Animal hosts Natural host Laboratory animals Rabbits Mice Rats Hamsters Newborn or suckling rodents Animal models for viral pathogenesis 4 Growth of v

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1 Principles of Virology Department of Molecular Genetics & Microbiology Univ ersity of Florida, Gainesv ille, FL 1 Outline Virus cultivation Assay of viruses Virus genetics 2 Virus isolation Evidence of virus infection Skin and mucous membrane lesions Digestive, respiratory or neurological disorders Immune dysfunct ion Specific organ failure: hepatitis or myocarditis Death Virus isolation Excreted or secreted material Blood Tissue Test for virus Identical host Substitute host Cell culture 3 The screen versions of these slides have full details of copyright and acknowledgements 1

2 Animal hosts Natural host Laboratory animals Rabbits Mice Rats Hamsters Newborn or suckling rodents Animal models for viral pathogenesis 4 Growth of virus on embryonated eggs 5 Cell culture Primary cell cultures Cell strains Cell lines 6 The screen versions of these slides have full details of copyright and acknowledgements 2

3 Primary cell culture + enzymes A culture of cells obtained from the original tissue that hav e been cultiv ated in vitro for the first time, and which hav e not been subcultured Time Top view Side view 7 Subculture Enzymes Time 8 Cell strains & cell lines Cumulative number of cells Immortalization Cell strain Cell line Degeneration and death Primary culture Time/passag e 9 The screen versions of these slides have full details of copyright and acknowledgements 3

4 Cell transformation Immortalization Loss of contact inhibition Anchorage independence Growth in soft agar Growth in suspension Tumor formation in immunodeficient mice 10 Comparison of cultured cell types Primary Heterogeneou s many cell types Closest to animal Technically difficult Cell strain Relatively homogeneous fewer cell types Further from animal Technically less difficult Continuous cell line Immortal Most homogeneous Genetically altered furthest from animal Technically least difficult Suspension or monolayer 11 Cultured cell morphologies Epithelial-like (human lung carcinoma, A549) Fibroblast like (baby hamster kidney, BHK) 12 The screen versions of these slides have full details of copyright and acknowledgements 4

5 Recognition of virus infection in culture Cytopathic effect (CPE) Morphological changes Inclusion bodies Hemabsorption 13 CPE: morphological changes Rounding Shrinkage Increased refractility Fusion Aggregation Loss of adherence Lysis 14 CPE: measles 15 The screen versions of these slides have full details of copyright and acknowledgements 5

6 CPE: Vaccinia Uninfected Infected 16 CPE: inclusion bodies Neuron Negri bodies Negri bodies 17 Hemabsorption Add red blood cells 18 The screen versions of these slides have full details of copyright and acknowledgements 6

7 Hemabsorption 19 Outline Virus cultivation Assay of viruses Virus genetics 20 Assay of viruses Biological Plaque assay Focus assay Endpoint method Physical Hemagglutination Particle count 21 The screen versions of these slides have full details of copyright and acknowledgements 7

8 Plaque formation 22 Plaque 23 Plaque assay 1:100 Virus 1:10 1:10 1:10 1:10 1:10 Serial dilution Plate 1 ml Plaques (100,000) (10,000) (1000) Titer = 1 x 10 7 pfu/ml 24 The screen versions of these slides have full details of copyright and acknowledgements 8

9 Plaque assay Titer = 2.4 x 10 8 pfu/ml 25 Focus formation Time 26 Focus assay 27 The screen versions of these slides have full details of copyright and acknowledgements 9

10 Endpoint titration Dilution replicat e samplings = Infected = Uninfected TCID 50 per ml (TCID = tissue culture infective dose) 28 Assay of viruses Biological Plaque assay Focus assay Endpoint method Physical Hemagglutination Particle count 29 Hemagglutination RBC 30 The screen versions of these slides have full details of copyright and acknowledgements 10

11 Hemagglutination test 1:8 Virus 1:2 1:2 1:2 1:2 1:2 Serial dilution Mix with red blood cells Side v iew Top view Titer = 32 HA units/ml 31 Hemagglutination test: Influenza virus 32 Direct particle count Beads (10 4 /ml) 1.5 x Virus 10 4 virus/ml (?) 10 beads => 1 ul 15 virus => 1.5 x 10 4 virus/ml 33 The screen versions of these slides have full details of copyright and acknowledgements 11

