1. Basic Techniques to Grow Viruses and Study Virus-HostInteractions

2. Growth of Viruses While it is easy to grow bacterial viruses, it is much more difficult and expensive to grow animal viruses Whole animals Embryonating eggs (the classic host for vaccineproduction) 3. Growth of Viruses, continued Organ culture - pieces of brain, gut, or trachea, etc.containing different cell types are grown in culture 4. Organ cultures Sections through tracheal organ cultures: (a) uninfected; (b) infected with a rhinovirus for 36 hours. Note the disorganization of the ciliated cells (uppermost layer) after infection. 5. Growth of Viruses, continued Cell or tissue culture this is where tissues areremoved from an organism and are grown invitro, usually in flasks Primary cultures are cells that have been directly derived from a tissue and placed in culture. Are differentiated They, like the tissue from which they werederived, have a limited life span. Most will grow attached to the flask as a monolayer ofcells one cell thick. 6. Making a primary cell line 7. Growth of Viruses, continued Cell lines Are dedifferentiated Are diploid Survive more passages than primary cell lines, buteventually die Immortalized cells or continuous cell lines are cells thathave a mutation or mutations that allow the cells to bepassaged many times, i.e. they dont have a limited lifespan. Are usually heteroploid Most were originally derived from a tumor. Most grow as monolayers, though a few grow insuspension. 8. Making a continuous cell line 9. Tissue Culture Cells 10. Growth of Viruses, continued When cells grow as monolayers, they can be used toquantify the number of animal viruses using a plaque assay. The virus is serially diluted in a liquid medium. For each dilution a set amount is added to a separateplate containing a monolayer of tissue culture cells andthe viruses in that solution are allowed to attach to thetissue culture cells. After attachment has been allowed to occur, a semi-solid medium is added to restrict the movement of newviruses produced so that only adjacent cells will beinfected. 11. Growth of Viruses, continued Where virus has infected the tissue culture cells, the infected cells will die causing the formation of a clear zone amongst the otherwise intact monolayer of cells This clear zone is called a plaque and it theoretically represents an area where one virus has infected a single tissue culture cell, has multiplied and been released, and has gone on to infect adjacent cells. The number of plaque forming units (pfu)/ml can be calculated based on the dilution of the original viral solution. The term pfu/ml is used rather than the number of viruses/ml because it is possible that occasionally more than one virus infects a single cell. Often the cells or plaques are stained to help in visualization of the plaques. 12. Serial dilutions 13. Animal Virus Plaque Assay 14. Plaque assay results 15. Basic Techniques to Study Viruses and Virus-Host Cell Interactions Serological and immunological methods these tests are often used for diagnosis of viral infections May assay directly for the virus (direct assay) May assay for antibodies, produced in the host, againstthe virus (indirect assay) Hemagglutination assay-a direct method to titer virus. Is based on the ability of some viruses to agglutinate RBCs. Virus is titered by making serial two-fold dilutions of the virus and determining the highest dilution of virus that causes agglutination of the RBCs. 16. Hemagglutination assay 17. Hemagglutination assay 18. Serological/Immunological Methods Hemagglutination-Inhibition Assay an indirect test for antibody against specific viruses that can agglutinate RBCs. Mix serial dilutions of patients sera with the virus that is thesuspected causative agent of the patients infection, andthen add RBCs. If the patient has antibodies specific to the virus, they willbind to the virus and prevent the virus from agglutinating theRBCs. 19. Hemagglutination inhibition assay 20. Serological/Immunological Methods Immunofluorescence may be either: direct and test for the presence of viral antigen in tissues or indirect and test for the presence of antibodies against a specific virus in a patients sera. This method uses an antibody with a fluorescent tag attached to it. With the direct test, the antibody that is tagged is an antibody against the virus that one is testing for. In the indirect test, the tagged antibody is an antibody against another antibody, i.e. anti-human IgG. The presence of the fluorescent tag is detected by looking under a fluorescent microscope. 21. Direct immunofluorescentantibody test 22. Indirect immunofluorescent antibody test 23. Immunofluorescence ?? 