VIROLGY LECTURE NOTES

OKOROR LAWRENCE (Phd) ASSOCIATE PROFESSOR, DEPARTMENT OF BIOLOGICAL SCIENCES, JOSEPH AYO BABALOLA UNIVERSITY, IKEJI ARAKEJI

MODULE 1

1. History of virology Virus growth requirement

Isolation of viruses Electron microscopy Discovery of bacteriophage Mammalian tissue culture Crystallization of viruses

HISTORY OF VIRUSES Viruses have been shown to cause a wide range of diseases in humans and animals which have made the well-known in the immediate past and in the present. Viruses cause diseases of closely knitted populations both in humans and in animals. Viruses can infect a wide range of organisms which include plants and animals and even bacteria and other microorganisms. The problem of viruses in humans will become unprecedented with the increase in human population and the quest for more food through more agricultural activities like animal husbandry and systems like monocropping. Viral diseases mostly occur on epidemic scale and a proper understanding of viruses and the diseases they cause is essential to be able to cope and arrest the situation.

Viral infections were first suspected in the 1892 when Dimitiri Iwanowsky working on the causative agent of the tobacco mosaic disease discovered that the bacteria free extract of the diseased tobacco leaves still causes tobacco mosaic disease. And toxins were suspected to be responsible for the tobacco mosaic disease. This went on until 1898 when Loffler and Frosch working on the foot and mouth disease (agent causing the rottening of the foot and mouth of cattle) of cattle and Beinjerinck working on tobacco mosaic diseases confirmed that the infection could be serially transmitted from bacteria free filtrates. They thereby confirm that by the rate of transmission of this infectious agent, it could not have been toxins or poisons since the agent was able to move from one plant to another and from one animal to another.

And the diseases due to toxins or poisons will be related to the quantity of dose given and does not increase during the course of illness. However, in the course of infection new infectious agents necessary for the spread of disease were produced hence the diseases were transmitted from one plant to another and from one animal to another. Multiplication is a principal phenomenon of living cells, both increment in the number of cells or the development of an organism. It is important to note that viruses are not cells. And early definitions of viruses have been to distinguish them from other infectious agent. And one of the main distinguishing factor has been that viruses are unable to multiply in chemically defined solution or synthetic medium.

Viruses will only multiply in the presence of a living host be it a cell or an organism and the host must be susceptible to the virus. The bacteria free extract could retain its infectivity for a long time but they could not be cultivated without a living host.. Viruses replicate inside cells and uses the hosts cell cellular environment. This includes the system of enzymes since viruses lack the Limans system, a system of enzyme for the production of enzymes which is a component of most prokaryotic cells. Since viruses lack most of the sub-cellular organelles.

The purification of the tobacco mosaic disease virus by Stanley in 1935 marked a major breakthrough in the study of virology. Since infected tobacco leaves are usually saturated with the virus as a result of viral multiplication. Quantities of infected leaves were grinded and made into aqueous solution, then the cellular materials were removed by filteration. It was believed that viruses may be proteins and that if the method of precipitating proteins are applied to viruses proteins may also be precipitated and retained their infectivity. Since they are protein they could salted out like proteins. This method was eventually used by Stanley to obtain viruses and it was to be discovered that viruses are nucleic acids and proteins.

More details about viruses came with invention of the electron microscope. Detailed structure of viruses were now possibly seen since they are much smaller than bacteria.

For sometimes plant viruses form the bases of virology researches but in 1915 Twort found that some viruses infect bacteria which was to be later confirmed by dHerelle in 1917 and they were called bacteriophages (phages for short). Little attention were paid to phages since they did not appear to be associated with any infection. They were later to contribute immensely to virology researches especially in area of viral replication where it was discovered that only the nucleic acid enters in to the bacteria during infection leaving all other appendages outside the cell. Hersey and Chase in 1952 proved that only the nucleic acid of an infectious phage enters into the cell during infection. The use of biochemistry radio isotopes was used in this study. Hersey and Chase labelled a phage nucleic acid with radioactive phosphorus and the outer protein shell with radioactive sulphur and the found that it is only the radioactive phosphorus that was found in the bacterium cell. This implied that it is only the nucleic acid that is involved in the replication of viruses and that the protein plays little or

non-significant role in replication as well as transfer of information from generation to generation.

