized during e 1st year.Developmental abnormalities that appear & progress during childhood & adolescence.E classic triad of rubella: cataract, cardiac abnormalities, & deafness.Infants may develop transient symptoms of growth retardation, rash, hepatosplenomegaly, jaundice, & meningoencephalitis. CNS involvement: > global & may lead to mental retardation with problems in balance & motor skills which may require institutionalization. TREATMENT & PREVENTIONNo specific treatment but can be prevented by childhood immunization with MMR.PICORNAVIRUSESPORTIONDETAILSIntroductionThey are two major groups in this virus class. They are:Enterovirus – inhabit inside the human alimentary canal.Rhinovirus – mainly at the nose and throat.Structure & CompositionThe picornaviruses are nonenveloped (naked), small (22 to 30 nm) icosahedral virions resistant to lipid solventsThe virus capsid is composed of 60 copies each of four viral proteins VP1–4, which form a quasi T = 3 icosahedral shell.The RNA strand consists of approximately 7,500 nucleotides and is covalently bonded to a noncapsid viral protein (VPg) at its 5′ end and to a polyadenylated tail at its 3′ end.This genome RNA serves as an mRNA and initiates the synthesis of virus macromolecules. Thus, this virus is a positive sense virus.Enterovirus and some rhinoviruses are stabilized by magnesium chloride against thermal inactivationEnteroviruses have a buoyant density in CsCl of about 1.33 to 1.34 g/ml Human rhinoviruses are about 1.38 to 1.42 g/ml. The viral capsid gives the picornaviruses their characteristic shape and size and protects the infectious viral RNA from hostile environments and host ribonucleases.Enteroviruses can survive for long periods in organic matter and are resistant to the low pH in the stomach (pH 3.0 to 5.0). By contrast, rhinoviruses are labile at this pH range. Picornaviruses are inactivated by pasteurization, boiling, formalin, and chlorine. ClassificationThe Picornaviridae family contains 9 genera:Enterovirus:Polioviruses.Coxsackieviruses ( Group A & B).Echoviruses. Enteroviruses.Rhinovirus: includes more than 100 antigenic types.Hepatovirus: Hepatitis A.Parechovirus: Parechovirus.Aphthovirus: Foot-and-mouth disease virus of cattle.Cardiovirus: Encephalomyocarditis virus of rodents.Replication Site: replication cycle occurs in the cytoplasm of cells:The virion attaches to specific receptor on the plasma membrane↓Receptor binding triggers a conformational change in the virion which results in release of the viral RNA into the cell cytosol↓Genome-linked protein (VPg) is removed from the viral RNA and associates with the ribosomes↓Translation occurs by a cap-independent mechanism, using the internal ribosome entry site ( IRES ) downstream from the 5’ end of the viral genome↓The infecting viral RNA is translated into a polyprotein that contains both proteins and essential replication proteins ↓RNA-dependent RNA polymerase are synthesized↓Viral RNA strand is copied ↓The complementary strand serves as a template for the synthesis of a new plus strand↓The coat precursor protein P1 is cleaved to form aggregates of VP0, VP3 and VP1↓When adequate concentration is reached, these ‘promoters’ assemble into pentamers that package plus-stranded VPg- RNA to form provirions↓The provirions are not infectious until a final cleavage changes VP0 to VP4 and VP2↓The virus are released when the cell disintegratesEnterovirus GroupPoliovirusIntroductionIt is acute infectious diseaseIts serious form affects the CNSThe destruction of motor neurons in the spinal cord results in flaccid paralysis Properties of the VirusGeneral PropertiesTypical EnterovirusThey are inactivated when heated at 55 ?C for 30 min Mg2+, 1 mol/L prevents its inactivationAnimal Susceptibility & Growth of VirusPolioviruses have a very restricted host range Most strains will infect monkeys when inoculated directly into the brain or the spinal cordPoliovirus requires a primate-specific membrane receptor for infection, thus, poliovirus only infect those of the primates and higher animalsAntigenic PropertiesThere are three antigenic types of poliovirusPathogenesis & PathologyPortal of entry – mouth↓Primary multiplication of the virus takes place in the oropharynx or intestine↓The virus is regularly present in the throat and in the stools before the onset of illness↓One week after infection there is little virus in the throat↓Virus continues to be excreted in the stools for several weeks even though high antibody levels are present in the blood↓Antibodies to the virus appear early in the disease, usually before paralysis occur↓The virus first multiplies in the tonsils, the lymph nodes of the neck, Peyer’s patches and the small intestines↓The CNS are invaded by the dissemination of the virus via hematogeneous spread↓Poliovirus can spread along the axons of peripheral nerves to the CNS, where it continues to progress along the fibres of the lower motor neurons, thus involve the spinal cord or the brain.