basic techniques to grow viruses and study virus-host interactions

Basic Techniques to Grow Basic Techniques to Grow Viruses and Study Virus-Host Viruses and Study Virus-Host

InteractionsInteractions

Growth of VirusesGrowth of Viruses While it is easy to grow bacterial viruses, it is While it is easy to grow bacterial viruses, it is

much more difficult and expensive to grow much more difficult and expensive to grow animal viruses animal viruses Whole animalsWhole animals Embryonating eggs (the classic host for vaccine Embryonating eggs (the classic host for vaccine

production)production)

Growth of Viruses, continuedGrowth of Viruses, continued

Organ culture - pieces of brain, gut, or trachea, etc. Organ culture - pieces of brain, gut, or trachea, etc. containing different cell types are grown in culturecontaining different cell types are grown in culture

Organ culturesOrgan 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.


Cell or tissue culture – this is where tissues are Cell or tissue culture – this is where tissues are removed from an organism and are grown “in vitro”, removed from an organism and are grown “in vitro”, usually in flasksusually in flasks

Primary cultures – are cells that have been directly derived Primary cultures – are cells that have been directly derived from a tissue and placed in culture. from a tissue and placed in culture.

Are differentiatedAre differentiated They, like the tissue from which they were derived, They, like the tissue from which they were derived,

have a limited life span. have a limited life span. Most will grow attached to the flask as a monolayer of Most will grow attached to the flask as a monolayer of

cells one cell thick.cells one cell thick.

Making a primary cell lineMaking a primary cell line


Cell linesCell lines Are dedifferentiatedAre dedifferentiated Are diploidAre diploid Survive more passages than primary cell lines, but Survive more passages than primary cell lines, but

eventually dieeventually die Immortalized cells or continuous cell lines – are cells that Immortalized cells or continuous cell lines – are cells that

have a mutation or mutations that allow the cells to be have a mutation or mutations that allow the cells to be passaged many times, i.e. they don’t have a limited life passaged many times, i.e. they don’t have a limited life span. span.

Are usually heteroploid Are usually heteroploid Most were originally derived from a tumor. Most were originally derived from a tumor. Most grow as monolayers, though a few grow in Most grow as monolayers, though a few grow in

suspension. suspension.

Making a continuous cell lineMaking a continuous cell line

Tissue Culture CellsTissue Culture Cells


When cells grow as monolayers, they can be used to When cells grow as monolayers, they can be used to quantify the number of animal viruses using a plaque quantify the number of animal viruses using a plaque assay.assay.

The virus is serially diluted in a liquid medium.The virus is serially diluted in a liquid medium. For each dilution a set amount is added to a separate For each dilution a set amount is added to a separate

plate containing a monolayer of tissue culture cells and plate containing a monolayer of tissue culture cells and the viruses in that solution are allowed to attach to the the viruses in that solution are allowed to attach to the tissue culture cells.tissue culture cells.

After attachment has been allowed to occur, a semi-After attachment has been allowed to occur, a semi-solid medium is added to solid medium is added to restrict the movementrestrict the movement of new of new viruses produced so that only adjacent cells will be viruses produced so that only adjacent cells will be infected.infected.


Where virus has infected the tissue culture cells, the infected Where virus has infected the tissue culture cells, the infected cells will die causing the formation of a clear zone amongst cells will die causing the formation of a clear zone amongst the otherwise intact monolayer of cellsthe otherwise intact monolayer of cells

This clear zone is called a This clear zone is called a plaque plaque and it theoretically and it theoretically represents an area where one virus has infected a single represents an area where one virus has infected a single tissue culture cell, has multiplied and been released, and has tissue culture cell, has multiplied and been released, and has gone on to infect adjacent cells.gone on to infect adjacent cells.

The number of The number of plaque forming units (pfu)/mlplaque forming units (pfu)/ml can be calculated can be calculated based on the dilution of the original viral solution.based on the dilution of the original viral solution.