The discovery of infection phage DNA The biological characteristic of viruses and shown that their behavious could best be explained within the general content of genetics. They could mutate, propagate themselves and yet maintain highly defined characteristics such as host specificity, size and shape. Like other organisms they always or nearly always, appeared to breed true. Direct chemical analysis showed that viruses contained nucleic acids as well as protein and a famous experiment by Hershey & Chase in 1952 established beyond doubt that the nucleic acid (DNA) of the bacteriophage was directly responsible for infection. The elegance of the Hershey & Chase experiment lay in the simplicity of approach and the fact that only a single, yet fundamentally important , question was asked. What part of the virus enters the bacterium? By this time the use of radioactive isotopes was well established in biochemistry and it was possible to label nucleic acid with 32P and protein with 35S Hershey & Chase made preparation of bacteriophage labelled with 32P and 35S, purified them and then used the labelled virus to infect non-radioactive bacterial cells (fig. 1.3). They found that the only radioactive element which entered the cell was 32P. The proteins labelled with 35S remained outside and could be removed from the bacterial without reducing the ability of the infected cells to produce progeny phage particles. The important implication here is that the replication of the phage is under the control of the nucleic acid from one generation to the next. Although this result might have been expected from earlier work by Avery, McCleod & McCarthy (1944) on the transformation of a non virulent strain of bacterial cells by the cells by the Hershy & Chase experiment left no doubt about the chemical nature of the genetic material. Their experiment opened the flood gates and led to our current knowledge of the biochemistry of genetics. The development of mammalian tissue culture These interest discoveries in the field of bacterial viruses had mainly development from the dedicated work of Max Delbruck and his collaborates in establishing the quantitative characteristics of bacteriophage replication. They stressed the ultimate importance of working with a simple, readily controlled system a d realised the importance of what is now called the one step growth cycles, when all the cells of a population are undergoing synchronous or nearly synchronous infection. Only from such an experimental system can one deduce the probable events in a single cell. Also, of course, the yield of bacteriophages from culture or bacteria was sufficient to allow accurate quantities and chemical analysis to be done on them. With animal viruses at that time no feasible way of studying the life cycle was available because of the complexity of the host organisms. Development in this field continued to be predominantly of clinical nature until around 1950. As is clear from the above comments on the growth of bacteriophages, a suitable experimental culture systems was required and the answer to the animal virologist problems arrived with the development of the techniques of tissue culture.

Tissue culture is basically simple, and modern developments have made it one of the most widely used tools of virus research. In brief, small samples from an organ such as a kidney are removed and the tissue in dissociated into individual cells or minute pieces. If these fragments are put into a glass or plastic bottle in a suitable nutrient solution or medium, the cells will adhere to the surface of the glass and multiply of form cells sheets, generally called monolayers (see fig. 3.2, p. 23). Some cells, if kept agitated,

Table 1.1

Composition of Eagles Medium

Component Inorganic salts Sodium chloride (NaCI) Calcium chloride (CaCl2) Magnesium sulphate (MgSo4 . 7H2O) Sodium phosphate (NaH2PO4. 2H2O) Ferric nitrate (Fe(NO3)3. 9H2O) Amino acids L-arginine hydrochloride L-cystine L-histidine hydrochloride DL- isoleucine DL- leucine DL-methionine DL-phenylalanine DL-threonine DL- tryptopham L-tryosine DL.valine

Concentration (mg/1) 6400.0 400.0 200.0 200.0 140.0 0.1

42.0 18.0 19.2 104.8 104.8 73.0 66.0 95.2 16.0 36.2 93.6

Vitamins Aneurine hydrochride Choline chloride Folic acid Inositol 2.0 2.0 2.0 3.5

Nicotinemide Calcium pantothenate Pyridoxal hydrochloride Riboflavin Miscellaneous Glucose L-Glutamine Phenol red Antibiotics Penicillin Streptomycin Calf serum

2.0 2.0 2.0 0.2

4500.0 292.0 15.0

200000 units 100000 5-10%

Will grow in suspension just as bacterial cells do. In many cases, cells have become so adopted to growing under these conditions that they have been maintained for years in continuous culture with frequent renewal of the medium. Media are complex solutions (tables 1.1) containing all the essential ingredients for supporting life, such as glucose, amino acids, vitamins, mineral salts and how concentrations of biological extracts such as serum . although simple in concepts, the practical problems involved in perfecting the techniques to a routine procedure involved years of dedicated and tenacious work. The biggest hazard is the adventitious presence of bacteria of fungi. Since these grow more rapidly then animals cells, the slightest contamination soon destroy the culture. Tissue culture was initially of great importance to the cancer research but in the early 1950s it became firmly established that animal viruses form a large variety of disease could be successfully grown in cultured cells. JohnEnders. Who received the Nobel Prize in 1954 for his pioneering studies on the growth of pliovirus, provided a further landmark in the history of virology. The repercussions of the development of the growth of animal viruses in tissue culture today with sustained vigour throughout virology . Tissues culture is currently used is diagnosis, vaccine production, the isolation of unknown agents and in fundamental studies of the replication and structure of viruses.

