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135 Cards in this Set

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True or False. There is more biological diversity within viruses than in all the rest of the bacterial, plant, and animal kingdoms put together.
TRUE!
What type of interactions determine the structure of virus particles, the synthesis and expression of virus genomes, and the effects of viruses on the host cell?
1. protein-protein
2. protein-nucleic acid
3. protein-lipid
What is a virus?
a submicroscopic, obligate intracellular parasite
A few groups of prokaryotic organisms have specialized intracellular parasitic life cycles and confound the definition of a virus. What are these organisms?
Rickettsiae and Chlamydiae---obligate intracellular parasitic BACTERIA which have evolved to be so cell-associated that they can exist outside the cells of their hosts for only a short period of time before losing viability
What constitutes a virus?
1. virus particles are produced from the assembly of PREFORMED components, whereas other agents grow from an increase in the integrated sum of their components and reproduce by division.
2. Virus particles (virions) themselves do NOT grow or undergo division
3. viruses LACK the genetic information that encodes apparatus necessary for the generation of metabolic energy or for protein synthesis (ribosomes)
True or False. NO known virus has the biochemical or genetic potential to generate the energy necessary to drive all biological processes.
TRUE!
Why are viruses absolutely dependent on the host cell?
because viruses do not have the biochemical or genetic potential to generate the energy necessary to drive all biological processes.
Are viruses alive?
one view is that inside the host cell viruses are alive, whereas outside it they are merely complex assemblages of metabolically inert chemicals.
True or False. All viruses are smaller than bacteria.
False. The largest virus known is 400nm in diameter, while the smallest bacteria are only 200 to 300nm long.
What is the largest virus known?
the Mimivirus, 400nm in diameter
True or False. There is more biological diversity within viruses than in all the rest of the bacterial, plant, and animal kingdoms put together.
TRUE!
What type of interactions determine the structure of virus particles, the synthesis and expression of virus genomes, and the effects of viruses on the host cell?
1. protein-protein
2. protein-nucleic acid
3. protein-lipid
What is a virus?
a submicroscopic, obligate intracellular parasite
A few groups of prokaryotic organisms have specialized intracellular parasitic life cycles and confound the definition of a virus. What are these organisms?
Rickettsiae and Chlamydiae---obligate intracellular parasitic BACTERIA which have evolved to be so cell-associated that they can exist outside the cells of their hosts for only a short period of time before losing viability
What constitutes a virus?
1. virus particles are produced from the assembly of PREFORMED components, whereas other agents grow from an increase in the integrated sum of their components and reproduce by division.
2. Virus particles (virions) themselves do NOT grow or undergo division
3. viruses LACK the genetic information that encodes apparatus necessary for the generation of metabolic energy or for protein synthesis (ribosomes)
True or False. NO known virus has the biochemical or genetic potential to generate the energy necessary to drive all biological processes.
TRUE!
Why are viruses absolutely dependent on the host cell?
because viruses do not have the biochemical or genetic potential to generate the energy necessary to drive all biological processes.
Are viruses alive?
one view is that inside the host cell viruses are alive, whereas outside it they are merely complex assemblages of metabolically inert chemicals.
True or False. All viruses are smaller than bacteria.
False. The largest virus known is 400nm in diameter, while the smallest bacteria are only 200 to 300nm long.
What is the largest virus known?
the Mimivirus, 400nm in diameter
True or False. There is more biological diversity within viruses than in all the rest of the bacterial, plant, and animal kingdoms put together.
TRUE!
What type of interactions determine the structure of virus particles, the synthesis and expression of virus genomes, and the effects of viruses on the host cell?
1. protein-protein
2. protein-nucleic acid
3. protein-lipid
What is a virus?
a submicroscopic, obligate intracellular parasite
A few groups of prokaryotic organisms have specialized intracellular parasitic life cycles and confound the definition of a virus. What are these organisms?
Rickettsiae and Chlamydiae---obligate intracellular parasitic BACTERIA which have evolved to be so cell-associated that they can exist outside the cells of their hosts for only a short period of time before losing viability
What constitutes a virus?
1. virus particles are produced from the assembly of PREFORMED components, whereas other agents grow from an increase in the integrated sum of their components and reproduce by division.