The term pfu/ml is used rather than the number of viruses/ml The term pfu/ml is used rather than the number of viruses/ml because it is possible that occasionally more than one virus because it is possible that occasionally more than one virus infects a single cell. infects a single cell.

Often the cells or plaques are stained to help in visualization Often the cells or plaques are stained to help in visualization of the plaques.of the plaques.

Serial dilutions Serial dilutions

Animal Virus Plaque AssayAnimal Virus Plaque Assay

Plaque assay resultsPlaque assay results

Basic Techniques to Study Viruses and Virus-Basic Techniques to Study Viruses and Virus-Host Cell InteractionsHost Cell Interactions

Serological and immunological methods – these Serological and immunological methods – these tests are often used for tests are often used for diagnosis diagnosis of viral infectionsof viral infections May assay directly for the virus (May assay directly for the virus (direct assaydirect assay)) May assay for antibodies, produced in the host, against May assay for antibodies, produced in the host, against

the virus (the virus (indirect assayindirect assay)) Hemagglutination assay-a Hemagglutination assay-a directdirect method to titer virus. method to titer virus.

Is based on the ability of some viruses to agglutinate RBCs. Is based on the ability of some viruses to agglutinate RBCs. Virus is titered by making serial two-fold dilutions of the virus and Virus is titered by making serial two-fold dilutions of the virus and

determining the highest dilution of virus that causes agglutination determining the highest dilution of virus that causes agglutination of the RBCs.of the RBCs.

Hemagglutination assayHemagglutination assay

Serological/Immunological Serological/Immunological MethodsMethods

Hemagglutination-Inhibition Assay – an Hemagglutination-Inhibition Assay – an indirect indirect test test for antibody against specific viruses that can for antibody against specific viruses that can agglutinate RBCs. agglutinate RBCs.

Mix serial dilutions of patient’s sera with the virus that is the Mix serial dilutions of patient’s sera with the virus that is the suspected causative agent of the patient’s infection, and suspected causative agent of the patient’s infection, and then add RBC’s. then add RBC’s.

If the patient has antibodies specific to the virus, they will If the patient has antibodies specific to the virus, they will bind to the virus and prevent the virus from agglutinating bind to the virus and prevent the virus from agglutinating the RBCs.the RBCs.

Hemagglutination inhibition Hemagglutination inhibition assayassay


Immunofluorescence – may be either:Immunofluorescence – may be either: directdirect and test for the presence of viral antigen in tissues or and test for the presence of viral antigen in tissues or indirectindirect and test for the presence of antibodies against a specific and test for the presence of antibodies against a specific

virus in a patients sera. virus in a patients sera.

This method uses an antibody with a fluorescent tag attached to This method uses an antibody with a fluorescent tag attached to it. it.

With the direct test, the antibody that is tagged is an antibody With the direct test, the antibody that is tagged is an antibody against the virus that one is testing for. against the virus that one is testing for.

In the indirect test, the tagged antibody is an antibody against In the indirect test, the tagged antibody is an antibody against another antibody, i.e. anti-human IgG. The presence of the another antibody, i.e. anti-human IgG. The presence of the fluorescent tag is detected by looking under a fluorescent fluorescent tag is detected by looking under a fluorescent microscope.microscope.

Direct immunofluorescent Direct immunofluorescent antibody testantibody test

Indirect immunofluorescent Indirect immunofluorescent antibody testantibody test

ImmunofluorescenceImmunofluorescence

?

?


ELISA (enzyme linked immunosorbent assay)ELISA (enzyme linked immunosorbent assay) Can either be direct (tests for virus) or indirect (tests for Can either be direct (tests for virus) or indirect (tests for

antibody to virus). antibody to virus). ELISA is similar to the immunofluorescent assays, but ELISA is similar to the immunofluorescent assays, but

differs in the type of molecule that is tagged to the differs in the type of molecule that is tagged to the antibodies that are used. antibodies that are used.

The molecule that is attached to an antibody in an The molecule that is attached to an antibody in an ELISA assay is an ELISA assay is an enzymeenzyme. .