Probably the most rapidly advancing area of virology today is the study of cancer-inducing or oncogenic viruses. In 1911, Peyton Rous discovered an agent, now known as the Rous sarcoma virus (RSA), which could produce sarcoma (solid tumours) in chickens. May viruses have since been found that are known as to be the immediate cause of tumour formation especially in chickens and rodents. This area of virology has recently been recognised by the award of the Noble Prize to three independent workers. Dulbecco, Temin and Baltimore, in 1975. Dulbecco and his colleagues had shown during the 1960s that certain small DNA viruses had alternative replication pathways. Usually these viruses replicate in and kill the

host cell resulting in cell lysis. Occasionally, however, the infection does not lead to cell lysis but to cell transformation as a result of the stable integration of the virus genome into the host cells chromosomes. On the other hand, the Rous sarcoma virus was shown to be an RNA virus and the way in which such viruses cause transformation of cells was a mystery until 1970 when Baltimore and Temin independently discovered a new type of enzyme called reverse transcriptase in RNA tumour viruses. Reverse transcriptase can make DNA from RNA molecules and hence also result in the stable integration of RNA virus genetic information into the host cell chromosomes. These discoveries have established that virology has an important role to play in the understanding of cancer as well as of the classical infectious diseases. Of great current interest is the possibility that some long-term degenerative diseases such as multiple sclerosis, schizophrenia, diabetes, rheumatoid arthritis and many other conditions may have a virus etiology and can result from complications arising from both virus infection and the immune system. During the 1950s a new brain disease called Kuru was recognised among the eastern highland cannibals of Stone Age New Guinea. This disease was a longterm degenerative condition and Gajdusck and his colleagues showed that it was passed on from generation to generation by the tribes unusual eating habit. They also isolated from the affected people a virus agent, similar to the agent which causes a brain disease in sheep known as scrapie. This group of viruses, often referred to as slow viruses. Are receiving a great deal of attention as they may be involved with a host of major degenerative human diseases. In summary, we see that virology has passed through three main phases. Firstly, it has been definitely proved that viruses are causative agents of many disease. Secondly, the particulate and molecular nature of viruses has been demonstrated. Finally, the principal event of virus replication and a detailed outline of their chemical structure have been established. During this time, virology has matured into a scientific discipline that has not only a sound fundamental basis but also important medical, agriculture and perhaps sociological application. From the quiet clinical search for the causative agent of disease during the early part of the century, we have recently passed through a more noisy, through exciting phase, illuminating the fundamental basis of biology and we can now approach the future from the firm foundation and understanding of the chemistry of viruses. The structural component Viruses are divided into two major groups or sub-phyla based on the type of nucleic acid present. Viruses particles contain only one molecule of nucleic acid; however, there are important exception to this rule and some viruses, such as influenza, contain a number of molecules of RNA, and in these the genome is said to be segmented. In all cases the nucleic acid is of one type, DNA or RNA, and is surrounded by an protein coat of shell. The protein coat is composed of a large number of structural units which consist of single polypeptide chains or a complex of different chains. Often these structural units are clustered in specific groups (Capsomeres) which are usually large enough to be resolved in electron micrographs. A good example of this is seen in the common human wart virus (papilloma virus group) shown in fig.

A virus particles consist of an ordered complex of viral nucleic acid and structure protein subunits. The entire structure is called the nucleocapsid. The term capsid is reserved for the protein shell which lacks the nucleic acid. In some large RNA viruses, the nucleocapsid is enshrouded by an envelope which is rich in glycoproteins and lipoprotein. These envelopes have meny of the characteristics of cell membranes and are derived from the host-cell membrane during the final stages of virus maturation.

The size and shape of viruses We have seen that the chief characteristic of viruses is their small size. They lie in the range between the largest protein molecules, such as haemocyanin (30 nm), and the smallest independent cells (300 X 500 nm) . Fig. 2.2 illustrates the range of sizes and shapes of some common viruses. The crystallisation of TMV and poliovirus emphasised the molecular characteristics of viruses and there is clearly a case for considering them as macromolecules.

