Dy. RAGHAVENDRA ™P. 2 Methods used in virology At a glance Virus Isolation and Culture Phage plaques ina lawn of bacterial cells? Separation of virus particles ina density gradient? Confocal Microscopy Virus-infected cells detected using a virus-specific antibody labelled with a Sluorescent dye + An endosome (labelled red) containing virus protein (abetted green) in an infected cell $ rely. Pines nd Aplications ok B, Caer od ees A Sins (07 hon Wey Som id ISON 978047003860 (HD) 78.0470. 02387 PB)METHODS USED IN VIROLOGY. tinued) At a glance (con Electron Microscopy E & images from eryo-clectron stained virus particles © microscopy? Electrophoretic Techniques Analysis of vi nucleic ae acids by agarose electrophoresis E § 3 Detection of virus proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis ® Analysis of virus transcription " Reet CheetaTa OOM Tore 14 'Plaques formed by porcine reproducti 5 i laques formed by porcine reproductive and respiratory syndrome virus in a cell culture From Lee and Yoo (2005) Journal of General Virology, 86, 3091. Reproduced by permission of the Society for General Microbiology and the author. 2plaques formed by phage MS2 in Escherichia coli cells Courtesy of Kathryn Newton. Separation of double-layered and triple-laycred particles of rotavirus in a cacsium chloride gradient From L6pez et al. (2005) Journal of Virology, 79, 184. Reproduced by permission of the American Society for Microbiology and the author. “Light micrograph of cells infected with influenza A virus treated with antibody labelled with a fluorescent dye Reproduced by permission of Argene SA, France. An endosome (labelled with a red fluorescent dye) containing rabies virus protein (labelled with green fluorescent protein) in the cytoplasm of an infected cell From Finke and Conzelmann (2005) Virus Research, 111, 120. Reproduced by permission of Elsevier Limited and the author. Negatively stained particles of an orbivirus. The bar represents 50 nm. From Attoui et al. (2005) Journal of General Virology, 86, 3409. Reproduced by permission of the Society for General Microbiology and the author. 7Reconstructed images from cryo-electron microscopy: measles virus nucleocapsid (left), echovirus type 12 ound to a fragment of its cell receptor (right) Courtesy of Dr. David Bhella (MRC Virology Unit, Glasgow). Reinterpretations of data in Bhella et al. (2004) ‘Journal of Molecular Biology, 340, 319 (by permission of Elsevier Limited) and Bhella et al. (2004) Journal of Biological Chemistry, 279, 8325 (by permission of The American Society for Biochemistry and Molecular Biology). ‘Rotavirus proteins from infected cells From Lépez et al. (2005) Journal of Virology, 79, 184. Reproduced by permission of the American Society for Microbiology and the author. SOrbivirus RNA segments separated by electrophoresis through an agarose gel From Attoui et al. (2005) Journal of General Virology, 86, 3409. Reproduced by permission of the Society for General Microbiology and the author. Analysis of varicella-zoster virus transcription using microarrays From Kennedy et al. (2005) Journal of General Virology, 86, 2673. Reproduced by permission of the Society for General Microbiology and the author. ‘i used Permit us to mention only some of them. Much of the 2.1 Introduction to methods focus of this chapter is on methods that are unique to in virology virology. Many of these methods are used, not only Methods used in virology are introduced carly inthis in virus research, but also in the diagnosis of virus aoe gn order to provide an appreciation of the nature of diseases of humans, animals and plans, Peo pigues that have been used to achieve our cur- _Intaly this chapter eould be skimmed to gtin an a esol knowledge and understanding of viruses. overview of is contents and thereafter used for fof remove fa huge subject and uses a wide range of erence, Details of the methods outined bere, and of methods, Many of the techniques of molecular biology ‘other methods important in virology, fill many vol~ sacrnvell biology are used, and constraints on space umes, some of which are listed at the end of the chapter.Fry 2.2 Cultivation of viruses Virologists need to be able to produce the objects of their study, so a wide range of procedures has been developed for cultivating viruses. Virus cultivation is also referred to as propagation or growth, all terms borrowed from horticulture! A few techniques have been developed for the cultivation of viruses in cell- free systems, but in the vast majority of cases it is necessary to supply the virus with appropriate cells in which it can replicate, Phages are supplied with bacterial cultures, plant viruses may be supplied with specially cultivated plants or with cultures of protoplasts (plant cells from which the cell wall has been removed), while animal viruses may be supplied with whole organisms, such as mice, eggs containing chick embryos (Figure 2.1) or insect larvae, For the most part, however, animal viruses are grown in cultured animal cells. 