The presence of the enzyme is detected by adding a The presence of the enzyme is detected by adding a substrate to the enzyme which when acted upon by the substrate to the enzyme which when acted upon by the enzyme produces a colored product. enzyme produces a colored product.

An indirect ELISA test is used to screen individuals for An indirect ELISA test is used to screen individuals for HIV infection.HIV infection.

Direct (sandwich) ELISADirect (sandwich) ELISA

(virus?)

Indirect ELISAIndirect ELISA

virusagainst virus?

Indirect versus direct (sandwich) Indirect versus direct (sandwich) ELISAELISA

ELISA (sandwich method to ELISA (sandwich method to detect Ag)detect Ag)

ELISA (indirect)ELISA (indirect)


Western immunoblot- Western immunoblot- A Western immunoblot can be either direct or indirect. A Western immunoblot can be either direct or indirect. The Western immunoblot analyzes a sample for a specific The Western immunoblot analyzes a sample for a specific

protein(s) (direct) or for antibodies against a specific protein(s) (direct) or for antibodies against a specific protein(s) (indirect).protein(s) (indirect).

The screening test to diagnose HIV is the indirect ELISA The screening test to diagnose HIV is the indirect ELISA test. test.

The indirect Western immunoblot is used to confirm a The indirect Western immunoblot is used to confirm a positive ELISA test.positive ELISA test.

Western immunoblotWestern immunoblot

Western BlotWestern Blot

Indirect Western immunoblot for Indirect Western immunoblot for HIV diagnosisHIV diagnosis

Indirect Western immunoblot Indirect Western immunoblot for HIV diagnosisfor HIV diagnosis


Ultrastructural studies – used for purification Ultrastructural studies – used for purification purposespurposes– Physical methodsPhysical methods

Size by filtration- molecular sieve chromatography. Uses a Size by filtration- molecular sieve chromatography. Uses a column filled with beads containing holes. column filled with beads containing holes.

Large molecules are excluded from the holes and come off the Large molecules are excluded from the holes and come off the column first.column first.

Small molecules enter the holes in the beads and therefore Small molecules enter the holes in the beads and therefore move slower down the column, coming off the column after move slower down the column, coming off the column after large molecules. large molecules.

Molecular Sieve ChromatographyMolecular Sieve Chromatography

Physical methodsPhysical methods CentrifugationCentrifugation

Can pellet materials (virus) by centrifugationCan pellet materials (virus) by centrifugation Equilibrium density gradient centrifugation – an inert Equilibrium density gradient centrifugation – an inert

material is used and it forms a density gradient during the material is used and it forms a density gradient during the centrifugation. Materials (virus) are forced down until they centrifugation. Materials (virus) are forced down until they reach a density that buoys them up.reach a density that buoys them up.

Rate-zonal centrifugation – similar to density gradient Rate-zonal centrifugation – similar to density gradient centrifugation, but uses a centrifugation, but uses a preformedpreformed gradient rather than gradient rather than generating a gradient during the centrifugation process.generating a gradient during the centrifugation process.

CentrifugationCentrifugation

Equilibrium density gradient Equilibrium density gradient centrifugationcentrifugation

Physical methodsPhysical methods Electrophoresis – materials are forced through a Electrophoresis – materials are forced through a

meshwork of matrix material (agarose or meshwork of matrix material (agarose or polyacrylamide) by an electric current. polyacrylamide) by an electric current.

Usually used for nucleic acids or proteins which are Usually used for nucleic acids or proteins which are separated on the basis of size, shape, and charge.separated on the basis of size, shape, and charge.

ElectrophoresisElectrophoresis

Physical methodsPhysical methods Affinity chromatography – Takes advantage of highly specific Affinity chromatography – Takes advantage of highly specific

binding interactions. binding interactions. A column is made with a material that has a specific receptor A column is made with a material that has a specific receptor

(binding interaction) for the substance you are trying to purify (binding interaction) for the substance you are trying to purify (for example the receptor for a particular virus).(for example the receptor for a particular virus).

