Viruses

1. Viruses Microbiology 221

2. Viruses • Obligate intracellular parasites • They are able to reproduce their life cycle only within the cell of their host • They usually have an external capsid composed of proteins • Inner core of nucleic acid( dsDNA, ssDNA,dsRNA, and ssRNA) • Specificity for the host

3. Classification of viruses • According to Baltimore classification, viruses are divided into the following seven classes: • dsDNA viruses • ssDNA viruses • dsRNA viruses • (+)-sense ssRNA viruses • (-)-sense ssRNA viruses • RNA reverse transcribing viruses • DNA reverse transcribing viruses • where "ds" represents "double strand" and "ss" denotes "single strand".  

4. Table of Viruses

5. Viral Capsids

6. Capsids • Capsids are made from protein subunits called capsomeres • In some viruses, the capsomeres are all the same Geometric shape in others the sub units vary

7. Antigenic spikes • Some viruses have molecules inserted into the outer covering of the virus • These may be glycoproteins • In some cases these serve as a means of attaching to the host cell • They are specific for one cell type

8. Capsid Shapes

9. Ebola – Shepherd’s Crook

10. Enveloped Virus

11. Envelopes • Lipid composition • Acquired from the host when the virus exits the cell • Provides a means for viruses to elude the immune system of the host by surrounding itself with the host envelope • Enveloped viruses may be more vulnerable to chemical agents(chlorine, hydrogen peroxide, and phenol) • They do not survive well on surfaces

12. Viral Life Cycle – Factors Influencing the Life Cycle • Nucleic acid • Enveloped or naked • Shape • Host

13. Host Range • Primates • Vertebrates ( birds) • Plants • Insects • Bacteria

14. Bacteriophages • Viruses that infect bacterial cells • Genome can be DNA or RNA • Bind to specific receptors that are proteins or carbohydrates in the bacterial cell wall

15. Bacteriophage structure

16. Bacteriophages

17. PhiX174 – Spherical Bacteriophage • Interesting to study because of its overlapping genes which is a model of efficiency

18. T- 4 Bacteriophage • Studied by Luria and Delbruck at Cold Spring Harbor • Ds DNA virus • 168, 800 base pairs • Phage life cycles studied by Luria and Delbruck

19. Filamentous phages • Fd • Filamentous • Circular ss DNA • Lies in the middle of the filament • Infects through the pilus • Create a symbiotic relationship with the host

20. M 13 • Used for Genetic engineering experiments

21. Bacteriophages Life Cycles • Lytic – They attach and enter the bacterial cell. Complete their life cycle by bursting the bacterial cell – “ lysis” • Lysogenic – They attach and enter the bacterial cell. The virus then integrates into the bacterial cell as a “prophage”

22. Process of Infection • Attachment • Injection • Hostile take over( lytic) • Integration( lysogenic) – Genetic control • Early genes/late genes • Replication of nucleic acid • Production of viral proteins • Assembly • Lysis

23. Lytic Life Cycle( Virulent) • The viral genome contains a promoter that attaches to host cell RNA Polymerase • Early genes code for those proteins that shut down the host, replicate nucleic acid, and code for vital proteins • Nuclease genes, are capable of digesting host DNA so that the bases can be used for the production of new virions • Late genes code for viral capsid and for those proteins that lead to lysis of the host cell

24. Lytic Cycle • Strict control • Do not want to lyse the cell prior to the completion of assembly • Usually only one virus in a cell at a time • Recombination occurs between the two viruses if more than one is present

25. Lysogenic Life Cycle • Bacteriophages that do not lyse the bacteria cell are referred to as temperate • Lysogenic bacteria contain a copy of the virus which is non infective and is known as the prophage • The prophage can remain inactive through many cell division • Bacteria can switch between lytic and lysogenic life styles • When the host is stressed or damaged by mutagens, this stimulates the prophase to excise itself and

26. Generalized Transduction • Any part of bacterial genome can be transferred • Occurs during lytic cycle • During viral assembly, fragments of host DNA mistakenly packaged into phage head • generalized transducing particle

27. Generalized transduction

28. Specialized transduction

29. Lambda Phage • Bacteriophage Lambda is a temperate phage meaning that it can undergo either a lytic or lysogenic cycle • The phage regulates this cycle genetically through a switch

30. Attachment • Bacteriophage Lambda binds to the target E. coli cell, the tail tip binding to a maltose receptor. • The linear phage genome is injected into the cell, and immediately circularises. • Transcription starts • There are two regulatory viral proteins, CI and Cro • CI and Cro compete for the operator promoter sites on the phage DNA • When the bacterial host is healthy CI accumulates and the lambda integrates into the bacterial genome and stays in this position .