2.2.1 Animal cell culture Animal cell culture techniques are well developed and most of the cells used are from continuous cell fines derived from humans and other animal species, Continuous cell fines consist of cells that have been immortalized, either in the laboratory or in the body (Figure 2.2); they can be subcultured indefinitely. The Sites into which viruses can be inoculated amniotic cavity yolk sae Figure 2.1 allan eaiy METHODS USED IN VIROLOGY. HeLa cell line is a widely used continuous gy, that was initiated in the middle of the 20th cec®* from cells taken from a cervical carcinoma." Sometimes it is difficult to find a cell line in which «Virus can replicate. For many years n0 suitable ce culture system could be found for hepatitis C virus, but eventually a human hepatoma cell line was found to support replication of an isolate of the virus. Cells are cultured in media that provide nutrients Most media are supplemented with animal serum, which contains substances that promote the growth of many cell lines. Other important roles for the medium are the maintenance of optimum osmotic pressure and pH for the cells. Viruses can be cultivated in cells growing on the surface of a variety of plastic vessels (Figure 2.3) with the cells bathed in the growth medium. Most cells grow on a plastic or glass surface as 2 single layer of cells, known as a monolayer. Alternatively the cells can be suspended in the medium, which is stirred to keep them in suspension. Contamination with bacteria and fungi can cause major problems in cell culture work; in order to minimize these problems work is normally done in 2 sterile cabinet (Figure 2.4) and most media contain antibiotics. Many cell types require a relatively high concentration of carbon dioxide, which can be supplied in a special incubator. Inoculation of an egg, Cultivotion of viruses in eggs containing chick embryos. Photograph courtesy of World Health Organisation. €‘Normal eels from human or animal | Primary cll culture ‘Cancer eels rom human or animal Ceolltine Immortalization using ‘ virus ora chemical ‘Continuous cell line Figure 2.2 Derivation of continuous cell tines of ‘human and animal cells. Most types of cell taken from the body do not grow well in culture, If cells from a primary culture can be subcultured they are growing as a cellline. They can be subcultured only a finite number of times untess they are immortalized, in which case they can be subcultured indefinitely as 2 continuous cell tine. Cancer cells are already immortalized, and continuous cell lines may be established from these without further treatment. 2.3 Isolation of viruses Many viruses can be isolated as a result oftheir ability to form discrete visible zones (plaques) in layers of host cells. Ifa confluent layer of cells is inoculated with virus at a concentration so that only a small proportion of the cells is infected, then plaques may form where areas of cells are killed or altered by the virus infection, Each plaque is formed when infection spreads radially from an infected cell to surrounding cells. Plaques can be formed by many animal viruses in monolayers if the cells are overlaid with agarose gel to maintain the progeny virus in a discrete zone (Figure 2.5). Plaques can also be formed by phages in Tawns of bacterial growth (Figure 2.6). It is generally assumed that a plaque is the result of the infection of a cell by a single virion, If this is the case then all virus produced from virus in the plaque should be a clone, in other words it should be genetically identical. This clone can be referred to as an isolate, and if it is distinct from all other isolates it can be referred to as a strain. This is analogous to the derivation of a bacterial strain from a colony on an agar plate ‘There is a possibility that a plaque might be derived from two or more virions s0, to increase the probability that a genetically pure strain of virus has been obtained, material from a plaque can be inoculated sr monolayers and virus can be derived from dual plaque. The virus is said to have been plaque purified, ‘When a virus is first isolated it may replicate poorly in cells in the laboratory, but after it has gone through ‘a number of replication cycles it may replicate more efficiently. Each time the virus is ‘sub-cultured” (to borrow a term from bacteriology) it is said to have been passaged. After a number of passages the virus may be genetically different to the original wild strain, in which case it is now a laboratory strain, 2.4 Centrifugation ‘After a virus has been propagated it is usually necessary to remove host cell debris and other contaminants before the virus particles can be used for laboratory studies, for incorporation into a vaccine, or for some ther purpose. Many virus purification procedures involve centrifugation; partial purification can be achieved by differential centrifugation and a higher degree of purity can be achieved by some form of density gradient centrifugation. 