2. Virus particles (virions) themselves do NOT grow or undergo division
3. viruses LACK the genetic information that encodes apparatus necessary for the generation of metabolic energy or for protein synthesis (ribosomes)
True or False. NO known virus has the biochemical or genetic potential to generate the energy necessary to drive all biological processes.
TRUE!
Why are viruses absolutely dependent on the host cell?
because viruses do not have the biochemical or genetic potential to generate the energy necessary to drive all biological processes.
Are viruses alive?
one view is that inside the host cell viruses are alive, whereas outside it they are merely complex assemblages of metabolically inert chemicals.
True or False. All viruses are smaller than bacteria.
False. The largest virus known is 400nm in diameter, while the smallest bacteria are only 200 to 300nm long.
What is the largest virus known?
the Mimivirus, 400nm in diameter
What are viroids?
very small (200-400 nucleotides), circular RNA molecules with a rod-like secondary structure
Do viroids have a capsule?
NO
Do viroids have an envelope?
NO
What type of diseases are viroids associated with?
associated with plant diseases
What are virusoids?
satellite, viroid-like molecules, somewhat larger than viroids (e.g., approx. 1000 nucleotides), which are dependent on the presence of virus replication for multiplication; they are packaged into virus capsids as passengers.
What are prions?
infectious agents genrally believed to consist of a single type of protein molecule with NO nucleic acid component
What diseases are prions associated with?
associated with 'slow' virus diseases such as Creutzfeldt-Jakob disease in humans, scapie in sheep, and bovine spongiform encepalopathy (BSE) in cattle
What is the key to understanding viruses?
understanding diversity
Why are viruses so biologically diverse?
This is the result of the success of viruses in parasitizing all known groups of living organisms, and understanding this diversity is the key to comprehending the interactions of viruses with their hosts.
True or False. Viruses have DNA and RNA.
FALSE! Viruses have either DNA OR RNA, NOT BOTH!
What was the first record of virus infection?
consists of a hieroglyph from Memphis, the capital of Egypt, drawn in approx. 3700 B.C., which depicts a temple priest showing typical clinical signs of paralytic poliomyelitis.
Pharaoh Ramses V
died in 1196 B.C., his body was mummified and is now in a Cairo museum, is believed to have succumbed to smallpox--a comparison between the pustular lesions on the face of the mummy and those of more recent patients is startling
When was smallpox endemic to China?
by 1000 B.C.
Who invented variolation in response to smallpox?
China
Edward Jenner
on May 14, 1796, he used cowpox-infected material obtained from the hand of Sarah Nemes, a milkmaid from his home village of Berkeley in Gloucestershire, England, to successfully vaccinate 8-year-old James Phipps.
When was vaccination against smallpox adopted worldwide?
during the 19th century
Antony van Leeuwenhoek
a Dutch merchant who constructed the first simple microscopes and identified bacteria as the 'animalcules' he saw in his specimens
Who jointly proposed the 'germ theory' of disease?
Robert Koch and Louis Pasteur
List Koch's postulates.
1. the agent must be present in every case of the disease
2. the agent must be isolated from the host and grown in vitro
3. the disease must be reproduced when a pure culture of the agent is inoculated into a healthy susceptible host
4. the same agent must be recovered once again from the experimentally infected host
What are Koch's postulates regarded as?
the proof that an infectious agent is responsible for a specific disease
Dimitri Iwanowski (1892)
showed that extracts from diseased tobacco plants could transmit disease to other plants after passage through ceramic filters fine enough to retain even the smallest bacteria
Martinus Beijerinick (1898)
confirmed and extended Iwanowski's result on tobacco mosaic virus (TMV) and was the first to develop the modern idea of the virus, wich he referred to as contagium vivum fluidum ('soluble living germ').
Freidrich Loeffler and Paul Frosch (1898)
showed that a similar agent (filterable agent) was responsible for food-and-mouth disease in cattle
Karl Landsteiner and Erwin Popper (1909)
showed that poliomyelitis was caused by a 'filterable agent'--the first human disease to be recognized as being caused by a virus
What was the first human disease to be recognized as being caused by a virus?
poliomyelitis
Frederick Twort (1915) and Felix d'Herelle (1917)
the first to recognize viruses that infect bacteria, which d'Herelle called bacteriophages ('eaters of bacteria')
In the 1930s and subsequent decades, pioneering virologists such as ________1___________, ________2_______, and many others used these viruses (bacteriophages) as model systems to investigate many aspects of virology, including virus structure.