It is very unlikely, however, that the larger viruses, especially those possessing envelopes, Will ever by crystallised since there is evidence that both the shape and size of these particles can very. It has been generally accepted therefore that the term particles weight is used rather then molecular weight and that the latter is confined to the weight of the individual covalently bonded components.

One of the most striking features of viruses is the manner in which the structural units are arranged and this feature has been used as a major criterion for classification. Small RNA viruses and most of the DNA viruses have a spherical shape in which the protein units are arrange in the form of an icosahedron. On the other hand, the nucleocapsids of some of the RNA viruses and of many plant viruses are elongated and the sub-units are arranged in a helical fashion. The electron micrographs shown in fig. 2.3 illustrate the range of particles shapes that are found among some common viruses.

Viruses consist of a nucleic acid (either DNA or RNA) associated with proteins encoded by the nucleic acid. The virus may also have a lipid bilayer membrane (or envelope) but this is acquired from the host cell, usually by budding through a host cell membrane. If a membrane is present, it must contain one or more viral proteins to act as ligands for receptors on the host cell. Many viruses encode a few structural proteins (those that make up the mature virus particle (or virion)) and perhaps an enzyme that participates in the replication of the viral genome. Other viruses can encode many more proteins, most of which do not end up in the mature virus but participate in some way in viral replication. Herpes virus is one of the more complicated viruses and has 90 genes. Since many viruses make few or no enzymes, they are dependent on host cell enzymes to produce more virus particles. Thus, virus structure and replication are fundamentally different from those of cellular organisms. Viral dependence on

the host cell for various aspects of the growth cycle has complicated the development of drugs since most drugs will inhibit cell growth as well as viral multiplication (because the same cell enzymes are used). Since a major reason to study viral metabolism is to find drugs that selectively inhibit the multiplication of viruses, we need to know when the virus uses its own proteins for part of its replication cycle - we can then try to develop drugs that inhibit the viral proteins (especially viral enzymes) specifically. In contrast to viruses, the much larger bacteria (figure 1) carry out their own metabolic processes and code for their own enzymes. Even when catalyzing similar reactions, bacterial enzymes differ from their eukaryotic homologs and can therefore be targeted by specific antibiotics. Like viruses, some bacteria (such as mycoplasma, rickettsia and chlamydia) can enter the cytoplasm of eukaryotic cells and become parasites. These small intracellular bacteria nevertheless provide all of the enzymes that are necessary for replication. Thus, mechanisms for control of bacteria, including those with a parasitic lifestyle, are more easily developed than for viruses. Control measures for microorganisms include capitalizing on our knowledge of: Whether they Growth Division Their have Whether Whether they on by sensitivity both they have have artificial binary to DNA ribosomes muramic acid media fission antibiotics and RNA Bacteria Mycoplasma Rickettsia Chlamydia Viruses Yes Yes No No No Yes Yes Yes Yes No Yes Yes Yes Yes No Yes Yes Yes Yes No Yes No Yes No No Yes Yes Yes Yes No

* The arenavirus family (an RNA virus family) appears to package ribosomes 'accidentally'.

Viruses infect all major groups of organisms: vertebrates, invertebrates, plants, fungi, bacteria but some viruses have a broader host range than others; however, none can cross the eukaryotic/prokaryotic boundary. Factors that affect host range include:

whether the virus can get into the host cell; that is, does it have the correct attachment protein to bind to a receptor on the cell surface? For example, HIV is largely restricted to cells that have the CD4 antigen on their surface (such as T4 cells).

if the virus can enter the cell, whether the appropriate cellular machinery is available for the virus to replicate; for example, some DNA viruses can only replicate in dividing cells which have high enough levels of deoxyribonucleotides for viral DNA synthesis. if the virus can replicate, whether infectious virus can get out of the cell and spread the infection.