A solution from which you wish to purify your virus is run A solution from which you wish to purify your virus is run through the column. through the column.

The virus binds to the receptor, but everything else is washed The virus binds to the receptor, but everything else is washed through the column. through the column.

Next you run a new solution through the column which changes Next you run a new solution through the column which changes the conditions (pH, ionic strength, etc.) in the column to those in the conditions (pH, ionic strength, etc.) in the column to those in which the specific virus-receptor interaction no longer occurs.which the specific virus-receptor interaction no longer occurs.

The virus will be eluted from the column.The virus will be eluted from the column.

Affinity chromatographyAffinity chromatography

Physical methodsPhysical methods X-ray crystallographyX-ray crystallography

Chemical methods – to determine the overall Chemical methods – to determine the overall composition and the nature of the nucleic acidcomposition and the nature of the nucleic acid

Electron microscopyElectron microscopy Whole mounts Whole mounts

+ staining (heavy metals)+ staining (heavy metals) - staining- staining

Ultrathin sectionsUltrathin sections


Molecular biology – often used to study the Molecular biology – often used to study the structure of the nucleic acidstructure of the nucleic acid Hybridization – to come together through Hybridization – to come together through

complementary base-pairing. complementary base-pairing. Can be used in identification. Can be used in identification. For in situ (or plaque) hybridization the tissue containing For in situ (or plaque) hybridization the tissue containing

the putative organism is treated to release the nucleic the putative organism is treated to release the nucleic acid which is then denatured to single strands. acid which is then denatured to single strands.

Labeled single-stranded DNA (a probe) Labeled single-stranded DNA (a probe) unique unique to the to the organism you are testing for is added and hybridization organism you are testing for is added and hybridization is allowed to occur. is allowed to occur.

Unbound probe is washed away and the presence of Unbound probe is washed away and the presence of bound probe is determined by the presence of the label. bound probe is determined by the presence of the label.

In situ hybridizationIn situ hybridization

Molecular BiologyMolecular Biology Polymerase chain reaction – used to amplify Polymerase chain reaction – used to amplify

something found in such small amounts that something found in such small amounts that without PCR it would be undetectable. without PCR it would be undetectable.

Uses two primers, one that binds to one strand of Uses two primers, one that binds to one strand of a double-stranded DNA molecule, and the other a double-stranded DNA molecule, and the other which binds to the other strand of the DNA which binds to the other strand of the DNA molecule, all four nucleotides and a thermostable molecule, all four nucleotides and a thermostable DNA polymerase. DNA polymerase.

The primers must be The primers must be uniqueunique to the DNA being to the DNA being amplified and they flank the region of the DNA to amplified and they flank the region of the DNA to be amplified.be amplified.

PCRPCR

The PCR reaction has three basic stepsThe PCR reaction has three basic steps Denature – when you denature DNA, you separate it Denature – when you denature DNA, you separate it

into single strands (SS). into single strands (SS). In the PCR reaction, this is accomplished by heating at 95In the PCR reaction, this is accomplished by heating at 9500 C C

for 15 seconds to 1 minute.for 15 seconds to 1 minute. The SS DNA generated will serve as templates for DNA The SS DNA generated will serve as templates for DNA

synthesis.synthesis. Anneal – to anneal is to come together through Anneal – to anneal is to come together through

complementary base-pairing (hybridization). complementary base-pairing (hybridization). During this stage in the PCR reaction the primers base-pair with During this stage in the PCR reaction the primers base-pair with

their complementary sequences on the SS template DNA their complementary sequences on the SS template DNA generated in the denaturation step of the reaction. generated in the denaturation step of the reaction.

PCRPCR

The primer concentration is in excess of the template The primer concentration is in excess of the template concentration. concentration.