1. Salvador Luria
2. Max Delbruck
What agents have been proven to be very important to our understanding of all types of viruses, including those of humans which are more difficult to propogate and study.
bacteriophages
The history of virology is the development of _____________.
experimental tools
What is virus classification based on?
Phenotypic characteristics:
1. morphology
2. nucleic acid type
3. mode of replication
4. host organisms
5. types of diseases they cause
Which taxonomic groups does not apply to viruses?
1. kingdom
2. phylum
3. class
David Baltimore
devised the Baltimore Classification system, which places viruses into one of seven groups. These groups are designated by Roman numerals and separate viruses based on their mode of replication and genome type
How often does the ICTV meet?
every four years
When was the International Committee on Taxonomy of Viruses established?
1966
What did the ICTV produce?
the first unified scheme for virus classification
What rules for virus taxonomy did the ICTV establish?
1. Latin binomial names are not used. No person's name should be used in nomenclature. Names should have international meaning.
2. A virus name should be meaningful and should consist of as few words as possible. Serial numbers or letters are not acceptable as names.
3. A virus species is a polythetic class of viruses that constitute a replicating lineage and occupy a particular ecological niche.
4. A genus is a group of virus species sharing common characters. Approval of a new genus is linked to the acceptance of a type of species.
5. A family is a group of genera with common characters. Approval of a new family is linked to the acceptance of a type of genus.
6. The names of virus orders, families, subfamilies, genera, and species should be written in italics with the first letter capitalized.
7. Other words are not capitalized unless they are proper nouns.
8. This format should only be used when official taxonomic entities are referred to---it is not possible to centrifuge the species Poliovirus, for example, but it is possible to centrifuge poliovirus.
9. Italics and capitalization are not used for vernacular usage, for acronyms, nor for adjectival forms
List the seven categories of the Baltimore Classification system.
1. Group I:double stranded DNA viruses
2. Group II:single stranded DNA viruses
3. Group III: double stranded RNA viruses
4. Group IV:positive sense single stranded RNA viruses
5. Group V: negative sense single stranded RNA viruses
6. Group VI: reverse transcribing RNA viruses
7. Group VII: reverse transcribing DNA viruses
ICTV classification:
Order (-?)
Family (-?)
Subfamily (-?)
Genus (-?)
Species (-?)
Order (-virales)
Family (-viridae)
Subfamily (-virinae)
Genus (-virus)
Species (-virus)
How many orders of viruses have been named to date?
THREE
How many families of viruses are known? How many species of viruses are known?
1. 80
2. 4000
What techniques are used to study viruses?
1. Living Host Systems
2. Cell Culture Methods
3. Serological/Immunological Methods
4. Ultrastructural Methods
5. Molecular Methods
Louis Pasteur (1881)
developed methods of producing attenuated virus preparations by progressively drying the spinal cords of rabbits experimentally infected with rabies which, when inoculated into other animals, would protect from challenge with virulent rabies virus
What did Pasteur do in 1885?
He inoculated a child, Joseph Meister, with rabies vaccine, the first artificially produced virus vaccine.
What was the first artificially produced virus vaccine?
rabies vaccine
Whole plants have been used to study the effects of plant viruses after infection ever since tobacco mosaic virus was first discovered by _____________.
Iwanowski
Walter Reed (1900)
through experimental transmission to mice, he demonstrated that yellow fever was caused by a virus spread by mosquitoes.
Max Theiler (1937)
propogated the yellow fever virus in chick embryos to produce an attenuated vaccine--the 17D strain--which is still in used today
What did Max Theilers method of using chick embryos lead to?
led investigators from the 1930s to the 1950s to develop animal systems to identify and propogate pathogenic viruses
What viruses will replicate in the living tissues of developing embryonated hen eggs?
1. Influenza virus
2. some poxviruses (e.g., Vaccinia virus
What were first used to propogate viruses in the early decades of the 20th century?
embryonated hen eggs
What was the first quantitative assay for a virus?
counting the 'pocks' chorioallantoic membrane of eggs
What uses do animal hosts have in virology?