VIRUS STRUCTURE
Viruses range in size from 20 nanometers in diameter, such as the Parvoviridae, to several hundred nanometers in length in the case of the filoviridae (Figure 1 and 2). All viruses contain a nucleic acid genome (RNA or DNA) and a protective protein coat (called the capsid). The nucleic acid genome plus the protective protein coat is called the nucleocapsid which may have icosahedral, helical or complex symmetry. Viruses may or may not have an envelope. Enveloped viruses obtain their envelope by budding through a host cell membrane. In some cases, the virus buds through the plasma membrane but in other cases the envelope may be derived from internal cell membranes such as those of the Golgi body or the nucleus. Some viruses bud through specialized parts of the plasma membrane of the host cell; for example, Ebola virus associates with lipid rafts that are rich in sphingomyelin, cholesterol and glypiated proteins. Poxviruses are exceptional in that they wrap themselves in host cell membranes using a mechanism that is different from the usual budding process used by other viruses. Enveloped viruses do not necessarily have to kill their host cell in order to be released, since they can bud out of the cell - a process that is not necessarily lethal to the cell - hence some budding viruses can set up persistent infections. Enveloped viruses are readily infectious only if the envelope is intact (since the viral attachment proteins which recognize the host cell receptors are in the viral envelope). This means that agents that damage the envelope, such as alcohols and detergents, reduce infectivity. VIRION NUCLEOCAPSID STRUCTURES Icosahedral symmetry An icosahedron is a Platonic solid with twenty faces (figure 3A) and 5:3:2 rotational symmetry (figure 3B). There are six five-fold axes of symmetry through which the icosahedron can be rotated passing through the vertices, ten 3-fold axes of symmetry passing though each face and fifteen two-fold axes of symmetry passing through the edges (figure 3B). There are twelve corners or vertices and 5-fold symmetry around vertices (figure 3C). The capsid shell is made of repeating subunits of viral protein (There may be one kind of subunit or several, according to the virus). All faces of the icosahedron are identical.

The nucleic acid is packaged inside the capsid shell and protected from the environment by the capsid (figure 3D). Proteins associate into structural units (this is what we see in the electron microscope or when we start to disassociate a capsid), the structural units are known as capsomers. Capsomers may contain one or several kinds of polypeptide chain. Capsomers at the 12 corners have a 5fold symmetry and interact with 5 neighboring capsomers, and are thus known as pentons or pentamers (figure 3E). Larger viruses contain more capsomers; extra capsomers are arranged in a regular array on the faces of the icosahedrons. They have six neighbors and are called hexons or hexamers (figure 3F). The size of an icosahedron depends on the size and number of capsomers; there will always be 12 pentons (at each corner) but the number of hexons increases with size (figure 3H). A good example of an icosahedral virus is human adenovirus which contains the usual twelve pentons plus two hundred and forty hexons (figure 3G and I). The symmetrical formation of hexagonal arrays on a flat face occurs in many situations; for example, in the packing of test tubes in a box (figure 3J). It can also be seen in the packing of the subunits of herpes virus, an enveloped icosahedral virus. In figure 3K, the external membrane of herpes simplex has been removed to reveal the nucleocapsid. Although icosahedrons are flat-faced (as in figure 3A), viral icosahedrons are usually round as seen in figure 3K. A good example of a small round icosahedron is a normal soccer ball (figure 3L). A larger icosahedron is a geodesic dome (figure 3M).

A Icosahedron: 20 triangular faces

B 5:3:2 rotational symmetry

C Five fold symmetry at vertices

D Nucleic acid is packaged inside the capsid

Image Dr J.Y. Sgro -

E Capsomers at the 12 corners have a 5-fold symmetry and interact with 5 neighboring capsomers, and are thus known as pentons (or pentamers).

F-i
Larger viruses contain more capsomers, extra capsomers are arranged in a regular array on the faces of the icosahedrons, these often have six neighbors and are called hexons

F-ii
Herpes nucleocapsid showing pentons at the vertices of the icosahedron
Zhou et al. Baylor College of Medicine Reference: Z. H. Zhou, B.V.V Prasad, J. Jakana, F.R. Rixon, W. Chiu Baylor College of Medicine, Journal of Molecular Biology

G Adenovirus symmetry

H Components of an icosahedral capsid

I Human adenovirus seen by negative staining 1995

Cape Town.

Dr Linda Stannard, University of

J Packing of uniform circular objects in a hexagonal array

K 3-D computer reconstruction from cryo-electron micrographs of herpes simplex virus capsids.
Rotating image.
National Institutes of Health

L The icosahedral shape of a soccer ball. Note that the ball consists of penton subunits (black) and hexon subunits (white)

M Geodesic dome Helical symmetry

Protein subunits can interact with each other and with the nucleic acid to form a coiled, ribbon like structure. The best studied virus with helical symmetry is the non-enveloped plant virus, tobacco mosaic virus (Figure 4 A-E). The helical nature of this virus is quite clear in negative staining electron micrographs since the virus forms a rigid rod-like structure. In enveloped, helically symmetrical viruses (e.g. influenza virus, rabies virus), the capsid is more flexible (and longer) and appears in negative stains rather like a telephone cord (figure 4H, I). Complex symmetry These are regular structures, but the nature of the symmetry is not fully understood. Examples include the poxviruses (Figure 5).