24. Serological/Immunological Methods ELISA (enzyme linked immunosorbent assay) Can either be direct (tests for virus) or indirect (tests for antibody to virus). ELISA is similar to the immunofluorescent assays, butdiffers in the type of molecule that is tagged to theantibodies that are used. The molecule that is attached to an antibody in an ELISA assay is an enzyme. The presence of the enzyme is detected by adding a substrate to the enzyme which when acted upon by the enzyme produces a colored product. An indirect ELISA test is used to screen individuals for HIV infection. 25. Direct (sandwich) ELISA (virus?) 26. Indirect ELISA virusagainst virus? 27. Indirect versus direct (sandwich) ELISA 28. ELISA (sandwich method to detect Ag) 29. ELISA (indirect) 30. Serological/Immunological Methods Western immunoblot- A Western immunoblot can be either direct or indirect. The Western immunoblot analyzes a sample for a specificprotein(s) (direct) or for antibodies against a specificprotein(s) (indirect). The screening test to diagnose HIV is the indirect ELISAtest. The indirect Western immunoblot is used to confirm apositive ELISA test. 31. Western immunoblot 32. Western Blot 33. Indirect Western immunoblot forHIV diagnosis 34. Indirect Western immunoblot for HIV diagnosis 35. Basic Techniques to Study Viruses and Virus-Host Cell Interactions Ultrastructural studies used for purification purposes Physical methods Size by filtration- molecular sieve chromatography. Uses a column filled with beads containing holes. Large molecules are excluded from the holes and come off the column first. Small molecules enter the holes in the beads and therefore move slower down the column, coming off the column after large molecules. 36. Molecular Sieve Chromatography 37. Physical methods Centrifugation Can pellet materials (virus) by centrifugation Equilibrium density gradient centrifugation an inertmaterial is used and it forms a density gradient during thecentrifugation. Materials (virus) are forced down until theyreach a density that buoys them up. Rate-zonal centrifugation similar to density gradientcentrifugation, but uses a preformed gradient rather thangenerating a gradient during the centrifugation process. 38. Centrifugation 39. Equilibrium density gradientcentrifugation 40. Physical methods Electrophoresis materials are forced through a meshwork of matrix material (agarose or polyacrylamide) by an electric current. Usually used for nucleic acids or proteins which are separated on the basis of size, shape, and charge. 41. Electrophoresis 42. Physical methods Affinity chromatography Takes advantage of highly specific binding interactions. A column is made with a material that has a specific receptor (binding interaction) for the substance you are trying to purify (for example the receptor for a particular virus). A solution from which you wish to purify your virus is run through the column. The virus binds to the receptor, but everything else is washed through the column. Next you run a new solution through the column which changes the conditions (pH, ionic strength, etc.) in the column to those in which the specific virus-receptor interaction no longer occurs. The virus will be eluted from the column. 43. Affinity chromatography 44. Physical methods X-ray crystallography Chemical methods to determine the overall composition and the nature of the nucleic acid Electron microscopy Whole mounts + staining (heavy metals) - staining Ultrathin sections 45. Basic Techniques to Study Viruses and Virus-Host Cell Interactions Molecular biology often used to study the structure of the nucleic acid Hybridization to come together through complementary base-pairing. Can be used in identification. For in situ (or plaque) hybridization the tissue containing the putative organism is treated to release the nucleic acid which is then denatured to single strands. Labeled single-stranded DNA (a probe) unique to the organism you are testing for is added and hybridization is allowed to occur. Unbound probe is washed away and the presence of bound probe is determined by the presence of the label. 46. In situ hybridization 47. Molecular Biology Polymerase chain reaction used to amplify something found in such small amounts that without PCR it would be undetectable. Uses two primers, one that binds to one strand of a double-stranded DNA molecule, and the other which binds to the other strand of the DNA molecule, all four nucleotides and a thermostable DNA polymerase. The primers must be unique to the DNA being amplified and they flank the region of the DNA to be amplified. 48. PCR The PCR reaction has three basic steps Denature when you denature DNA, you separate it into single strands (SS). In the PCR reaction, this is accomplished by heating at 950 Cfor 15 seconds to 1 minute. The SS DNA generated will serve as templates for DNAsynthesis. Anneal to anneal is to come together through complementary base-pairing (hybridization). During this stage in the PCR reaction the primers base-pair withtheir complementary sequences on the SS template DNAgenerated in the denaturation step of the reaction. 