↓The virus invades certain types of nerve cells, and in the process of its intracellular multiplication it may damage or completely destroy these cells↓PARALYSISClinical findingsIncubation period: 7 – 14 days.Mild DiseaseMost common form of the diseaseMinor illnessCharacterized by fever, malaise, drowsiness, headache, vomiting, constipation, sore throatNonparalytis Poliomyelitis (Aseptic Meningitis)The disease lasts for 2 – 10 daysStiff neck and severe headacheParalytic PoliomyelitisFlaccid paralysisIncoordination secondary to painful spasms of nonparalyzed muscles may also occurProgressive Postpoliomyelitis Muscle AtrophyMuscle wastingIt is not a consequence of persistent viral infection, but rather a result of physiologic and aging changes in paralytic patients already burdened by loss of neuromuscular functionsLab DiagnosisThroat swabs.Specimen should be kept frozen.CPE appear in 3 – 6 days.PCR.Require molecular techniques to differentiate between the wild and the vaccine type.ImmunityImmunity is permanent to the virus type causing infection.Antibody mediated.Passive immunity is transferred from mother to child: maternal antibodies gradually disappear within 6 months.Virus-neutralizing antibodies forms soon after exposure to the virus, often before the onset of illness, and persists for life: the VP1 surface protein of poliovirus is responsible for the formation of the neutralizing antibodies.EpidemiologyPoliomyelitis has three epidemiologic phases: Endemic.Epidemic.Vaccine era The prevaccine pattern include the improves systems of hygiene and sanitation in cooler climates.The diseases occur in all age groups, but more prevalent in children.Adult is less susceptible to the infection because of the acquired immunity.Humans are the only known reservoir hosts. Prevention & ControlBoth live-virus and killed-virus vaccine are available:Salk vaccine ( formalinized vaccine) is prepared from virus grown in monkey kidney cultures.Killed-virus vaccine: induces humoral antibodies but does not induce local intestinal immunity. Thus, the virus can still replicate inside the gut. Oral live attenuated vaccine: The live polio vaccine infects, multiplies, and immunizes.The vaccine induce the production of Ig M, Ig G & Ig A antibodies in the intestines (systemic and gut immunity). Both killed-virus and live-virus vaccine induce antibodies and protect the CNS from subsequent invasion by wild virusLimiting factors r e Interference: If the alimentary tract is infected by other enterovirus at the time the vaccine is given, the establishment of polio infection and immunity may be blocked. This phenomenon usually occurs at places where enterovirus infection is common. Pregnancy is neither an indication for nor a contraindication to required immunization.Live-virus vaccine should not be administered to immunodeficient or immunosuppressed individuals or their household contacts.Only killed-virus (Salk) vaccine is to be used. There are no antiviral drugs are prepared for the treatment of poliovirus.COSACKIE VIRUSESPORTIONDETAILSIntroductionA large subgroup of the enterovirusThey are divided into two groups ; A & BHaving different pathogenicity potential for miceThey produce variety illnesses to human, such as aseptic meningitis, respiratory and undifferentiated febrile illnessesGroup A: Herpangina (vesicular pharyngitis), hand-foot-and-mouth disease & acute hemorrhagic conjunctivitisGroup B: Pleurodynia, myocarditis, pericarditis, severe generalized disease of infantsA number of A and B serotypes can give rise to meningoencephalitis and paralysisCoxsackie virus group B are the most commonly identified causative agents of viral heart disease in humans.The coxsackie virus are more pathogenic than echovirusProperties of the VirusCertain strain can grow in monkey kidney cell culture Some Group A strains grow in human amnion and human embryonic lung fibroblast cellsPathogenesis & PathologyVirus has been recovered form the blood at initial stages of infection in humansVirus is also found in the stool for 5 – 6 weeksVirus distribution is similar to that of other enterovirusClinical FindingsIncubation period = 2 – 9 daysClinical manifestation = diverseDiseaseCausative VirusClinical ManifestationsAseptic meningitisall types of Group B coxsackie virussome strains of Group A coxsackie virusFever, malaise, headache, nausea, abdominal pain are common early symptomsMild muscle weakness – suggestive for paralytic poliomyelitisPatients almost recover completely from nonpolio virus paresisHerpanginasome