2.4.1 Differential centrifugation Differential centrifugation involves alternating cycles of low-speed centrifugation, after which most of the virus is still in the supernatant, and high-speed centrifugation, after which the virus is in the pellet (Figure 2.7). 2.4.2 Density gradient centrifugation Density gradient centrifugation involves centrifuging particles (such as virions) or molecules (such as nucleicCC RR Rley g “Figure 2.4 Cell culture work. Precautions to avoid contamination include working in a sterile cabinet and wearing Gloves and mask. Photograph courtesy of Novartis Vaccines. Hcell monolayer inoculated with virus. agarose overlay l ‘A cell monolayer is inoculated with vinas and overlaid with agarose, ‘Plaques formed by West Nile virus in a cell ‘monolayer oth flasks were inoculated with virus. Plaque formation in the flask onthe right has been | inhibited by West Nile virus-specific antibody. Courtesy of Dr. Elieen Ostiund, National Veterinary Services Laboratories, Department of Agriculture, Figure 2.5 . Method for production of plaques by animal viruses. © Faure 2. “Grady. 3 acids) in a solution of increasing concentration, and therefore density. The solutes used have high solubility sucrose is commonly used. There are two major cate- gories of density gradient centrifugation: rate zonal and ‘equilibrium (isopycnic) centrifugation (Figure 2.8). In rate zonal centrifugation a particle moves through the gradient at a rate determined by its sedimentation coefficient, a value that depends principally on its size. Homogeneous particles, such as identical virions, should move as a sharp band that can be harvested after the band has moved part way through the gradient, “Plaques formed by a phage in a bacterial lawn. The control plate on the left was inoculated with only the “bacterial host. The plate on the right was inoculated with phage and bacterial host. Photograph courtesy of Philip In equilibrium centrifugation a concentration of solute is selected to ensure that the density at the bottom of the gradient is greater than that of the particles/molecules to be purified. A particle/molecule suspended in the gradient moves to a point where the gradient density is the same as its own density. This technique enables the determination of the buoyant densities of nucleic acids and of virions. Buoyant densities of virions determined in gradients of caesium chloride are used as criteria in the characterization of viruses.ats ees CL decant supernatant i tion of virus containing host ferential centrifugation. & crude prepara contin at teatime! Wirons fy cireta pane ie nutes) followed by high-speed/Long praure ast Pele aur ei ee agate tea. 30000 9/20 minute 1 lowed by His sped. si ae i n be repei Hee ane Canon oe a2 haus) TiBiegde ca otal time Se oy pute virus is resuspended in a small volume Final pellet con Fulte 28 Pusication of virions by density gradient sanfugation. & partly purified preparation of virus is further purified in a density gradient. Rate zonal on pagtion involves layering the preparsting on top of a pre-formed Fr the geal centrifugation can often be don: Starting with a suspension of the impure vires rare solution the gradient material the gradient is formed during centrifugation5 Structural ‘investigations of cells and virions 2.5.1 Light microscopy ‘The sizes of most virions are beyond the limits of Taotution of light microscopes, but light microscopy has “useful applications in detecting virusinfecect ‘cells, for example by observing cytopathic effests ection 2.7.2) or by detecting a fluorescent dye linked to antibody molecul les that have bound to a virus anti- en (Guorescence microscopy), Confocal microscopy is proving to be especially Yaluable in virology. The principle of this technique 's the use of @ pinhole to exclude light from out. of-focus regions of the specimen, Most confocal microscopes scan the specimen with a laser, producing exceptionally clear images of thick specimens and of Aluorescing specimens. Furthermore, ‘optical slices’ of 8 specimen can be collected and used to create a three- dimensional representation. The techniques can be used with live cells and can be applied to investigations of protein trafficking, with the virus or cell protein under investigation carrying a suitable label, e.g. green fluorescent protein (a jellyfish protein). 