FIVE BASIC STRUCTURAL FORMS OF VIRUSES IN NATURE

Naked icosahedral e.g. poliovirus, adenovirus, hepatitis A virus Naked helical e.g. tobacco mosaic virus. So far no human viruses with this structure are known Enveloped icosahedral e.g. herpes virus, yellow fever virus, rubella virus Enveloped helical e.g. rabies virus, influenza virus, parainfluenza virus, mumps virus, measles virus Complex e.g. poxvirus

(Figure 6)

UNCONVENTIONAL AGENTS
There are also the 'unconventional agents' sometimes known as 'unconventional viruses' or 'atypical viruses' - Up to now, the main kinds that have been studied are viroids and prions. VIROIDS Viroids contain RNA only. They are small (less than 400 nucleotides), single stranded, circular RNAs. The RNAs are not packaged, do not appear to code for any proteins, and so far have only been shown to be associated with plant disease. However, there are some suggestions that somewhat similar agents may possibly be involved in some human diseases. Hepatitis delta virus At present, the only known human disease agent to resemble viroids is hepatitis delta virus (HDV). In some ways HDV (also called hepatitis delta agent) appears to be intermediate between 'classical viruses' and viroids. HDV has a very small RNA genome (~1700

nucleotides) compared to most viruses, although it is somewhat larger than viroids. However, features of HDV's nucleic acid sequence and structure are similar to some viroids. HDV differs from viroids in that it codes for a protein (various forms of the hepatitis delta antigen). Unlike the viroids, it is packaged. However, it differs from true viruses in that it does not code for its own attachment protein. The RNA is encapsidated by the hepatitis delta antigen, and HDV acts as a parasite on the unrelated hepatitis B virus (HBV), using HBV envelopes containing the hepatitis B attachment protein (HBsAg). PRIONS Prions contain protein only (although this is somewhat controversial). They are small, proteinaceous particles and there is controversy as to whether they contain any nucleic acid, but if there is any, there is very little, and almost certainly not enough to code for protein: Examples of prion-caused human diseases are Kuru, Creutzfeldt-Jakob disease and Gerstmann-Straussler syndrome. Prions also cause scrapie in sheep.

Virus classification Problems of nomenclature and classification are often a stumbling block to students who wish to bridge the gap between the physical and biological disciplines. In this section the principles involved in a modern approach to classification of viruses will be outlined. Historically, many viruses were named after the description of the disease they caused. Hence the agent causing foot-and-mouth disease was called foot-and-mouth disease virus (FMDV). Similarly, TMV is a direct description of the pathogenic effect that the virus has on tobacco plants. The naming of many other plant viruses has followed this principle: sugar beet curly top virus (SBCTV), cranberry false blossom virus (CFBV) and potato witchs broom virus (PWBV). Traditional or trivial names of human diseases, followed by the word virus, are commonly used, such as measles, mumps, smallpox, influenza and rabies. Still others have been named after their discoverer , for example Rous sarcoma virus (RSV), or locality of occurrence or discovery, such as Fiji disease, Semliki Forest disease, Newcastle disease. However, the student should not let this apparent state of anarchy deter him, since the situation has arisen mainly because the true nature of viruses has been defined only relatively. The chief obstacle in classifying viruses has been the assumption the viruses are organisms and various attempts have been made to classify them on the Linnean binomial system analogous to the schemes used with plants and animals. Here, however, the purpose of classification is t group together into meaningful categories the organisms that are closely related Although Linnaeus developed this method of classification about 120 years before Darwin formulated the theory of evolution, the schemes does reveal evolutionary and phylogenetic relationships. At present, there is no reason to believe that viruses from a single group of organisms, all members of which have a common ancestor, and virologists have had to find another framework on which to develop a logical and scientifically sound classification. In contrast, the classification and nomenclature of chemicals has been based generally on structure. Elements are classified according to electronic configuration in the Period Tables.

At a slightly higher level of chemical complexity, carbon compounds are classified named on the basis of structural and functional groups.

and

The discovery that viruses are particular introduced the possibility of classifying them on their chemical, physical and structural characteristics rather then of their biological properties alone. The international Committee for the Nomenclature of Viruses favours a scheme in which all viruses with similar chemical and structural chemical and structural properties are grouped together regardless of whether they infect plants, animals or bacteria. They also suggest that a Latinised binomial system to used in accordance with the systematics of other organisms. However, viruses failed in this method of classification and taxonomy. Hence early virologists proposed a method for viral taxonomy and nomenclature which was then accepted internationally. Based on this, viruses were classed according to their structurewhether or not they possess envelopes, symmetry, whether is a DNA or RNA virus. Viruses were then classified into classes which takes the suffix virales, farmilies takes the suffix viridae, sub-family takes the suffix virinae while both the genus and species takes the suffix virus respectively.