1. to produce viruses that cannot be effectively studied in vitro (e.g., hepatitis B virus)
2. to study the pathogenesis of virus infections (e.g., coxsackieviruses)
3. to test vaccine safety (e.g., oral poliovirus vaccine)
Why are animal hosts increasingly being discarded (in regards to studying viruses)?
1. Breeding and maintenance of animals infected with pathogenic viruses is expensive
2. Whole animals are complex systems in which it is sometimes difficult to discern events
3. results obtained are not always reproducible due to host variation
4. unnecessary or wasteful use of experimental animals is morally repungent
5. they are rapidly being overtaken by 'modern science'--cell culture and molecular biology
In the cell culture method, eukaryotic cells are grown ___________.
in vitro
When did cell culture begin?
early in the 20th century with whole-organ cultures, then progressed to methods involving individual cells
What two types of cells are used in cell culture?
1. primary cell cultures
2. immortalized cell lines
John Enders (1949) and colleagues
propogated poliovirus in primary human cell cultures
Renato Dulbecco (1952)
was the first to quantify accurately animal viruses using a plaque assay
In the 1950s and 1960s, what did the acheivement of John Enders and his colleagues lead to?
the identification and isolation of many viruses and their association with human diseases
How are plaque assays performed?
Plaque assays are performed by applying a suitable dilution of a virus preparation to a confluent or semiconfluent adherent monolayer of susceptible cells. After allowing time for virus attachment to and infection of the cells, liquid medium is replaced by a semisolid culture medium containing a polymer such as agarose or carboxymethyl cellulose, which restricts diffusion of virus particles from infected cells. Only direct cell-to-cell spread can occur, resulting in localized destruction of the monolayer. After a suitable period, the medium is usually removed and the cells stained to make the holes in the monolayer (plaques) more easily visible. Each plaque therefore results from infection by a single plaque-forming unit (p.f.u.).
Renato Delbaco history
Dulbecco is a pivotal figure in the history of tumor virus research. An Italian immigrant with an M.D., Dulbecco came to the U.S. in order to learn research biology that could be applied to medicine. He spent a postdoctoral fellowship with Salvador Luria, one of the founders of the phage group, in the late 1940s. There he worked side by side with James Watson, then a graduate student. Dulbecco modified techniques used by the bacteriophage researchers and applied them to animal viruses, which had more relevance to human disease. His "plaque assay" made laboratory research on the genetics of tumor viruses practical and opened the door to the flood of tumor virus research in the 1960s and 1970s. Dulbecco shared the 1975 Nobel Prize in Physiology or Medicine with David Baltimore and Howard Temin, two researchers who independently isolated, using tumor viruses, the paradigm-shattering enzyme reverse transcriptase.
George Hirst (1941)
observed haemagglutination of red blood cells by influenza virus
What did George Hirst's observation of haemagglutination of red blood cells prove to be?
an important tool in the study of not only influenza but also several other groups of viruses--for example, rubella virus
What can Serological/Immunological methods be used for?
measuring the titre (i.e., relative amount) of virus present in any preparation and to determine the antigenic type of the virus
Haemagglutination Assay
This assay takes advantage of the fact that many viruses contain proteins that can bind red blood cells (erythrocytes). Examples of viral proteins with the ability to bind to red blood cells include the influenza virus hemagglutinin (HA) protein, which binds to sialic acid residues on the cell surface. Normally, red blood cells will fall to the bottom of a culture well, forming a sharp dot. However, if viruses are present, the red cells become bound to the virus particles in a lattice or network (this happens because a single virus can bind multiple red blood cells). This lattice then coats the well. The assay is fast (30 minutes).
How does the haemagglutination assay work?
after a viral titre is determined, antisera can be determined. If the antibodies in the serum bind to the virus it will prevent the virus from binding red blood cells (RBCs). Different serotypes of virus can be differentiated in this way.
In the 1960s and subsequent years, many improved detection methods for viruses were developed. List them.