2.5.2 Electron microscopy Many investigations of the structure of virions or of virus-infected cells involve electron microscopy. Large magnifications are achievable wit th a transmission electron microscope but the specimen, whether it is 2 sspention of irons or an ulate setion of ‘irweinfected cell, must be Weated so that deta can be visualized Negative saning techniques generate contrast by using heavy-melarcomtaning. eompounds, sueh ss potassium [Link] ammonium melyb- Se Ineecron microg of ios ths peat 3s dark areas around the visions, allowing the ‘ver virion shape and size to be determined, Further ftructral deta may be apparent ithe stains penetrate any crevices onthe von surface or any hollows within the virion, Negative staining techniques have generated ‘many high quality electron micrographs, bathe teh niques have limitations, including structural distortions, resulting from drying. MST cs Cryo-electron microscopy recent. techniques are more In these a wet specimen is rapidly cooled {0 a temperature below —160°C, freezing the water a8 a glasslike material, as in the method outlined in Figure 2.9. The images are recorded while the specimen is frozen. They require computer process- ing in order to extract maximum detail, and data from multiple images are processed to reconstruct three-dimensional images of virus particles, This may involve averaging many identical copies or combin- ing images into three-dimensional density maps (tomo- grams) 2.5.3 X-ray crystallography X-ray crystallography is another technique that revealing detailed information about the three dimensional structures of virions (and DNA, proteins and DNA-protein complexes). This technique requires the production of a crystal of the virions or molecules under study. The crystal is placed in a beam of X- rays, which are diffracted by repeating arrangements of molecules/atoms in the crystal, Analysis of the \iffraction pattern allows the relative positions of these ‘molecules/atoms to be determined. Other techniques that are providing useful informa- tion about the structure of viruses are nuclear magnetic resonance and atomic force microscopy. 2.6 Electrophoretic techniques Mixtures of proteins or nucleic acids can be sepa- rated by electrophoresis in a gel composed of agarose or polyacrylamide. In most electrophoretic techniques each protein or nucleic acid forms a band in the gel. Electrophoresis can be performed under condi- tions where the rate of movement through the gel depends on molecular weight. The molecular weights Of the protein or nucleic acid molecules can be es mated by comparing the positions of the bands wi Positions of bands formed by molecules of known ‘molecular weight electrophoresed in the same gel. The lechnique for estimating molecular weights of proteins is polyacrylamide gel electrophoresis in the presence of the detergent sodium dodecyl sulphate (SDS-PAGI Figure 2.10),‘okey es i ian 9 se ypecinen bs appli (othe fil. ‘ae pl ph ino nese device “The grid is blotted with filter paper to produce very thin aqueous layers ross the holes. “The forceps are dropped into a bath of ethane slush held in a container af Fiquid nitrogen, Rapid cooling causes vitrification fof the sample Figure 2.9 Preparatic Perce ksueeae ace in pair of forceps which are locked n of @ specimen for cryo-electron microscopy. Modified, with the permission of the authors ‘and the American Society for Microbiology, from Baker, Olson and Fuller (1999) Microbiology and Molecular Bioloay Reviews, 63, 862. ‘The patterns of nucleic acids and proteins after clectrophoretic separation may be immobilized by transfer (blotting) onto a membrane. If the molecules are DNA the technique is known as Southern blotting, named after Edwin Southern; if the molecules are RNA the technique is known as northern blotting, and if the molecules are protein the technique is known as western blotting! 2.7 Detection of viruses and virus components A wide range of techniques has been developed for the detection of viruses and virus components and ‘many of them are used in laboratories involved with diagnosis of virus diseases. The techniques can be arranged in four categories: detection of (1) virions, (2) virus infectivity, (3) virus antigens and (4) vires nucleic acids. 2.7.1 Detection of virions Specimens can be negatively stained (Section 2.5) and examined in an electron microscope for the presence of virions. Limitations to this approach are the high costs of the equipment and limited sensitiv the minimum detectable concentration of virions is about 10%/ml. An example of an application of this technique is the examination of faeces from a patient with gastroenteritis for the presence of rotavirus particles.Pada ATP ATL ued Juneton of aking pet and — resolving el Aye ont ves —— rolcolr weights of protein standards 20K 6K, mak oo. 45K Bik 213k Mask 65K | Figure 2.10 Separation of proteins and estimation oftheir molecular weights using SDS-PAGE. Lanes 1 and 3 contain | standard proteins of known molecular weight. Lane 2 contains the four capsid proteins of a picornavrus. 