Group A Coxsackie virusAbrupt onset of fever and sore throat with discrete vesicles on the posterior half of the palate, pharynx, tonsils or tongueSelf limitedFrequent in small childrenHand-foot-and-mouth diseaseCoxsackie virus A16Oral and pharyngeal ulcerationsVesicular rash of the palms and soles that may spread to the arms and legsVesicles heal without crusting (clinically differentiated from the vesicles of herpesvirus and poxvirus)The virus can be isolated from stool, pharyngeal secretions and vesicular fluid.PleurodyniaGroup B virusesFever, stabbing chest pain that lasts for 2 days to 2 weeksPreceded by malaise, headache and anorexiaAbdominal pain occurs in half of the casesRecover is complete bur relapses are commonMyocarditisGroup B virusesIt is a serious diseaseMay cause inflammation of the heart or the pericardiumMay cause permanent heart damage to any age groupPersistent viral infections of the heart may also occur, sustaining chronic inflammationRespiratory Tract InfectionsA number of Coxsackie virusCause common colds & undifferentiated febrile illnessesGeneralized Disease of InfantsGroup B virusesExtremely fatal diseaseSimultaneous viral infections of various organsIn severe cases – myocarditis or pericarditis can occur first 8 days of lifeCan be preceded by a brief episode of diarrhea and anorexiaCan be acquired transplacentallyAlthough the GIT is the primary site of replication for enterovirus, they do not cause marked disease there.Certain Group A virus are responsible for diarrhea in childrenLaboratory DiagnosisRecovery of VirusViruses can be isolated from throat washings during the first few days of illness and the from the stools the first few weeksCoxsackievirus A21 – can be found in the nasal secretionsIn aseptic meningitis, strains can be recovered from the CSF as well as the GITNucleic Acid DetectionReverse transcription –PCR are reactive (detect many serotypes) and specificReal time PCRSerologyNeutralizing antibodies appear early during the course of infection, tend to be specific for the infecting virus and persist for yearsSerum antibodies can be detected by IFImmunityNeutralizing antibodies are transferred from mother to childAdults tend to have more types of antibodies against Coxsackievirus than do children, indicating that multiple experiences with these viruses are common and increasingly so with ageEpidemiologyCoxsackievirus are more frequently encountered during the summer and early fallMany children develop antibodies against these viruses which indicates infectionControlThere are no antiviral drugs or vaccines available for the treatment of CoxsackievirusOTHER ENTEROVIRUSESPORTIONDETAILSIntroductionEchovirus (Enteric Cytopathic Human Orphan viruses).They primarily infect the human enteric tract.Clinical FindingsTo establish etiologic association of an enterovirus with disease, the following criteria are used:There is much higher rate of recovery of virus from patients with the disease than from healthy individuals of the same age and socioeconomic level in the same area at the same timeAntibodies against the virus develop during the course of infectionThe virus can be isolated from body fluids or tissues manifesting the lesionsMany echovirus infections are associated with aseptic meningitisRashes are more common in young childrenEnterovirusClinical manifestationEnterovirus 70Chief cause of acute hemorrhagic conjunctivitisHas a sudden onset of subconjuctival hemorrhageMost common in adultsComplete recovery is a ruleThe virus is highly communicable and spreads rapidly under crowded or unhygienic conditionsEnterovirus 71Presents with meningitis, encephalitis, and paralysis resembling poliomyelitisMain causes of CNS diseaseLab DiagnosisIt is impossible in an individual case to diagnose an echovirus infection on clinical grounds.However, in epidemic situations echovirus must be consideredSummer outbreaks of aseptic meningitisSummer epidemics, especially in young children, of a febrile illness with rashDiagnosis depend on:Lab testNucleic acid detection assays, PCRViral isolation from throat swabs, rectal swabs, CSFInfection with 2 or more viruses may occur.EpidemiologySame like other enterovirusUsually occur during summer and early autumnHigh prevalence in young childrenControlAvoid contact with patients exhibiting acute febrile illness esp, those with a rashThere are no antiviral drugs or vaccine available for the treatment of any enterovirus diseasesRHINOVIRUSESPORTIONDETAILSIntroductionCommon cold viruses which can be isolated from nasal, throat and oral secretions.Cause upper respiratory tract infections with common cold syndrome.ClassificationThere are more than 100 speciesHuman rhinoviruses can be divided into two groups : major & minor