1. Virology 1.4-Virus Cultivation and Assays How do you grow viruses and then determine how many there are in a sample? (Physical, Chemical, Biological approaches)

2. Initial Detection Usually symptoms in infected host But rarely detection of unusual appearance of cultured cells Body fluids or culture medium tested on uninfected host

3. Cultivation Maintained in laboratory by serial passage through intact organisms or cell cultures (or in the freezer) Laboratory stocks expanded for bulk harvesting Virus maintained in the lab will not have the same properties as the virus from nature

5. Direct particle counts-a physical method  Limited usefulness  Internal standard  Electron microscopy or or cytometry cytometry

6. Example Mix virus stock with known concentration of virus-sized plastic spheres. Count # virions & spheres per EM field. From the ratio of spheres to virus, calculate virus “concentration”. Are the virus particles “alive”, “dead”, “pseudovirions” or just “empties” (e.g. bromoviridae)?

7. Example of particle counts Direct Counts of Viruses in Natural Waters and Laboratory Cultures by Epifluorescence Microscopy, by Kilian P. Hennes and Curtis A. Suttle © 1995 American Society of Limnology and Oceanography. American Society of Limnology and OceanographyAmerican Society of Limnology and Oceanography http://www.azonano.com/article.aspx?ArticleID=2476 http://www.applitechpharma.com/en/product.asp?id=223

8. Chemical Measurement By nucleic acid (genome) concentration: The MW of the genome must be known to use this method. Determine DNA or RNA amount Divide by “molecular weight” of one genome Virus particle count can be approximated.

9. Example A solution contains 1 ug of pure DNA. Each virus genome contains 1 fg of DNA. 1 ug pure DNA/ 1 fg DNA per copy = 1,000,000,000 copies

10. qPCR is another way to measure the number of genomes

11. Hemagglutination assay Based on Hemagglutinin (HA) protein Causes RBCs to agglutinate agglutinate Coat well bottom evenly evenly HA titer Frequently used with influenza A

12. Biological Methods: Infectivity assays Measurement of key biological activity Ability to cause an infection detects “live” viruses (signs, symptoms, death, etc.)

13. Better methods to measure infections How many infections are caused by a How many infections are caused by a defined amount of my sample? defined amount of my sample? Two types of assays used Two types of assays used Dilution (quantal) Dilution (quantal) Titration (preferred method-quantitative) Titration (preferred method-quantitative)

14. Dilution or quantal assays Dilution End Point (DEP) Infectious Dose 50% (ID 50 ) n/10 individuals infected

15. Dilution end point Highest (or greatest) dilution of sample that still shows infectivity Cartoon illustrates methodology with cultured cells on a plate (not all the methodology with cultured cells on a plate (not all the data) data)

16. More data from the experiment SampleDilutionLogDilution # Infected Wells Total # OfWells%Infected None04848100 1/10003394881 1/100004144829 1/10000053486 1/100000060480

17. Graphic data from a similar experiment ID 50 or TCID 50

18. Titration  Most common quantitative assay  “Titer” used as noun or verb  More accurate count  All cells in sample must get the same treatment  Involves same geometry

19. Titration invented for lytic bacteriophage  Allow adsorption of phage to occur in a small volume  Dilute mixture  Plate samples in top agar  Incubate 8-16 hrs Holes in the lawn will form = plaques

20.  Plaque forming unit (PFU)  Provides a count of infectious units in sample  Phage titer = dilution x average plaque count/volume plated

21. Phage plaques  101 plaques per plate  0.1 ml plated  1 x 10 -5 dilution  Titer = ?  PFU = plaque forming units Additional examples of phage plaques

22. Animal virus titration  Technically difficult (use of live animals)  No comparable system to bacteria at first  Type of data depends on host-virus interaction  Some viruses could be roughly titrated in eggs

23. Use of eggs to quantitate virus

24. Animal virus titration  Dependent upon ability to culture host cells in vitro (1948-)  Dulbecco adapts plaque assay to animal viruses 1952 At Caltech I continued to work with phages for a few years. One day I was told by Delbrück that a rich citizen had given Caltech a fund for work in the animal virus field. He asked me whether I was interested. My medical background and the experience gained in Levi's laboratory came back to me and I accepted. After visiting the major centers of animal virus work in the US I set out to discover the way to assay animal viruses by a plaque technique, similar to that used for phages, using cell cultures. Within less than a year, I worked out such a method, which opened up animal virology to quantitative work. I used the technique for studying the biological properties of poliovirus. These successes brought me an appointment first to associate professor, then to full professor at Caltech.

