《微生物生物学》课程书籍文献：《Principles of Molecular Virology》PDF电子书（Molecular Virology 4th Edition，Alan J. Cann）

10Principles of Molecular Virology increasingly widespread application in other types of serological assays (e.g.,ELISAs) toincrease their reproducibility,sensitivity,and specificity It would be inappropriate here to devote too much discussion to the techni- cal details of what is also a very rapidly expanding field of knowledge;however, I strongly recommend that readers who are not familiar with the techniques mentioned above should familiarize themselves thoroughly with this subject by reading one or more of the texts given in the Further Reading for this chapter Time spent in this way will repay readers throughout their reading of the rest of this book. (a)Complement fixation test (b)Immunofluorescence 1 Direct: Indirect: ▲A not "fixed'haemolysis occurs (c)ELISA (d)Western blot Direct: Indirect: 00 Col product Nitrocellulose or nylon membrane Key: ▲▲Virus antigen Primary antibody

increasingly widespread application in other types of serological assays (e.g., ELISAs) to increase their reproducibility, sensitivity, and specificity. It would be inappropriate here to devote too much discussion to the techni￾cal details of what is also a very rapidly expanding field of knowledge; however, I strongly recommend that readers who are not familiar with the techniques mentioned above should familiarize themselves thoroughly with this subject by reading one or more of the texts given in the Further Reading for this chapter. Time spent in this way will repay readers throughout their reading of the rest of this book. 10 Principles of Molecular Virology (a) Complement fixation test (1) (2) Antibody present, complement ‘fixed’, no haemolysis Antibody absent, complement not ‘fixed’, haemolysis occurs (b) Immunofluorescence Direct: Indirect: (d) Western blot Direct: Indirect: Nitrocellulose or nylon membrane (c) ELISA Colourless substrate Coloured product Virus antigen Primary antibody Secondary antibody ‘Detector’ molecule: complement, enzyme, radioisotope, or fluorescent dye ‘Sensitized’ (antibody-coated) red blood cell Key:

Introduction Figure 1.2 It is difficult to overestimate the importance of serological techniques in virology.The four assays illustrated by the diagrams in this figure have been used for many years and are of widespread value.(a)The test works on the basis that complement is sequestered by antigen-antibody complexes.'Sen- sitized'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.(b)Immunofluorescence is performed using derivatized antibod- ies containing a covalently linked fluorescent molecule that emits a characteristi- cally 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 immuno Huorescence a second antbody reactive to the antivirus antibody carries the marker tion of particular virus proteins (e.g.,in the nucleus or in the cytoplasm).(c) Enzyme-linked immunosorbent assays(ELISAs)are a rapid and sensitive means of iden- tifying or quantifying small amounts of virus antigens or antivirus antibodies.Eithe 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 ver 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 (d)Western blotting is used to analyse a specific virus protein from a complex mixture of antigens.Virus antigen-containing preparations(particles,infected cells,or clini- cal materials)are subjected to electrophoresis on a polyacrylamide gel.Proteins from the gel are then transferred to a nitrocellulose or nylon membrane and immobi- lized in their relative positions from the gel.Specific antigens are detected by allow ing the membrane to react with antibodies directed against the antigen of interest. By using samples containing proteins of known sizes in known amounts,the appar- ent molecular weight and relative amounts of antigen in the test samples can be determined