INTERNATIONAL CLASSIFICATION OF VIRUSES Primary characteristics used in classification Viruses are classified according to the nature of their genome and their structure VIRAL CLASSIFICATION RNA or DNA single-stranded or doublestranded non-segmented or segmented

Nucleic acid

linear or circular if genome is single stranded RNA, can it function as mRNA? whether genome is diploid (such as in retroviruses) symmetry (icosahedral, helical, complex)

Virion structure

enveloped or not enveloped number of capsomers

CULTIVATION OF VIRUSES Viruses are obligate pathogen that cannot be cultivated on synthetic media as is done for bacteria. They require a living system in order to multiply. In the laboratory such living systems could be in the form laboratory animals, embryos (Chicken embyonated eggs is most commonly used), cells could be cultured and maintained in the laboratory for the purpose of propagating viruses.

Laboratory animals are inoculated with the virus following different routes which could be intradermally, intraperitoneally depending on the virus. Laboratory animals used include suckling mice, mice, rats, guinea pigs, monkeys etc. Then the animals are observed for the symptoms of the disease caused by the virus, some viruses may cause the animal to go comatous while others may kill the animal. However, 50% of the laboratory animals used in cultivating the virus must show these symptoms and this is called the lethal dose 50 (LD 50) LD50 of any virus can now be calculated mathematically. It should be noted that the symptom a virus will show is specific to the virus but some viruses will have similar symptoms.

For the fact the cumbersome nature of reading results from laboratory animals as well as their maintenance the use of cultivating viruses in chickens embronated eggs begin to gain ground. Different embryos have been used in the past but It was discovered that chickens or hens embryos are

easily maintained in the laboratory. The inoculation of hens embronated eggs follows different route depending on the type of virus and the viruses have different effects in different sites of the eggs. Viruses could be inoculated into chorionallantoinc membrane, allantoinc cavity, amniotic cavity or yolk sac. The site for different viruses could be as follows: Chorioallantoic mem_brane (CAM)

visible lesions called pocks. Each infectious virus particle forms one pock. e.g. Variola, Vaccinia virus _Allantoic cavity Influenza virus (vaccine production) & paramyxoviruses _Amniotic cavity primary isolation of Influenza virus _Yolk sac Chlamydia, Rickettsia & some viruses. The embryonated eggs are candled daily to see that to see that chicken embryo are inside.

An egg containing chickens embryo is exemplified either by the direct presence of the developed embryo or by observing for extensive veination.

Though this method is no longer popularly used in the laboratory because of the development of the tissue culture technique, it still has several advantages over the use of laboratory animals and other methods of viral cultivation which includes: Fertile chicken eggs provide a convenient, space-

saving incubator for many kinds of animal viruses. Different viruses can be injected into an egg at different sites and the egg can be easily observed for viral replication throughout the development of the chicken embryo. Eggs are incubated for 7 to 12 days before the inoculation of the virus.

Harvesting the virus is the process of obtaining the virus from the eggs after several days of incubation at 37oC. The use of eggs in the cultivation of viruses is still very important in bulk virus production as in antigen and vaccine production.

The use of the tissue culture technique is now widely used for the propagation of viruses because of the ease in the maintenance of cell lines in the laboratory. Organs and tissues are treated with trypsin to separate the cells. The cells are now provided with adequate growth medium in specialised bottles called the tissue culture bottles and incubated. As the cells grow they form a confluent sheath of cells called a monolayer. Monolayers are formed when the cells reach the body of the bottle and stop growing therefore resulting in only one sheath of cells in a process called the contact inhibition. Cancerous cells have lost the contact inhibition and therefore cannot form the monolayers instead form multilayers.