25. Primary cell culture

26. Terminology Organ culture, Explant culture, cell culture Cell culture Primary cell cultures cell strains cell lines Adherent Contact inhibition Passage Secondary culture

27. TerminologySelection Senescence or crisis Transformation (aka cell transformation) ImmortalTumorigenic Continuous cell lines Euploid/aneuploid Growth factor dependent/independent

28. Formation of a cell line “Crisis” “Immortal” Continuous cell line

29. HeLa Cells Henrietta Lacks-1951 Cervical tumor First highly prolific human cell line

30. Animal virus plaques HSV on Vero cells Polio on Hela cells HSV on Vero cells Polio on Hela cells Vital stain-taken up by living cells

31. Antiviral resistance measured by plaque assay Wild type AndMutantInfluenzaVirus Effect of Ribavirin ©2003 by National Academy of Sciences

32. Transforming viruses  Viruses that cause transformation in host cells  “ viral transformation”  Transformed cells on a plate form a clump = “focus”  Focus assay, expressed in Focus Forming Units or FFU

33. Focus Assays Loss of contact inhibition Immortalized Able to move more freely Cytoskeleton and other organelles change appearance Biochemical changes

34. Cytopathic Effect or CPE The viruses are cytopathic-not lytic infected cells are really sick Misshapen Abnormal Fused (Syncytium) Inclusion bodies

35. Limitations  Titration methods error-prone  100% error?  Measure only those particles that can cause a detectable infection in the system used  Does not count particles that do not cause infection

36. Efficiency of Plating or EOP  Absolute EOP- titer/number of virions  Relative EOP- ratio of virus titer on two hosts  Particle/Pfu ratio- inverse of absolute EOP Variable but always more than 1.0!!! Variable but always more than 1.0!!! Some particles in the preparation Some particles in the preparation cannot start an infection. cannot start an infection.

37. Multiplicity of infection (m.o.i.)  Experimental parameter  Ratio of infectious units to cells in an experiment  Necessary to ensure that each cell has a chance to collide with a virus  Poisson distribution The more virus you use in the experiment (high moi)-the more likely it is that each cell will encounter a virus. Common sense!!!!

38. Titration Statistics-Poisson Distribution  Allows calculation of probability that any given cell in a sample will come in contact with n virus particles.  Moi must be known  Equation P(k) = e -m m k /k! P(k) probability that any cell will be infected with k particles m= moi k! = factorial of k

39. Poisson Distribution P(k) values moi k13510 00.370.050.010.00 10.370.150.030.00 20.180.220.080.00 30.060.220.140.01 40.020.170.180.02 50.000.100.180.04 60.000.050.150.06 70.000.020.100.09 80.000.000.070.11 90.000.000.040.13 100.000.000.020.13

40. Plant virus assays “One sick plant” TMV in tobacco-systemic infection Signs: Mottling with borders-mosaic ChlorosisRugosity

41. Different host-different reaction TMV on pinto bean leaves Localized infection Local-lesion host Very tricky assay, dependent on host plant status

42. Local lesions and assay Local lesion assay Alfalfa mosaic virus on P. vulgaris Half-leaf only is shown Right: TMV on Samsun tobacco NN Left: mock-inoculated

43. Utility of plaque assays  D’Herelle discovers bacteriophage plaques by 1921; viruses must be particulate  Delbruck decides to use phage as the atom of biology in the 1930’s  Ellis and Delbruck demonstrate the one step growth curve in 1939  Later, Luria and Delbruck use plaque assays to demonstrate that mutations are spontaneous

44. One Step Growth Total Phage Extracellular Phage Latent or eclipse phase Intracellular accumulation phase Time after Infection Number of Infectious Particles Lysis

45. Interpretation  Phage are fundamentally different from their hosts because they have a different growth curve.  “Eclipse” or “latent” phase  “burst” as phage are released from infected cells

46. Plaque Assay Dose-Response Animal viruses and phage display single hit kinetics with few exceptions.

47. Some additional reading and some advertisements http://www.iul-instruments.eu/pdf/233_VirusQuantificationWhitePaper.pdf http://people.ucalgary.ca/~ceri/cmmb421prot/virus%20assay.html http://courses.bio.indiana.edu/M430-Taylor/assay.html