1. Complement fixation tests
2. Radioimmunoassays
3. Immunofluorescence
4. Enzyme-linked immunosorbent assays (ELISAs)
5. Radioimmune precipitation
6. Western blot assays
Complement Fixation Test
(book): The complement fixation test works on the basis that complement is sequestered by antigenantibody complexes. Sensitized antibody-coated red blood cells, known amounts of complement, a virus antigen, and the serum to be tested are added to the wells of a multiwell plate. In the absence of antibodies to the virus antigen, free complement is present which causes lysis of the sensitized red blood cells (haemolysis). If, however, the test serum contains a sufficiently high titre of antivirus antibodies, then no free complement remains and haemolysis does not occur. Titrating the test serum by means of serial dilutions allows a quantitative measurement of the amount of antivirus antibody present to be made.
(lecture): In essence the test consists of two antigen-antibody reactions, one of which is the indicator system. The first reaction, between a known virus antigen and a specific antibody takes place in the presence of a predetermined amount of complement. The complement is removed or "fixed" by the antigen-antibody complex. The second antigen-antibody reaction consists of reacting sheep rbc with haemolysin. When this indicator system is added to the reactants, the sensitized rbcs will only lyse in the presence of free complement.
Immunofluorescence
(book): Immunofluorescence is performed using derivatized antibodies containing a covalently linked fluorescent molecule that emits a characteristically coloured light when illuminated by light of a different wavelength, such as rhodamine (red) or fluorescein (green). In direct immunofluorescence, the antivirus antibody itself is conjugated to the fluorescent marker, whereas in indirect immunofluorescence a second antibody reactive to the antivirus antibody carries the marker. Immunofluorescence can be used not only to identify virus-infected cells in populations of cells or in tissue sections but also to determine the subcellular localization of particular virus proteins (e.g., in the nucleus or in the cytoplasm).
(lecture):
•Direct:
–A fluorescently labeled antibody is bound to viral preparation or sectioned tissue sample. Ab is against the virus
•Indirect:
–Ab (e.g. rabbit) against the virus is bound. Next an fluorscently labeled secondary antibody (e.g. goat) against the primary rabbit antibody is bound to it.
•Common to Both:
–Virus is visualized by fluorescence microscopy. though the virus or viral antigen are too small to see, the emitted light will be visible.
•FISA also possible (fluorescent immunosorbant assays), see ELISA
Enzyme-linked Immunosorbent Assays (ELISAs)
(book): Enzyme-linked immunosorbent assays (ELISAs) are a rapid and sensitive means of identifying or quantifying small amounts of virus antigens or antivirus antibodies. Either an antigen (in the case of an ELISA to detect antibodies) or antibody (in the case of an antigen ELISA) is bound to the surface of a multiwell plate. An antibody specific for the test antigen, which has been conjugated with an enzyme molecule (such as alkaline phosphatase or horseradish peroxidase), is then added. As with immunofluorescence, ELISA assays may rely on direct or indirect detection of the test antigen. During a short incubation, a colourless substrate for the enzyme is converted to a coloured product, thus amplifying the signal produced by a very small amount of antigen. The intensity of the product can easily be measured in a specialized spectrophotometer (plate reader). ELISA assays can be mechanized and are therefore suitable for routine tests on large numbers of clinical samples.
(lecture):
•Virus or viral antigen is coated onto wells of multi-well plates
•Direct: Antibody against the viral antigen is added. Antibody is linked to an enzyme
•Indirect: enzyme-linked IIº antibody against the Iº antibody against the viral antigen is added. Antibody is linked to an enzyme.
•Applies to both:
•Plates are washed to remove excess antibody.
•Solutions replaced with substrates that the linked enzyme (e.g. alkaline phosphatase) will cause to form a spectrophotometrically detectable product.
•Wells are read automatically in a plate reader.
Western Blot Assays
(book)Western blotting is used to analyse a specific virus protein from a complex mixture of antigens. Virus antigen-containing preparations (particles, infected cells, or clinical materials) are subjected to electrophoresis on a polyacrylamide gel. Proteins from the gel are then transferred to a nitrocellulose or nylon membrane and immobilized in their relative positions from the gel. Specific antigens are detected by allowing the membrane to react with antibodies directed against the antigen of interest. By using samples containing proteins of known sizes in known amounts, the apparent molecular weight and relative amounts of antigen in the test samples can be determined.
