General Characteristics of Viruses

1. General Characteristics of Viruses • Acellular entities – virions • Reproduce only inside living cells • Single type of nucleic acid • Double or single stranded DNA or RNA • Is a group characteristic • Orthomyxoviruses – SS RNA • Herpesviruses – DS DNA • Protein capsids • Envelope

2. Exist as Distinct Entities Only in Extracellular Form Virion Infects Cell

3. Exist as Distinct Entities Only in Extracellular Form Virion Breaks Down

4. Exist as Distinct Entities Only in Extracellular Form Progeny Components Produced

5. Exist as Distinct Entities Only in Extracellular Form Progeny Assembled & Released

6. Theories on Origin of Viruses • Regressive hypothesis - represent ultimate in intracellular parasitism - descended from more complex life forms - have lost everything they don’t need - no intermediates (?) - weakens theory

7. Theories on Origin of Viruses • Outlaw messenger hypothesis - viruses were originally cellular DNA or RNA components that somehow developed ability to move from one cell to another • Originated along with self-replicating molecules and evolved along with cells.

8. Virus Cultivation • Cultivation vs tests involving replication • Bacterial viruses (bacteriophage or phage) • Broth cultures of host bacteria • Animal viruses • Animals • Embryonated eggs • Tissue cultures – cytopathic effects (CPE)

9. Fig. 16.1

10. Tissue Culture Trypsin Individual cells Tissue Liquid media Monolayer

11. Tissue Culture Types • Primary cell cultures • Derived from tissues/eggs • Limited number of “passes” • Maximal sensitivity • Cell lines • Derived from tumors • Pass indefinitely • Ordinarily less sensitive than primary • Hep, HeLa, • Both grow in tubes, plates, bottles

12. Isolation, Purification & Assay • Process Source – tissue, swabs, stools, insects, environmental samples • Cleanup – suspend in diluent with high level of antibiotics - centrifuge • Innoculate supernatant into animals, eggs or tissue cultures • Observe for death or CPE • Pass • Purify • Centrifugation – differential or density gradient • Precipitation – ammonium sulphate • Denaturation of contaminants

13. Differential Centrifugation

14. Density Gradient Centrifugation

15. Virus Assays • Direct counting – EM • Hemagglutination assay • Plaque assay • ID or LD50

16. Hemagglutination Assay • Some animal viruses bind to red blood cells • Use this property to “quantitate” viruses Virus added & diluted 2 4 8 16 32 64 32 Hemagglutinating Units Constant RBC added

17. Animal Virus Plaque Assays Grow monolayer Inoculate/incubate Add MM w agar inc Overlay agar contains dye that stains living cells

18. How to Make a 1:1000000 dilution • Take 1 ml of material – add it to 1000 liters of diluent – need a bath tub to do it • Take 0.0000001 ml of material and add it to 1 ml of diluent – don’t make pipettes that small • Make serial 10 fold dilutions – takes 0.1 ml of material and 6 ml of diluent

19. Serial Dilutions 1 2 3 4 5 6 7 9 ml of diluent in each tube 1 ml of virus suspension in first tube (1:10) Shake 1 ml from tube 1 into tube 2 (1:100) Shake 1 ml from tube 2 into tube 3 (1:1000) …

20. Dil -1 TMTC TMTC TMTC -2 TMTC TMTC TMTC -3 TMTC 276 TMTC -4 178 215 143 -5 63 27 49 -6 7 1 9 -7 1 0 0 0.1 ml of inocula (178+215+143)/3 = 179X104X101 =1.8x107pfu/ml

21. Virus Structure • Virion – virus particle • Capsomeres – protein subunits protomers • Capsomeres assembled to make the capsid • Nucleic acid & proteins – nucleocapsid • Envelope - external lipid layer • Complex viruses

22. Virus Structure Icosahedral Complex 10-400 nm

23. Virus Structure • Symmetry • Helical • Icosahedral

24. Nucleic Acid • Range from just enough to code for 3-4 proteins to more than 100 proteins. • DNA – linear or circular, DS or SS • + strand – serve directly as mRNA • - strand – complement serves as mRNA • Orthomyxovirus – segmented genome