receptor groupsMajor receptor groups – they use the intercellular adhesion molecule-1 (ICAM-1) as a receptorMinor receptor groups – utilize the low-density lipoprotein receptor (LDLR) family Properties of the VirusGeneral PropertiesRhinoviruses are picornavirus similar to enterovirusDiffer from enterovirus by having buoyant density in CsCl = 1.40 g/ml and acid labileVirions are unstable under the pH 5 – 6Complete inactivation at pH 3.0Rhinoviruses are more thermostable than enterovirusesAnimal Susceptibility & Growth of VirusThese viruses are only infectious for humans, gibbons and chimpanzeesMost grow better at 33° C which is similar to the temperature of the nasopharynx in human, than at 37° CAntigenic PropertyMore than 100 serotypes are knownPathogenesis & PathologyThe virus enters the upper respiratory tract – high titers of virus in nasal secretions as early as 2 – 4 days after infection↓Replication is limited to the surface epithelium of the nasal mucosa↓Edema & infiltration of the nasal mucosa↓Nasal secretion increases in quantityRhinoviruses rarely causes lower respiratory tract infectionSometime after infection, virus titers fall even though the infection persists!Clinical FindingsIncubation period = 2 days – 4 daysAcute illness usually lasts for 7 daysNonproductive cough may persists for 2 – 3 weeksSymptomsSneezing, nasal obstruction, nasal discharge, sore throatHeadache, mild cough, malaise, chilly sensationThere is little of no feverThe nasal and nasopharyngeal mucosa become red and swollenThe sense of smell become less keenSecondary bacterial infection may produce acute otitis medis, sinusitis, bronchitis, pneumonitis, esp in childrenImmunityNeutralizing antibodies to the infecting virus develops in serum and secretions of most personAntibodies develop 7 – 21 days after infectionThe appearance of neutralizing antibodies in nasal secretions is parallel to the serum antibodiesEpidemiologythis disease occurs throughout the worldIn temperate climates the highest attack rates are in early fall and late springPrevalence rates are lower in summerMode of transmission – close contact, by means of virus-contaminated respiratory secretionsRhinoviruses can survive in the environment for several hoursIn a single outbreak, multiple serotypes of rhinoviruses can cause infectionsTreatment & ControlNo specific prevention methods are available The development of vaccine against rhinoviruses are very unlikely because it is difficult to grow the virus in a culture media, the fleeting immunity and the different serotypes that cause infectionAntiviral drugs are thought to be the most likely control measure for rhinoviruses because of problem in developing the vaccineA 5- day course of high doses of intranasal interferon -α STERILIZATION & DISINFECTIONPortion Details DefinitionSterilizationThe total inactivation (killing) of all forms of all microbial life in terms of the organism’s ability to reproduceDisinfectionThe killing or removal of organisms capable of producing an infectionChemical disinfectantA substance that is used to kill organisms in inanimate surfaces but it is too toxic to be applied directly to tissues.AntisepsisThe prevention of sepsis (putrefaction), either by removal or killing of the organisms (-cidal) or by opposing their growth (-static)Chemical antisepticA substance that can be applied topically to living tissuesDegermingRemoval of microbes from a limited areaSanitizationLower microbial counts on eating utensilsMeasurement of DeathMethodIt is estimated by plotting the bacterial count on a semi-log scale against the time of exposure to the antimicrobial actionProperties of Death rate CurveIt is either logarithmic or exponential curveOne log decrease indicates 90% of the population is killedThe initial lag or shoulder may be due to either multihit inactivation dynamics or to spore germinationThe final lag is due to either to the presence of mixed population of sensitive or resistant organism or to protection from the antimicrobial action e.g. the presence of organic material.Types of inactivationSingle-hit inactivation- damaging one target molecule is enough to kill the organismMulti-hit inactivation- damaging more than one target is necessary to inactivate an organism.Factors affecting antimicrobial treatmentNumber of microbesEnvironment e.g. organic matter, temperature, biofilmsTime of exposureMicrobial characteristicsActions of Antimicrobial AgentsAlternation of membrane permeabilityDamage to proteinsDamage to nucleic acidSterilizationTypes of sterilizationPhysical methodsChemical methodsHeatDry heatMoist heatEthylene oxideBeta propiolactoneAldehydesFormaldehydeGlutaraldehydeIonizing radiationGamma raysMechanismFiltration removes microbeLow temperature inhibits microbial