Who introduced the complement fixation test?
Wasserman in 1909 for syphilis serology
What criteria do complement fixation tests meet?
1. it is convenient and rapid to perform
2. the demand on equipment and reagents is small
3. large variety of test antigens are available
Radioimmunoassays (RIAs)
*Either a radioactive antibody or a radioactive antigen is used
•Antigen-antibody complex that is formed is captured on a filter
•Filter is washed to remove radioactivity that is not part of the antibody-antigen complex
•Filter is placed in a scintillation vial for measurement of radioactivity.
What are the advantages/disadvantages of radioimmunoassays (RIAs)
Advantages:
–Extremely sensitive
–Very quantitative
–Simple, fast

Disadvantages:
–Radioisotope hazards
Radioimmune Precipitation
•All proteins in a virally infected cell or tissue are radioactively labeled (usually 35S-Met is the radiolabel)
•All protein in the cell (or tissue) is solublized with detergent
•Since only the antigen will bind to the antibody, it will be the only protein forming an antibody-antigen complex and only it will be large enough to be spun down by centrifugation
•Precipitate in bottom of tube is then resuspended in electrophoresis buffer and then subjected to SDS-PAGE (Polyacrylamide Gel Electrophoresis)
•After electrophoresis the gel is dried and the radioactive band is detected by autoradiography
What are the advantages/disadvantages of radioimmune precipitation?
Advantages:
–Identifies protein antigen by its molecular weight and quantitatively indicates the amount of antigen that was present
Disadvantage:
–Radioisotope use
Western Blot
•it involves SDS-PAGE electrophoresis of proteins commonly solubilized from cells or tissue infected with virus
•In this case however the proteins were not previously radiolabeled.
•After electrophoresis of proteins the bands of protein in the gel are transferred by electro-blotting onto a nylon membrane.
•Next the membrane is exposed to the anti-viral antibody.
•When DIRECT detection is used the antibody is labeled (e.g. 125I radiolabel). With INDIRECT detection (more common) the primary antibody is bound by a secondary antibody that is labeled. This secondary antibody can enable detection by
–125I radiolabel
–Fluorescent-label, or
–most commonly, Enzyme-linkage.
•In the case of enzyme-linked detection, the colored reaction product is deposited directly onto membrane and seen as a purple band on the white membrane. Alternatively, in another form of this technique, in an unique reaction, white light is produced and used to expose an X-ray film placed over the membrane in a darkroom.
Polyclonal Antibodies
•Live or attenuated virus or purified viral antigens can be injected into whole animals.
•Attenuation often involves heat destruction of the virus.
•To increase the immune response, the antigen is usually injected with an adjuvant that irritates the site of injection stimulating the immune response.
•Two or more booster shots are usually given prior to obtaining whole blood from the animal.
•Whole blood is then allowed to clot and the serum containing antibodies and other serum proteins is obtained.
•When necessary, the antibody fraction can be purified away from other serum proteins.
•Under exacting conditions, the other non-antigenic antibodies can be purified away as well.
•Polyclonal antibodies typically recognize multiple epitopes on the virus or viral antigen
Monoclonal Antibodies
Monoclonal antibodies are produced by immunization of an animal with an antigen that usually contains a complex mixture of epitopes. Immature B-cells are later prepared from the spleen of the animal, and these are fused with a myeloma cell line, resulting in the formation of transformed cells continuously secreting antibodies. A small proportion of these will make a single type of antibody (a monoclonal antibody) against the desired epitope. Recently, in vitro molecular techniques have been developed to speed up the selection of monoclonal antibodies, although these have not yet replaced the original approach.
What three areas can ultrastructural studies be considered under?
1. physical methods
2. chemical methods
3. electron microscopy
When did physical measurements of virus particles begin?
began in the 1930s with the earliest determinations of their proportions by filtration through colloidal membranes of various pore sizes. Led to the first estimates of the size of virus particles.