31. Genes needed to establish lysogeny cI yes cII yes cIII yes Genes needed for maintenance of lysogeny cI yes cII no cIII no Control of lysogeny and lytic cycle

32. Insertion sequences

33. Genetic Control of Lytic and Lysogenic Phage

34. Lytic vs. Lysogenic • When Cro is low, CI maintains the lysogenic life style • When Cro accumulates due to damage of the host DNA or other unsuitable environmental conditions, the virus switches to the lytic life style • This activates promoters for phage replication and protein synthesis • Serves as a model for understanding viral infectivity and genetic control

35. Integration • Integration of bacteriophage lambda requires one phage-encoded protein - Int, which is the integrase - and one bacterial protein - IHF, which is IntegrationHostFactor. • Both of these proteins bind to sites on the P and P' arms of attP to form a complex in which the central conserved 15 bp elements of attP and attB are properly aligned. • The integrase enzyme carries out all of the steps of the recombination reaction, which includes a short 7 bp branch migration.

36. Enzymes and Recombination • The strand exchange reaction involves staggered cuts that are 6 to 8 bp apart within the recognition sequence. • All of the strand cleavage and re-joining reactions proceed through a series of transesterification reactions like those mediated by type I topoisomerases.

37. Excision of bacteriophages • Excision of bacteriophage lambda requires two phage-encoded proteins: • Int (again!) and Xis, which is an excisionase. It also requires several bacterial proteins. • In addition to IHF, a protein called Fis is required. • All of these proteins bind to sites on the P and P' arms of attL and attR forming a complex in which the central conserved 15 bp elements of attL and attR are properly aligned to promote excision of the prophage.

38. Lytic Cycle • Lytic Lifestyle • The 'late early' transcripts -genes for replication of the lambda genome. • The lambda genome is replicated in preparation for daughter phage production. • Transcription from the R' promoter can now extend to produce mRNA for the lysis and the structural proteins. • Structural proteins and phage genomes self assemble into new phage particles. • Lytic proteins build sufficiently far in concentration to cause cell lysis, and the mature phage particles escape.

39. Bacteriophage growth curve

40. Plaque Assay • Eclipse Period- Penetration through biosynthesis • Latent-Spans from penetration up to the point of phage release

41. Lambda and Plaques • The plaque produced by Lambda had a different appearance on the Petri Dish. • It is considered to be turbid rather than clear • The turbidiy is the result of the growth of phage immune lysogens in the plaque • The agar surface contains a ratio of about a phage /107 bacteria

42. Plaque assay

43. Methodology • Grow bacteria to log phase • Prepare dilutions of bacteriophage • Mix bacteria with viruses or overlay bacteria with suspension of viral particles • Incubate • Count infectious particles based upon the number of plaques • Plaques are clear areas indicating lysis of infected cells

44. Plaque Assay

45. MOI • Average number of phages /bacterium • After several lytic cycles the MOI( multiplicity of infection) gets higher due to the release of phage particles

46. Horizontal Transfer of Genes • The transfer of genes or blocks of genes ( pathogenicity islands) from bacterium to bacterium or virus to bacteria or virus to virus • Has resulted in many changes in microbes that have led to increase in pathogenicity and accumulation of virulence genes, not just resistance

47. Streptococcus pyogenes • There are 15 prophages that have been identified in E. coli • These prophages belong to the group Siphoridae • All but one of these produce a toxin • In both strep and staph – the prophage is found at the site of recombination

48. Virulence and Streptococcus pyogenes • Streptococcal pyrogenic exotoxins(SPE) contribute to the diverse symptoms of a streptococcal infection. • These antigens compare to Staphylococcal antigens of the same type. • The A + C genes coding for these toxins were horizontally transferred from strain to strain by a lysogenic bacteriophage. • In addition the genes contributed by the phages produce hyaluronidase, mitogenic factor, and leukocyte( WBC) toxins

49. Pathogens with bacteriophages that cause complications • Corynebacterium diphtheriae • Vibrio cholerae • enterotoxogenic E. coli • Staphylococcus aureus • Clostridium botulinum • Staphylococcus aureus and Streptococcus pyogenes: Toxic shock syndrome

50. Toxic shock