Introduction 11 Figure 1.2 It is difficult to overestimate the importance of serological techniques in virology.The four assays illustrated by the diagrams in this figure have been used for many years and are of widespread value. (a) The complement fixation test works on the basis that complement is sequestered by antigen–antibody complexes. ‘Sen￾sitized’ 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. (b) Immunofluorescence is performed using derivatized antibod￾ies containing a covalently linked fluorescent molecule that emits a characteristi￾cally 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 immuno- fluorescence a second antibody reactive to the antivirus antibody carries the marker. Immunofluorescence can be used not only to identify virus-infected cells in popu￾lations of cells or in tissue sections but also to determine the subcellular localiza￾tion of particular virus proteins (e.g., in the nucleus or in the cytoplasm). (c) Enzyme-linked immunosorbent assays (ELISAs) are a rapid and sensitive means of iden￾tifying 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. (d) Western blotting is used to analyse a specific virus protein from a complex mixture of antigens.Virus antigen-containing preparations (particles, infected cells, or clini￾cal materials) are subjected to electrophoresis on a polyacrylamide gel. Proteins from the gel are then transferred to a nitrocellulose or nylon membrane and immobi￾lized in their relative positions from the gel. Specific antigens are detected by allow￾ing the membrane to react with antibodies directed against the antigen of interest. By using samples containing proteins of known sizes in known amounts, the appar￾ent molecular weight and relative amounts of antigen in the test samples can be determined

12Principles of Molecular Virology of epitopes) Splen cells○ CELL FUSION gepShbi8omattmotisos 日ョ 日 otbdy 日日白 aeaa ◇ Figure 1.3 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 mon- oclonal antibodies,although these have not yet replaced the original approach shown here. ULTRASTRUCTURAL STUDIES Ultrastructural studies can be considered under three areas:physical methods. chemical methods,and electron microscopy.Physical measurements of virus parti- cles began in the 1930s with the earliest determinations of their proportions by filtration through colloidal membranes of various pore sizes.Experiments of this

12 Principles of Molecular Virology CELL FUSION Immunize animals with antigen (complex mixture of epitopes) Apply selective medium to fusions, grow on hybridomas Test supernatant for antibody specificity Biologically clone cells by limiting dilution Grow up antibody secreting cells and isolate antibody from medium Spleen cells Immortal B-cell line Figure 1.3 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 mon￾oclonal antibodies, although these have not yet replaced the original approach shown here. ULTRASTRUCTURAL STUDIES Ultrastructural studies can be considered under three areas: physical methods, chemical methods, and electron microscopy. Physical measurements of virus parti￾cles began in the 1930s with the earliest determinations of their proportions by filtration through colloidal membranes of various pore sizes. Experiments of this

Introduction sort led to the first(rather inaccurate)estimates of the size of virus particles.The accuracy of these estimates was improved greatly by studies of the sedimentation properties of viruses in ultracentrifuges in the 1960s(Figure 1.4).Differential cen trifugation proved to be of great use in obtaining purified and highly concentrated preparations of many different viruses,free of contamination from host cell com- ponents,that can be subjected to chemical analysis.The relative density of virus Centrifugal force Purified virus 40 Density of Ultraviolet absorption 10 Figure 1.4 A number of different sedimentation techniques can be used to study viruses.In rate-zonal centrifugation (shown here),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 deter- mine 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

sort led to the first (rather inaccurate) estimates of the size of virus particles. The accuracy of these estimates was improved greatly by studies of the sedimentation properties of viruses in ultracentrifuges in the 1960s (Figure 1.4). Differential cen￾trifugation proved to be of great use in obtaining purified and highly concentrated preparations of many different viruses, free of contamination from host cell com￾ponents, that can be subjected to chemical analysis. The relative density of virus Introduction 13 Centrifugal force Optical density (260 nm) Sucrose % 50 40 30 20 10 Density of sucrose Ultraviolet absorption Purified virus Figure 1.4 A number of different sedimentation techniques can be used to study viruses. In rate-zonal centrifugation (shown here), 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 deter￾mine 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