The excess primer concentration ensures that the chances of The excess primer concentration ensures that the chances of the primers base-pairing with their complementary sequences the primers base-pairing with their complementary sequences on the template DNA are higher than that of the complementary on the template DNA are higher than that of the complementary SS DNA templates base-pairing back together.SS DNA templates base-pairing back together.

The annealing temperature used should ensure that annealing The annealing temperature used should ensure that annealing will occur only with DNA sequences that are completely will occur only with DNA sequences that are completely complementary. WHY?complementary. WHY?

The annealing temperature depends upon the lengths and The annealing temperature depends upon the lengths and sequences of the primers. The longer the primers and the more sequences of the primers. The longer the primers and the more Gs and Cs in the sequence, the higher the annealing Gs and Cs in the sequence, the higher the annealing temperature. WHY?temperature. WHY?

The annealing time is usually 15 seconds to 1 minute.The annealing time is usually 15 seconds to 1 minute.

PCRPCR

Extension – during this stage of the PCR reaction, the Extension – during this stage of the PCR reaction, the DNA polymerase will use dNTPs to synthesize DNA DNA polymerase will use dNTPs to synthesize DNA complementary to the template DNA. complementary to the template DNA. To do this DNA polymerase extends the primers that annealed To do this DNA polymerase extends the primers that annealed

in the annealing step of the reaction.in the annealing step of the reaction. The temperature used is 72The temperature used is 720 0 C since this is the optimum C since this is the optimum

reaction temperature for the thermostable polymerase that is reaction temperature for the thermostable polymerase that is used in PCR. Why is a thermostable polymerase used?used in PCR. Why is a thermostable polymerase used?

The extension time is usually 15 seconds to 1 minute.The extension time is usually 15 seconds to 1 minute.

The combination of denaturation, annealing, and The combination of denaturation, annealing, and extension constitute 1 cycle in a PCR reaction.extension constitute 1 cycle in a PCR reaction.

PCRPCR

Most PCR reactions use 25 to 30 of these Most PCR reactions use 25 to 30 of these cycles to amplify the target DNA up to a cycles to amplify the target DNA up to a million times the starting concentration.million times the starting concentration.

PCRPCR

Molecular BiologyMolecular Biology DNA sequencing – used to determine the actual DNA DNA sequencing – used to determine the actual DNA

sequence of an organism. Using a computer, one can sequence of an organism. Using a computer, one can predict protein sequences and functions based on the predict protein sequences and functions based on the nucleic acid data.nucleic acid data. The most commonly used sequencing method is the dideoxy The most commonly used sequencing method is the dideoxy

method. method. This method uses dideoxy nucleotide triphosphates (ddNTPs) This method uses dideoxy nucleotide triphosphates (ddNTPs)

which have an H on the 3’ carbon of the ribose sugar instead of which have an H on the 3’ carbon of the ribose sugar instead of the normal OH found in deoxynucleotides (dNTPs). the normal OH found in deoxynucleotides (dNTPs).

Dideoxynucleotides are chain terminators. Dideoxynucleotides are chain terminators. In a synthesis reaction, if a dideoxynucleotide is added instead of In a synthesis reaction, if a dideoxynucleotide is added instead of

the normal deoxynucleotide, the synthesis stops at that point the normal deoxynucleotide, the synthesis stops at that point because the 3’OH necessary for the addition of the next nucleotide because the 3’OH necessary for the addition of the next nucleotide is absent.is absent.

Deoxy versus dideoxyDeoxy versus dideoxy

DNA synthesisDNA synthesis

DNA sequencing continuedDNA sequencing continued In the dideoxy method of sequencing, the template DNA that is to In the dideoxy method of sequencing, the template DNA that is to

be sequenced is mixed with a primer complementary to the be sequenced is mixed with a primer complementary to the template DNA and the four normal deoxynucleotides, one of which template DNA and the four normal deoxynucleotides, one of which is radioactively labeled for subsequent visualization purposes. is radioactively labeled for subsequent visualization purposes.