growthRefrigerationDeep freezingLyophilizationHigh pressures denatures proteinDesiccation prevents metabolismOsmotic pressure cause plasmolysisRadiation damages DNAAll are alkylating agentsMethodsDry heatKills organisms by oxidationE.g. by direct flaming,incinerators, hot air oven Ethylene oxideAdvantages: Highly microbicidalHigh penetrating powerIt is used to sterilize heat sensitive equipmentsDisadvantages:An explosive gasHighly toxic gasNeed a special chamber to perform sterilization.Moist heatAutoclave A device that produces steam at high pressures and temperature in a closed chamber e.g.121° CThe type of the autoclave depends on how the air is removed. Downward displacement autoclaveHigh prevacuum autoclaveAdvantages : Local release of latent heat of evaporation at points of steam condensation on the articles being sterilizedGood penetration of steamBeta propiolactoneAn alkylating agent that hydrolyze very quickly to acrylic acidUsed to sterilize serum and other biological agentsUsed to kill microorganisms in the preparation of killed antigen vaccines. AldehydesGood –cidal activity even against sporesBoth aldehydes are toxic and may cause hypersensitivityDisinfect fibre-optic endoscopesIonizing Radiation ( gamma radiation) Are lethal to DNAGreat penetrating power, even of sealed packsSources: radioactive isotopes, such as Coblt-60Disadvantages: expensive and need extra precautionSterilization ProcessInvolves the steps:Designing the sterilization processTesting the processTesting the processDesigning the sterilization processBioburdenInitial contamination levelIncreased bioburden means increased time required to achieve a given level of sterility assuranceThus, precleaning before sterilization is important.Sterility assuranceIt is the probability that the organism may survive through a sterilizing processThe required level is dependent on the intended use of the articles10-6 is required for aseptic procedures10-12 is required for canned food10-3 is required for the articles that are going to come in contact with the skin or the mucus membraneRate of biocidal actionTo calculate the time required to achieve a certain level of sterility assurance we must know the decimal reduction time (D value)The time required to reduce the microbial population by 90% at a certain temperatureTesting the processPhysical testingTemperature and pressure measurement on record chartsAir leak test for pre-vacuum autoclavesChemical testingBrowne’s tubesThe tubes that are put in the pack to be sterilized The chemical liquid indicator will change color from red to green when exposed to the required temperature for the required timeBowie-Dick’s TestPlacing autoclave sheets with markings that change color in the pack and look for a uniform color change in the test sheet indicating the appropriate penetration of steam to the packs center.This test measures the sterilizer’s ability to remove all the air from the chamber in a pre-determined period of time. Testing the processBacteriological spore testSpore strips of the heat resistant Bacillus staerothermophilus They are included in different parts of the autoclave in a packAfter the completion of the sterilization process, they are cultured in suitable broth.Disinfection & AntisepsisObjectivesTo reduce the count of pathogenic organisms in a potential source of infection to below that required to cause infectionTypesCleansing (degerming)Reduces the number of organismRemoves organic matter, dirt, grease that might protect the organism from inactivationDisinfection by physical methodHeatingPasteurization Holder type process - 63°C for 30 minutesFlash process - 72°C for 20 secBoiling at 100°CCan kill most vegetative organism within one minuteBut spores might need more than 20 minutesLow temperature steam disinfectionIt is more effective if the steam is combined with formaldehyde.Thydillization Used for heat sensitive culture mediaIt is a successive procedurePls read notes,he3 ~UV lightMost efficient wavelength at 260nmTo decontaminate open spaces and surfacesDisadvantages – poor penetration. FiltrationFiltration heat-labile liquidsDepth filterConsists of granular material bonded into thick layer containing twisted channels of small diameterMilipore membrane filtersPorous membranes made of cellulose acetate or nitrate, polycarbonate etcThey replaced the depth filtersFiltration of air using HEPA filterUsed in operating theatres or in microbial safety hoodsDisinfection and antisepsis with chemicalsCleaning is a prerequisite before chemical disinfection because organic matter and dirt can interfere with their action and help protect the organism against inactivationFew chemicals are sporicidal. ................
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