2.7.2 Detection of infectivity using cell cultures icate in host cells. ‘Those virions that do have this ability are said to be ind the term ‘infectivity’ is used to denote the capacity of a virus to replicate. Virions may be non-infective because they lack part of the genome or because they have been damaged, ‘To determine whether a sample or a specimen con- tains infective virus it can be inoculated into a culture of cells, or a host organism, known to support the repli cation of the virus suspected of being present. After incubation of an inoculated cell culture at an appropri- fate temperature it can be examined by light microscopy for characteristic changes in the appearance of the cells resulting from virus-induced damage. A change of this type is known as a cytopathic effect (CPE); examples of, CPEs induced by poliovirus and herpes simplex virus, can be seen in Figure 2.11. The poliovirus-infected cells have shrunk and become rounded, while a multi- nucleated giant cell known as a syneytium (plural syn ‘ytia) has been formed by the fusion of membranes of herpes simplex virus-infected cell. ‘The quantity of infective virus in a specimen or a preparation can be determined (Section 2.8). 2.7.3 Detection of virus antigens Virus antigens can be detected using virus-specific antisera or monoclonal antibodies. In most techniques. Fr)ETHODS USED IN VIROLOGY Vv Figure 2.11 Gytopatic effets coused by replication of palais ‘and herpes simplex virus in cultures of Vero (monkey Taney ces. (a) Uninfected Vero cells. (b) Infected with potorit, {G), (6) Infected with herpes simplex virus, {@), (0) and (4) were viewed at » 400 magaification:(€) wit ‘vewed at x 100 magnification. The cells were stained (2) snatonyiin and eosin, Reproduced by permission of Dr Mara-Lucs Raez (University of S20 Paulo) and the ‘American Society for Hticrobiology MicrobeLibrary. Direct Tet toe et : second sry | Saba aniivrs antibody ft» tabol ‘ vausarigon JN, } rantogn JN Figure 2.12 Principles of tess to detect virus antigns. the specimen {s teated with antivirus antibody. In an Haare test a labelled second antibody detects any anti-viral antibody that has bound to antigen- igen it imal ss produced by injecting virus antigen inl ani Pres and the second antibody, is produced by tmetobulin from the fist animal species positive results are indicated by detecting the present eesy label, which may be attached either 10 the anti- spon an Oras anuibady (direct tests) or 10 a second antibody jnjecint Gadirect tests) (Figure 2.12). The anti-virus antibody into * secon 1d anisnal species:Pee Ua Se UUN Le kOn aes} Antibodies can have many types of label attached ‘and the labels can be detected using a range of methods. Some types of label and some methods for detecting them are listed in Table 2.1. 2.7.4 Detection of virus nucleic acids 2.7.4.a Hybridization ‘Virus genomes or virus messenger RNAs (mRNAs) may be detected using sequence-specific DNA probes ‘carrying appropriate labels (Figure 2.13). Some of the labels that are used for antibody detection can be used to label the probes (Table 2.1). Hybridization may take place on the surface of a membrane afier Southem blotting (DNA) or northem blotting (RNA) (Section 2.6). Thin sections of tissue may be probed for the presence of specific nucleic acids, in which case the technique is known as in situ hybrid target nucleic acid XTc Table 2.1 Motecules used to label antibodies (and nucleic acids) and techniques used to detect them Label Detection technique Enzyme Enzyme-linked immunosorbent assay Fluorescent Fluorescence microscopy Fluorimetry Gots Electron microscopy Radio-active Autoradiography [Link] Polymerase chain reaction (PCR) When a sample is likely to contain a low number of copies of a virus nucleic acid the probability of detection can be increased by amplifying virus DNA using a PCR, while RNA can be copied to label @ leTeTeTe Tt eTet xt __. hybridization Figure 2.13 Detection ofo specfic nucleic acid (DNA or RA) using o labelled DNA probe.RT (reverse transcriptase)- using « RT jigonucleotige primers dures require olig nets FO oAn amplified product cat viral sequences. followed specific 10 VIE Trophoresis in an agarose gel, follo. detected by ee trocellulose membrane, which is fer 10 il by ee with a labelled probe. incapare are also PCR techniques available for deter- rere number of copies of a specific nucleic acid rine mple. Real-time PCR is commonly used for this purpose. In this technique the increase in DNA concentration during the PCR is monitored using flu- orescent labels; the larger the initial copy number of DNA, the sooner a significant increase in fluorescence is observed. The PCR cycle at which the fluorescent signal passes a defined threshold is determined and this gives an estimate of the starting copy number. An example is given in Figure 2.14. NA and amplified PR, The PI 2.8 Infectivity assays An infectivity assay measures the titre (the concentra tion) of infective virus in a specimen or a preparation. oss METHODS USED IN VIROLOGY 10¥f 107/108 r0%/so* 10/10/10 