12 Direct particle count 34 Comparison of quantitative methods Method Direct electron microscope count Quantal infectiv ity assay in eggs Quantal infectiv ity assay by plaque formation Hemagglutination assay Amount (per ml) EM particles 10 9 egg ID pfu 10 3 HA units 35 One step growth experiment Virus 1) Infect T=0, moi = 10 2) various times T=3 T=6 T=9 T=12 T=0 T=24 T=48 3) Titer 1x10 7 1x10 6 1x10 6 5x10 7 3x10 8 7x10 8 1x10 9 Virus yield (pfu/cell) Time (hr) 36 The screen versions of these slides have full details of copyright and acknowledgements 12

13 One step growth experiment 37 Outline Virus cultiv ation Assay of v iruses Virus genetics 38 Genetic principles Mutation Selection Recombination 39 The screen versions of these slides have full details of copyright and acknowledgements 13

14 Scope of virus genetics Natural ev olution of v iruses Clinical management of v irus infections Experimental v irology 40 Clinical significance of genetics Antigenic v ariation in HIV and influenza Impact on v accination Drug resistance in herpes and HIV Rev ersion of attenuation in polio v accine Engineered v accines Temperature sensitiv e influenza (Flumist) 41 Mutation RNA v irus polymerases lack proofreading function RNA v iruses mutate more frequently than DNA v iruses RNA v irus "quasi species" are adaptable 42 The screen versions of these slides have full details of copyright and acknowledgements 14

15 Mutant phenotypes Conditional lethal Host range Temperature sensitiv e Drug dependence Drug resistance Plaque morphology 43 Host range wt hr Permissiv e Non-permissiv e wt hr Grow virus No growth Phenotype virus 44 Host range subtypes Natural Identified by brute force or serendipity Affect only a few v irus genes Engineered Deliberately target a specific v irus gene 45 The screen versions of these slides have full details of copyright and acknowledgements 15

16 Engineered host range Permissiv e Non-permissiv e No growth Grow virus Phenotype virus 46 Temperature sensitivity wt ts Low temp High temp wt ts Grow virus No growth Phenotype virus 47 Temperature sensitive mutants Genotype Single amino acid substitutions Mechanism Protein unstable, non-functional at an high temperature Protein stable, functional at a low temperature Isolation Random mutagenesis Brute force screening for growth at two temperatures Targeting possible Advantages Accesses any essential virus gene using a single set of protocols Cold sensitive mutants a rare but useful alternative 48 The screen versions of these slides have full details of copyright and acknowledgements 16

17 Temperature sensitivity wt 31 o C wt 40 o C ts 31 o C ts 40 o C 49 Antiviral drugs & virus genetics Some drugs target similar enzymes in different viruses Phosphonoacetic acid (DNA polymerases) Bromodeoxyuridine (thymidine kinases) Most drugs are virus and gene specific Guanidine (polio 2C NTPase) Acyclovir (Herpes simplex virus thymidin e kinase, DNA polymerase) Amantidine (Influenza virus M2 protein) Isatin-beta-thiosemicarbazone (viral transcription) 50 Uses of drug resistance/dependence Identify drug target or mechanism of action Amantidine resistance in influenza Selectable markers Recombination mapping Assessment of genetic protocols Selection of recombinant v iruses in rev erse genetic protocols 51 The screen versions of these slides have full details of copyright and acknowledgements 17

18 Drug resistance wt -IBT wt +IBT mut -IBT mut +IBT 52 Drug dependence wt -IBT wt +IBT mut -IBT mut +IBT 53 Recombination and reassortment DNA viruses Breaking and joining Non-segmented RNA viruses Copy choice Segmented RNA viruses Reassortment Impact Intrinsically interesting Virus evolution Laboratory virology 54 The screen versions of these slides have full details of copyright and acknowledgements 18

19 DNA virus recombination 55 RNA virus recombination Copy choice 56 RNA virus reassortment 57 The screen versions of these slides have full details of copyright and acknowledgements 19

20 Reverse genetics Engineer mutations in v irus genes Application Study of gene function Engineering attenuated v iral v accines Gene therapy The screen versions of these slides have full details of copyright and acknowledgements 20