This mixture is then splint into four different tubes that are labeled 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 A, C, G, and T. Each tube is then “spiked” with a different dideoxynucleotide (ddATP for tube A, ddCTP for tube C, ddGTT dideoxynucleotide (ddATP for tube A, ddCTP for tube C, ddGTT for tube G, or ddTTP for tube T). for tube G, or ddTTP for tube T).

DNA polymerase is added and using the DNA template and its’ DNA polymerase is added and using the DNA template and its’ complementary primer, the synthesis of new strands of DNA complementary primer, the synthesis of new strands of DNA complementary to the template begins. complementary to the template begins.

Occasionally a dideoxynucleotide is added instead of the normal Occasionally a dideoxynucleotide is added instead of the normal deoxynucleotide and synthesis of that strand is terminated at that deoxynucleotide and synthesis of that strand is terminated at that point.point.

DNA sequencing continuedDNA sequencing continued In the tube containing ddATP, some percentage of newly In the tube containing ddATP, some percentage of newly

synthesized molecules will get a ddATP in each place that there is synthesized molecules will get a ddATP in each place that there is a T in the template DNA. a T in the template DNA.

The result is a set of new DNA molecules in tube A, each of which The result is a set of new DNA molecules in tube A, each of which ends in an A.ends in an A.

A similar type of reaction occurs in the three other tubes to result 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 in molecules that end in C, G, and T in tubes C, G, and T respectively.respectively.

After the synthesis reactions are complete, the products of the four After the synthesis reactions are complete, the products of the four different tubes are loaded onto four adjacent lane of a different tubes are loaded onto four adjacent lane of a polyacrylamide gel and the different fragments are separated by polyacrylamide gel and the different fragments are separated by size.size.

The sequencing gel is able to resolve fragments that differ in size The sequencing gel is able to resolve fragments that differ in size from each other by only one base.from each other by only one base.

DNA sequencing continuedDNA sequencing continued

After electrophoresis to separate the fragments by size, the After electrophoresis to separate the fragments by size, the fragments are visualized by exposing the gel to fragments are visualized by exposing the gel to photographic film (Remember that one nucleotide was photographic film (Remember that one nucleotide was radioactively labeled).radioactively labeled).

All fragments in lane A will end in an A, fragments in lane C 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, 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.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 sequence of the DNA is read from the gel by starting at the bottom and reading upward.the bottom and reading upward.

Dideoxy DNA SequencingDideoxy DNA Sequencing

DNA sequencingDNA sequencing

DNA sequencingDNA sequencing

Automated DNA sequencing – in automated DNA sequencing a Automated DNA sequencing – in automated DNA sequencing a radioactive deoxynucleotide is not used and all four dideoxy radioactive deoxynucleotide is not used and all four dideoxy reactions are done in a single tube. reactions are done in a single tube.

This is possible because each dideoxynucleotide is labeled with This is possible because each dideoxynucleotide is labeled with a different flourescent dye. a different flourescent dye.

Therefore the dye present in each synthesized fragment Therefore the dye present in each synthesized fragment corresponds to the dye attached to the dideoxynucleotide that corresponds to the dye attached to the dideoxynucleotide that was added to terminate the synthesis of that particular fragment.was added to terminate the synthesis of that particular fragment.

The contents of the single tube reaction are loaded onto a single The contents of the single tube reaction are loaded onto a single lane of a gel (or capillary) and electrophoresis is done. lane of a gel (or capillary) and electrophoresis is done.

A flourimeter and computer are hooked up to the gel (or A flourimeter and computer are hooked up to the gel (or capillary) and they detect and record the dye attached to the capillary) and they detect and record the dye attached to the fragments as they come off the gel.fragments as they come off the gel.

The sequence is determined by the order of the dyes coming off The sequence is determined by the order of the dyes coming off the gel.the gel.

Automated DNA sequencingAutomated DNA sequencing

basic techniques to grow viruses and study virus-host interactions

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culture slide

infected cells

growth of viruses

adjacent cells

immortalized cells

primary cell line slide

study viruses

single tissue culture