Samples are inoculated into suit response can be observed if infective sins 9 Suitable hosts might be animals, bacterial, plant or animal cells. into two classes: quantitative and table hosts, in y Plants oF cultures of infectivity assays fa quantal,” 2.8.1 Quantitative assays Quantitative assays are those in which response can be any one of a series of values, such ay ¢ number of plaques (Section 2.3). A plague asay canbe carried out with any virus that can form plaques, giving an estimate of the concentration of infective vine plaque-forming units (pfu). ‘The assay is done using cells grown in Petri dishes, or other appropriate containers, under conditions tha will allow the formation of Plaques. Cells are inocu. lated with standard volumes of virus dilutions. After incubation, dishes that received high dilutions of vires may have no plaques or very few plaques, while dishes that received low dilutions of virus may have very large numbers of plaques or all the cells may have lysed. each host Teitial nomber of copies of HBV DNA jes of HBV DNA Figure 2.14 Quantification of hepatitis 8 virus (HBV) DNA by real-time PCR. When Sion sie oo Sy ie te sample, fluorescence starts to increase at an ext cle number. The increase stars 3 Fe dps "gles with decreasing numbers of HBV ONA copies. Data from Ho et cl. (2003) Journ! arleee + 387; by permission of the Society for General Microbiology and the authors.® AAgaAg Ik 0000¢ © 100 gost i bo ask 1 Ao Ao10* iu 5800000 ©) Contr! wo 10r4040%104 Control ‘az 29000000 WOO000065 10 woe Virws dilution 0? 108 10 ne TCD is presenti tis ition "Figure 2:15 Example ofo TCIDsp assy. (a) Tenfld dilutions ofthe virus were inoculated into cell cultures gronn in Dishes are selected that have plaque numbers within ‘a certain range, c.g. 30-300. The plaques are counted ‘and the concentration of virus in the sample (pfu/m!) is calculated. 2.8.2 Quantal assays In a quantal assay each inoculated subject cither responds or it does not; for example, an inoculated Cell culture either develops a CPE or it does not; an Jnoculated animal either dies or it remains healthy. The tim of the assay is to find the virus dose that produces ‘response in 50 per cent of inoculated subjects. In ‘ell culture assays this dose is known as the TCIDso (the dose that infects 50 per cent of inoculated tissve cultures). In animal assays the dose is known as the Dap, and where the virus infection kills the animal the he wells of a plate Each wel received 1 ml virus suspension, except forthe contol wells, which each receh vel re: Each ¥ . i i ved tml tent. (b) After incubation the cell culture in each well was scored “or : for CPE. (c) The results of the assay. dose is known as the LDyg (the dose that is lethal for 50 per cent of inoculated animals). ‘The outcome of a TCIDs determination can be used to estimate a virus titre in pfu, or vice versa, using the formula 1 TCID39 = 0.7 pfu formula applies only if the method used to visualize plaques does not alter the yield of virus. ‘An example of a TCIDso determination is depicted in Figure 2.15. It is clear that the dilution of virus that contains ‘one TCID39 lies between 10°? and 10~*, Several meth ‘ods have been developed for estimating this end point, fone of these, the Reed-Muench method, is shown in the box.A Prents OR Leg The Reed-Muench method for estimating TCIDso Virus Infections Observed v. i iiss 5 aes served values Cumulative values! Infection inoculated Positive Negative Positve Negaive "ition 107 88 8 0 u 102 78 7 16 mAs 100 103 58 5 3 Lely 38 oa 9 4 9/13 6 10 38 3 5 s 4 9 4/13 31 10 1s 1 7 10% os ; ss Mae § 0 8 0 24 0/24 0 ‘ie came vs ar des yang pth obsered va inte dct of aro 2k fon a tember of poses fee caste vale oo wal for be coal ‘% infection is the infection ratio converted to a percentage. eee ee It had already been determined that the dilution of i € i virus that contains one TCIDsq li 3 10", so the end point canbe expressed ss 10-0, whoo ish alue tbe edad. % infection at next dilution above 50% — 50 '% infection at next dilution above 50% — % infection at next dilution below 50% 1ogio dilution factor a(S=2 31 05 End point = 10~@#0 = 10-34, ile. 1 ml of a 10% dilution contains one TCIDso of virus ie. 1-ml of a 1/3200 dilution contains one TCIDso of virus. G.2is the antilogarithm of 0.5) ‘The concentration of virus in the undiluted suspension is 3.2 x 10° TCIDso/ml. directly equivalent to 2 plaque assay as the number ‘A similar approach can be used to assay the infec- tivity of any virus that causes the formation of dis crete lesions. A number of animal viruses, including smallpox virus and other poxviruses, can cause the Formation of pocks after inoculation onto the chorioal- Tantoic membrane of a chick embryo (Figure 2.16). Similarly, many plant viruses cause the formation of Tesions after they have been rubbed on the surface of leaves (Figure 2.17), though the titration is not of sites on.a leaf through which a virus can enter is limited. 