25. Nucleic Acid

26. Envelope • Outer layer • Common in animal viruses • Derived from host cell membrane and viral proteins • Many are disrupted by ether or ethanol

27. Virus Structure

28. Vaccinia Structure

29. Viral Taxonomy • Nature of host – bacteria, plant, animal • Nucleic acid – RNA/DNA, SS/DS • Capsid symmetry • Envelope • Diameter of virion or nucleocapsid • # capsomers • Immunologic properties • Gene number and map • Intracellular location of replication • DNA intermediates & RT • Type of virus release • Disease

31. Chap 17 - Bacteriophage Bacterio (Bacteria) + Phage (eat) • 1915 Frederick Twort isolated viruses that infected bacteria • Phage served as a model for virus infection until polio grown in tissue culture in 1949 • Some carry virulence factors • Some have high level of host specificity & are used to type bacteria • Potential for use in treating bacterial infections

32. Phage Typing • Different strains of same species of bacteria differ in their susceptibility to different strains of phage • Assemble “libraries” of different phage types and bacterial strains • Set up test “grids” Bact Phage Type strain A B C D E F 1 + O O + + + 2 + O O O O + 3 + + + O O O • Useful in linking isolates (e.g. common source outbreaks)

33. Both Both Archae Archae Archae

34. One-step Growth Curve • 1939 – Delbruck & Ellis – served as model for virus infection • Synchronize phage cycles – infect then dilute to delay 2nd generation • Eclipse period – no complete virus • Latent period - no free virus • Rise period – virus being released from cells • Burst size - # viruses/cell Complete Free Virus in cell but not released Virus not made

35. One-step Growth Curve

36. Initial Stages in T4 Phage Infection Table 16.2 • Bind to receptors on cell surface • Receptors may be proteins, teichoic acids, lipopolysacchides, flagella & pili • Responsible for host specificity

37. Initial Stages in Phage PRD1 Infection Infects Pseudomonads & Enterbacteriaceae

38. Synthesis of Phage Proteins & Nucleic Acids Switch from early – late transcription involves shift from host – viral sigma factors

39. Synthesis of Phage Proteins & Nucleic Acids • T4 phage model. • Bacterial RNA pol transcribes early viral mRNA. • Early proteins involved in shutting down host cell processes – including digestion of host genome - and replication of viral NA. • T4 DNA contains hydroxymethyl cytosine instead of C – protects from host REs • Late mRNA codes for progeny structural proteins, assembly proteins & lytic proteins

40. Assembly & Release

41. ΦX174 Replication • SS DNA virus - icosahedral • Infects E. coli • Genome is SS DNA circular + Strand (same a mRNA) • 5386 nucleotides • Overlapping genes

42. ΦX174Genome

43. ΦX174 Replication Bacterial enzymes (e.g. primase, DNA polymerase, gyrase & ligase) Replicative Form (DS DNA) Viral DNA + SS Formation of RF involves RNA primers

44. X174 Replication (Rolling Circle) Progeny synthesized using - strand as template

45. X174 Replication (Rolling Circle)

46. ΦX174 Replication Rolling Circle Replication Progeny DNA Replicative Form (DS DNA) Viral mRNA

48. Temperate Phage & Lysogeny • Temporate vs virulent phage • Lytic vs lysogenic infection • Lysogens – bacteria with temporate phage • Prophage • Induction • Cryptic virus lost ability to be induced • Temperate phage may alter host phenotype – increase pathogenicity and change surface proteins to block infection by other phage

49. Temperate vs Virulent Phage Provirus Lysogen Cell lysis is result of viral products

50. λ Phage • Infects E coli • Establish 2 mutually exclusive types of infection • Lytic - virulent • Lysogenic -temperate • Lytic infection • Viral structural proteins are produced • Host cell is lysed • Lysogenic infection • Structural proteins not produced • Phage genome incorporated into host - prophage • Replicates along with it • Advantageous when low nutrients or high MOI