How was the accuracy of the size of virus particles improved upon?
by studies of sedimentation properties of viruses in ultracentrifuges in the 1960s
Differential Centrifugation
A number of different sedimentation techniques can be used to study viruses. In rate-zonal centrifugation, virus particles are applied to the top of a preformed density gradient, i.e., a sucrose or salt solution of increasing density from the top to the bottom of the tube (top of figure). After a period of time in an ultracentrifuge, the gradient is separated into a number of fractions, which are analysed for the presence of virus particles. In the figure, the nucleic acid of the virus genome is detected by its absorption of ultraviolet light (below). This method can be used both to purify virus particles or nucleic acids or to determine their sedimentation characteristics. In equilibrium or isopycnic centrifugation, the sample is present in a homologous mixture containing a dense salt such as caesium chloride. A density gradient forms in the tube during centrifugation, and the sample forms a band at a position in the tube equivalent to its own density. This method can thus be used to determine the density of virus particles and is commonly used to purify plasmid DNA.
List some physical methods.
1. Spectroscopy
2. Electrophoresis
3. X-ray diffraction by crystalline forms of purified virus
Spectroscopy
The physical properties of viruses can be determined by spectroscopy, using either ultraviolet light to examine the nucleic acid content of the particle or visible light to determine its light-scattering properties.
Electrophoresis
Electrophoresis of intact virus particles has yielded some limited information, but electrophoretic analysis of individual virion proteins by gel electrophoresis, and particularly also of nucleic acid genomes, has been far more valuable.
X-ray Diffraction by Crystalline Forms
the most important method for the elucidation of virus structures has been the use of x-ray diffraction by crystalline forms of purified virus. This technique permits determination of the structure of virions at an atomic level.
The complete structures of many viruses, representative of many of the major groups, have now been determined at a resolution of a few ______________.
angstroms
What has the advancement of the use of ultrastructural methods improved?
has improved our understanding of the function of the virus particle considerably, however, a number of viruses have proven to be resistant to this type of investigation
What procedure is increasingly being used to determine anatomic structure of all kinds of molecules, including proteins and nucleic acids.
Nuclear magnetic resonance (NMR)
What is the limitation of nuclear magnetic resonance?
only relatively small molecule can be analyzed before the signals obtained become so confusing that they are impossible to decipher with current technology
At present, the upper size limit for NMR restricts its use to molecules with a molecular weight of less than about ________1______ to _________2_________, considerably less than even the smallest virus particles.
1. 30,000
2. 40,000
What can chemical investigation be used to determine?
1. the overall composition of viruses
2. the nature of the nucleic acid that comprises the virus genome
3. the construction of the particle and the way in which individual components relate to each other in the capsid
List some chemical methods.
•Stepwise disruption of particles.
•Electrostatic interactions
•Non-ionic, hydrophobic interactions.
•Proteins which interact with lipids.
•Surface labelling.
·Cross-linking reagents.
Denaturation of TMV
The structure and stability of virus particles can be examined by progressive denaturation or renaturation studies. At any particular ionic strength, the purified capsid protein of tobacco mosaic virus (TMV) spontaneously assembles into different structures, dependent on the pH of the solution. At a pH of around 6.0, the particles formed have a helical structure very similar to infectious virus particles. As the pH is increased to about 7.0, disk-like structures are formed. At even higher pH values, individual capsid monomers fail to assemble into more complex structures
What can progressive denaturation be used for?
1. revealing fundamental structure
2. observe alteration or loss of antigenic sites on the surface of particles
What can proteins exposed on the surface be labeled with?
various compounds (e.g., iodine)
What are labels on viruses used for?
to indicate which parts of the protein are exposed and which are protected inside the particle or by lipid membranes.
What are cross-linking reagents such as psoralens or newer synthetic agents with side-arms of specific lengths used for?
to determine the spatial relationship of proteins and nucleic acids in intact viruses.
What limitations did the electron microscope help the light microscope overcome?
the inablility to resolve individual virus particles owing to physical restraints caused by the wavelength of visible light illumination and the optics of the instruments
When was the first electron micrograph of a virus (TMV) published?
1939
Over subsequent years, techniques were developed that allowed the direct examination of viruses at magnifications of over _________________ times.
100,000
What are the two fundamental types of electron microscopes?
1. transmission electron microscope (TEM)
2. scanning electron microscope (SEM)
What two fundamental types of information can be obtained by electron microscopy?
1. the absolute number of virus particles present in any preparation (total number)
2. the appearance and structure of the virions