2.8.3 One-step growth curve ‘A-common type of experiment that involves assaying infectivity. is:a single-burst experiment, which ‘a one-step growth curve (Figure 2.18). virus iit provides data for© Figure 2.16 Virus-induced pocks on the chorioallan- © ‘toic membrane of chick embryo, From Buxton and Fraser (1977) Animal Microbiology, Volume 2, Blackwell © Scientific Publications. Reproduced by permission of ‘the publisher. ‘The first one-step growth curves produced were for phages, but they have since been produced for other viruses. Infectious virus titre (ogo pfu) cclipse petiod Time Figure 2.18 One-step growth curve. The eclipse period is the period during which no intracellular infectious Virus can be recovered; infectious nucleic acid might be recoverable [Link] cases, The burst size is the average yield of infectious virus per cell Fora period (the eclipse period), the titre of infective virus remains constant. During the eclipse period the virus is replicating, but no virus has been released from infected cells, so each infected cell gives rise to one | plaque. As infected cells begin to dic and release virus ions resulting fro Figure 2. with ster 1m infection with potato virus ¥, The leaf on the right is a control inoculated Copyright INRA, France/Didier Spire; reproduced with permission, Sethe titre begins to rise, and when all the infected cells have lysed the titre levels off. “The procedure for deriving a one-step growth curve is now outlined. «A suspension of virions is mixed with a suspension af host cells. Ik is necessary to ensure that, as far fs possible, all the cells are infected simultaneously, fo the number of virions must greatly exceed the umber of cells. The ratio of virions to cells is known fe the multiplicity of infection (m.0.i.). In a one-step growth experiment an m.o.. between S and 10 is commonly used. « Adsorption of virions to cells is allowed to proceed for a suitable period (e.g. 2 minutes), and then adsorption is stopped by, for example, greatly diluting the mixture and/or by adding antivirus antiserum. « A sample is taken from the suspension at intervals tintl lysis is complete. For phages the experiment is tisually complete in Tess than one hour. For animal viruses, however, the timescale is measured in hours Or days, reflecting the much slower growth ra'es Of eukaryotic cells and the longer times taken for replication of animal viruses. ° «A plaque assay is carried out on each sample. «The logarithm of the virus tte is plotted agains time to obtain the one-step growth curve (Figure 2.18). provides valuable informa: is type of experiment This tyPe Or le particular tion about the replication cyole of a virus 1 Te Host cell system, The average yield of infectious virus peer cell (the burst size) can te calculated from the formula final virus titre final virus jnitial virus titre burst size noted that the burst size is an averse ignificant variable as there usually rst size between cells. One factor size is the physiological Te one study with a phage, a Durst ‘growing bacteria were ‘obtained with resting It should be of what may be asi much variation in that may affect state of the cells. bacteria. METHODS USED IN VIROLOGY 2.9 Virus genetics 2.9.1 Genome sequencing Determination of the sequence of bases in a DN, Molecule is usually done using the deoxy chan termination method developed by Fred Sanger and his colleagues at Cambridge. The sequence of the genome of phage yX174 DNA was determined by this group six years after the first determination of a DNA sequence Which itself was from a virus: 12 base pairs (bp) of phage lambda DNA (Figure 2.19). With the help of computer programmes such as Artemis and BLAST much useful information can be derived from virus genome sequences. @ Open reading frames (ORFs) can be found. The ORFs can be translated into the amino acid sequences of the virus proteins, and these may allow characteristics and functions of the proteins to be deduced. Hydrophobic sequences may indicate membrane association; the sequence Gly~Asp—Asp may indicate RNA-dependent RNA polymerase activity. «Sequences that regulate gene expression, eB. Dro- moters and enhancers, can be identified. « During an outbreak of virus disease, such as foot and mouth disease or influenza, sequences of virus wolates can be comipared. This can provide useful epidemiological information such as the source of the virus responsible and whether more than one strain is responsible for the outbreak. « Phylogenetic trees can be constructed (Section 10.2.1). 