49. PCR The primer concentration is in excess of the template concentration. The excess primer concentration ensures that the chances of the primers base-pairing with their complementary sequences on the template DNA are higher than that of the complementary SS DNA templates base-pairing back together. The annealing temperature used should ensure that annealing will occur only with DNA sequences that are completely complementary. WHY? The annealing temperature depends upon the lengths and sequences of the primers. The longer the primers and the more Gs and Cs in the sequence, the higher the annealing temperature. WHY? The annealing time is usually 15 seconds to 1 minute. 50. PCR Extension during this stage of the PCR reaction, the DNA polymerase will use dNTPs to synthesize DNA complementary to the template DNA. To do this DNA polymerase extends the primers that annealedin the annealing step of the reaction. The temperature used is 720 C since this is the optimumreaction temperature for the thermostable polymerase that isused in PCR. Why is a thermostable polymerase used? The extension time is usually 15 seconds to 1 minute. The combination of denaturation, annealing, and extension constitute 1 cycle in a PCR reaction. 51. PCR Most PCR reactions use 25 to 30 of these cycles to amplify the target DNA up to a million times the starting concentration. 52. PCR 53. PCR 54. Molecular Biology DNA sequencing used to determine the actual DNA sequence of an organism. Using a computer, one can predict protein sequences and functions based on the nucleic acid data. The most commonly used sequencing method is the dideoxymethod. This method uses dideoxy nucleotide triphosphates (ddNTPs) which have an H on the 3 carbon of the ribose sugar instead of the normal OH found in deoxynucleotides (dNTPs). Dideoxynucleotides are chain terminators. In a synthesis reaction, if a dideoxynucleotide is added instead of the normal deoxynucleotide, the synthesis stops at that point because the 3OH necessary for the addition of the next nucleotide is absent. 55. Deoxy versus dideoxy 56. DNA synthesis 57. DNA sequencing continued In the dideoxy method of sequencing, the template DNA that is to be sequenced is mixed with a primer complementary to the template DNA and the four normal deoxynucleotides, one of which is radioactively labeled for subsequent visualization purposes. This mixture is then splint into four different tubes that are labeled A, C, G, and T. Each tube is then spiked with a different dideoxynucleotide (ddATP for tube A, ddCTP for tube C, ddGTT for tube G, or ddTTP for tube T). DNA polymerase is added and using the DNA template and its complementary primer, the synthesis of new strands of DNA complementary to the template begins. Occasionally a dideoxynucleotide is added instead of the normal deoxynucleotide and synthesis of that strand is terminated at that point. 58. DNA sequencing continued In the tube containing ddATP, some percentage of newly synthesized molecules will get a ddATP in each place that there is a T in the template DNA. The result is a set of new DNA molecules in tube A, each of which ends in an A. A similar type of reaction occurs in the three other tubes to result in molecules that end in C, G, and T in tubes C, G, and T respectively. After the synthesis reactions are complete, the products of the four different tubes are loaded onto four adjacent lane of a polyacrylamide gel and the different fragments are separated by size. The sequencing gel is able to resolve fragments that differ in size from each other by only one base. 59. DNA sequencing continued After electrophoresis to separate the fragments by size, the fragments are visualized by exposing the gel to photographic film (Remember that one nucleotide was radioactively labeled). All fragments in lane A will end in an A, fragments in lane C will all end in a C, fragments in lane G will all end in a G, and fragments in lane T will all end in a T. The sequence of the DNA is read from the gel by starting at the bottom and reading upward. 60. Dideoxy DNA Sequencing 61. DNA sequencing 62. DNA sequencing Automated DNA sequencing in automated DNA sequencing a radioactive deoxynucleotide is not used and all four dideoxy reactions are done in a single tube. This is possible because each dideoxynucleotide is labeled with a different flourescent dye. Therefore the dye present in each synthesized fragment corresponds to the dye attached to the dideoxynucleotide that was added to terminate the synthesis of that particular fragment. The contents of the single tube reaction are loaded onto a single lane of a gel (or capillary) and electrophoresis is done. A flourimeter and computer are hooked up to the gel (or capillary) and they detect and record the dye attached to the fragments as they come off the gel. The sequence is determined by the order of the dyes coming off the gel. 63. Automated DNA sequencing