1Principles of Molecular Virology particles,measured in solutions of sucrose or CsCl,is also a characteristic feature. revealing information about the proportions of nucleic acid and protein in the particles. The physical properties of viruses can be determined by spectroscopy,using either ultraviolet light to examine the nucleic acid content of the particle or visibl light to determine its light-scattering properties.Electrophoresis of intact virus par- ticles has yielded some limited info nation,but electrophoretic analysis of indi- vidual virion proteins by gel electrophoresis,and particularly also of nucleic acid genomes(Chapter 3),has been far more valuable.However,by far the most impor- tant method for the elucidation of virus structures has been the use of x-ray dif fraction 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 majo groups,have now been determined at a resolution of a few angstroms (A)(see Chapter 2).This advancement has improved our understanding of the functions of the virus particle considerably;however,a number of viruses have proven to be resistant to this type of investigation,a fact that highlights some of the problems inherent in this otherwise powerful technique.One problem is that the virus must first be purified to a high degree;otherwise,specific information on the virus cannot be gathered.This presuppo oses that adequate quantities of the virus can be propagated in culture or obtained from infected tissues or patients and that a method is available to purify virus particles without loss of structural integrity.In a number of important cases,this requirement rules out further study (e.g.,hepa titis C virus).The purified virus must also be able to form paracrystalline arrays large enough to cause significant diffraction of the radiation source.For some viruses,this is relatively straightforward,and crystals big enough to see with the naked eye and which diffract strongly are easily formed.This is particularly true for a number of plant viruses,such as tobacco mosaic virus(which was first crys tallized by Wendell Stanley in 1935)and turnip yellow mosaic virus (TYMV),the structures of which were among the first to be determined during the 1950s.It is significant that these two viruse s represent the two fundamental types of virus par ticle:helical in the case ofTMV and icosahedral for TYMV (see Chapter 2).In many cases,however,only microscopic crystals can be prepared.A partial answer to this problem is to use ever more powerful radiation sources that allow good data to be collected from small crystals.Powerful synchotron sources that generate intense beams of radiation have been built during the last few decades and are now used extensively for this purpose;however,there is a limit beyond which this brute force approach fails to yield further benefit.A number of important viruses stead- fastly refuse to crystallize;this is a particularly common problem with irregularly shaped viruses-for example,those which have an outer lipid envelope-and to date no complete high-resolution atomic structure has vet been determined for many viruses of this type (e.g.,HIV).Modifications of the basic diffraction

particles, measured in solutions of sucrose or CsCl, is also a characteristic feature, revealing information about the proportions of nucleic acid and protein in the particles. 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 of intact virus par￾ticles has yielded some limited information, but electrophoretic analysis of indi￾vidual virion proteins by gel electrophoresis, and particularly also of nucleic acid genomes (Chapter 3), has been far more valuable. However, by far the most impor￾tant method for the elucidation of virus structures has been the use of x-ray dif￾fraction 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 (Å) (see Chapter 2 ).This advancement has improved our understanding of the functions of the virus particle considerably; however, a number of viruses have proven to be resistant to this type of investigation, a fact that highlights some of the problems inherent in this otherwise powerful technique. One problem is that the virus must first be purified to a high degree; otherwise, specific information on the virus cannot be gathered. This presupposes that adequate quantities of the virus can be propagated in culture or obtained from infected tissues or patients and that a method is available to purify virus particles without loss of structural integrity. In a number of important cases, this requirement rules out further study (e.g., hepa￾titis C virus). The purified virus must also be able to form paracrystalline arrays large enough to cause significant diffraction of the radiation source. For some viruses, this is relatively straightforward, and crystals big enough to see with the naked eye and which diffract strongly are easily formed. This is particularly true for a number of plant viruses, such as tobacco mosaic virus (which was first crys￾tallized by Wendell Stanley in 1935) and turnip yellow mosaic virus (TYMV), the structures of which were among the first to be determined during the 1950s. It is significant that these two viruses represent the two fundamental types of virus par￾ticle: helical in the case of TMV and icosahedral for TYMV (see Chapter 2). In many cases, however, only microscopic crystals can be prepared. A partial answer to this problem is to use ever more powerful radiation sources that allow good data to be collected from small crystals. Powerful synchotron sources that generate intense beams of radiation have been built during the last few decades and are now used extensively for this purpose; however, there is a limit beyond which this brute force approach fails to yield further benefit. A number of important viruses stead￾fastly refuse to crystallize; this is a particularly common problem with irregularly shaped viruses—for example, those which have an outer lipid envelope—and to date no complete high-resolution atomic structure has yet been determined for many viruses of this type (e.g., HIV). Modifications of the basic diffraction 14 Principles of Molecular Virology