2.9.2 Genome manipulation ‘The wide range of techniques availabe forthe manipy- Jation of nucleic acids can be applied to virus genome "These techniques include the isolation of specific frag soe of genomes using restriction endonucleases: the cloning of fragments in bacterial plasmids and the introduction, of site-specific: mutations info WE genomes. ‘The natural processes of recombination andcytomegalovirus (229,354 bp) Lambda (48,502 bp) Lambda $569») (2 bp) Year S77 Tow [Te frat genome sequenced [ie first DNA sequenced Figure 2.19. Time line depicting some landmarks in viral genome seq smentous brown alga Ectocarpus si ‘The host of E5V-1 is the marine fila The first DNA ever sequenced was 1 sequence of this virus was published. Not to scale, reassortment (see Chapter 20) that produce new com- binations of virus genes can be harnessed to produce new viral genotypes in the laboratory. 2.9.3 Investigation of gene function and expression ‘The function of a gene may be deduced if its expression is blocked, and many studies of gene function involve the creation of a virus with a mutated gene. Many of the mutants are unable to replicate under conditions in which the wild-type virus replicates and are known fas conditional lethal mutants. Most studies have used temperature-sensitive mutants, which are unable to replicate at temperatures at which the wild-type Mimivirus (1,181,404 bp) Esy-l Hisvenl (235,593 bp) 1990 2007 Zoo or] aos | son | sna juencing. Lambda, MS2 and yX174 are phages. lculosus. The host of mimivirus fs an amoeba. "bp of the genome of phage lambda. Eleven years later the complete genome virus replicates (non-permissive temperatures), but are able to replicate at other temperatures (permissive temperatures). ‘Techniques for introducing mutations into DNA are well established and have been applied to the study of DNA viruses for some time. More recently, techniques for manipulating the genomes of RNA viruses have been developed. These techniques are referred to as reverse genetics, and they involve the reverse transcription of the RNA genome to DNA, mutation of the DNA, then transcription back to RNA. Reverse genetics are discussed further in Chapters 14 and 15. ‘Another way in which the expression of @ gene can be blocked is by RNA silencing, or RNA interference (RNAi). This approach exploits a cellEc Ree UNUC OLA ai defence mechanism (described in Section 9.2.3) that can destroy virus MRNAS alter induction by specific sRNA molecules. Short sequences of dsRNA ean be used to inhibit the expression of virus genes, and hence to investigate the functions of those genes. Yet another approach to the investigation of gene function involves the introduction of the gene into cells in which it is transiently expressed and changes in the cells are ‘monitored, As well as the production of virus mutants there are other ways in which viruses ean be genetically modified in order to aid their study. A sequence encoding a tag, such as green fluorescent protein, can be appended (0 the gene for a virus protein so that the distribution Gf the protein in infected cells can be monitored (Figure 2.20). ‘The expression of virus genes in infected cells ean ‘be monitored with the aid of DNA microarrays. A DNA microarray consists of a substrate, such as a glass slide, Figure 2.20° A syncytium (giant cell) infected with a ‘genetically modified HIV. The gene for enhanced green fluorescent protein has ‘been inserted into the virus ‘genome. The distribution of the fluorescence indicates the: distribution of the labelled virus protein. From Miller et al. (2004) Joumal of Virology, 78, 10803; ~ feproduced by permission of the American Society for Microbiology ‘and the authors. with hundreds, thousands or even millions of small spots of DNA attiched, Each spot contains a speci DNA probe and specific DNA or RNA molecules in a sample are detected by hybridization to specific spots, Microarray technology allows investigators to mon- itor the expression of hundreds or thousands oF genes, and is thus ideal for studies of viruses with large genomes such as the herpesviruses and the large DNA phages. Virus mRNAs are detected by copying RNA from infected cells to DNA using a reverse transerip- tase, amplifying the DNA with a PCR, labelling it with a fluorescent dye and adding it to a microarray. The probes that bind DNA from the sample are detected by scanning with'a laser at a wavelength that excites the Muorescent dye. DNA microarrays can also be used to investigate the expression of host cell genes that are relevant to viruses. Cell surface molecules used by viruses as receptors have been identified using microarrays. Learning outcomes By the end of thus chapter you should be able to © outline methods for © cultivation of viruse © purification of viruses; 6 detection of viruses and their components; 6 assay of virus infectivity; © investigation of virus gene function; ‘* assess the vahic of virus genome sequencing, Sources of further information Books Cann A. J., editor(1999) Virus Culture: a Practical Ap- proach, Oxford University Press Coleman W. B, and ‘Tsongalis G. J. (2006) Molecular Diag nosties for the Clinical Labaratorian, 2nd edition, Humana,