PURIFICATION OF VIRUSES Preparation of virus harvests Many of the biological characteristic of viruses can be determined on preparations which contain substantial amounts of host-cells debris and products. Analysis of properties such as infectivity, haemagglutination, serotype and virulence can be done directly of fluids isolated from infected tissues. For chemical analysis, however, it is essential to isolate the virus practices in a pure state. As we have seen, bacterial and animal-cell cultures are convenient sources of susceptible cells and initial concentration of virus, the intact in the range 10 5 -1010 p.f.u./ml can often be obtained. With plant viruses, the intact plant can be a useful reservoir of virus. Often, the leaves retain the newly formed virus particles and a suitable harvest can obtained by grinding of homogenising them in aqueous buffers. In contract, the use of intact

animals for the preparation of pure virus is of little value, since the intact animals for the preparation of pure virus is of little value, since the problems of purification are considerably increased. Some animals viruses grow well in hens eggs and these are used extensively in the study of influenza virus. In general, a virus particles and other virus products. The essential problem of purification is to remove all the host-cell material without damaging or inactivating the virus. The problem is immense in quantitative terms. Since the amount of cellular material is often many thousands of times greater than the actual mass of virus particles. Also, the chemical natures of both the cell products and virus particles are very similar. Purification schemes generally depend on two main characteristics. First, viruses behave as through they were very large proteins and therefore the techniques used for the purification of proteins are of value. Secondly, viruses often possess a highly defined size, shape and density and therefore techniques which can effect a separation of the basis of these properties are important.

Differential centrifugation Viruses are smaller than most sub-cellular organelles and the first step in purification is generally centrifugation in order to remove the bulk of cellular debris, including nuclei, mitochondria and cell membrane fragment. Some viruses such as TMV can be highly purified by repeated cycles of slow (500-10000 r.p.m) and high (50000-60000 r.p.m) speed centrifugation. For most animals viruses, however, a combination of several methods is required before an acceptable degree of purity is achieved.

Precipitation procedures Salting out by ammonium sulphated has long been used in the isolation of proteins and enzymes and has had wide application in the initial stages of virus purification. The production of even milligram quantities of most viruses usually involves an initial volume of a few litres of virus harvest and precipitation procedures can readily effect a rapid means of concentration. Recently, zinc acetate has become a popular precipitant of zinc hydroxide forms which efficiently co-precipitates virus and other protein present. This precipitate readily dissolves in a buffer containing ethylenediamine NNNN-tetra-acetic acids. The techniques has been successfully applied as a concentration step in the purification of rabies virus and enterovirueses.

Gel filtration After concentration of the virus suspension to a convenient volume, the next steps depend on more refined techniques for separate in molecules of different sizes and shapes. The techniques of gel filtration or exclusion chromatography has been used exclusion

chromatography has been used extensively in the separation of many virus particles. The commercially available Sepharose gels have well defined pore sizes which only allow molecules below a certain size to penetrate the interior of the gel beads. Large particles are excluded and therefore pass rapidly through a column of gel. A wide range of gel is available which allows separation of particles in the molecular weigh range 10 2-107 . Sepharose 2B, which has an exclusion limit of 20X10 6 daltons, is suitable for viral studies. Many viruses are not completely excluded from sepharose gels and hence gel filtration can be used for estimating the relative sizes of different virus particles. When gel filtration is used in purification procedures, it is advantageous to select a grade of gel which will not completely exclude the virus particle. In this way, it is possible to separate the virus from any large or smaller particles which are present. On the other hand, if a gel type is selected from which the virus is completely excluded, then no separation from contaminating larger particles will be effected. Gel filtration has been used in the purification of a bovine enterovirus and fig Sucrose-gradient sedimentation This techniques involves sedimentation of a sucrose whose viscosity, density and concentration increases in a linear fashion from the top to the bottom of the centrifuge tube. Centrifugation is generally carried out at approximately 30000-40000 r.p.m. and the gradient is then fractionated into small volumes by elution via a small hole pierced in the bottom of the tube as show in fig 4.2. if reference particles of known sedimentation coefficient (s) are available, fairly accurate estimation of the S value of unknown practices; they have a sedimentation coefficient of 80 S. Viruses have sedimentation coefficients between120 S and 100 S except for a few small viruses like brome grass masaic virus (86 S), broad bean mottle virus (84S) and the bacteriophages such as Q (84S) and f2 phage (80S). The sedimentation rate of particles depends not only of their molecular weights, but also on their shape and density and hence the techniques is extremely valuable in separating particles which have the same or similar sizes. Many viruses produce practices that do not contain the nucleic acid molecule but are the same sizes as the infectious virus. These non-infectious empty particles can be easily separated from the virus particles by sucrose-gradient sedimentation (see & 5.14) since they have vastly different S values.

Isophycnic centrifugation Solution of heavy metal salts such as caesium chloride or caesium sulphate will from linear density gradient when centrifuged for long period at high speeds. Since the exact nature of the gradients depends on the initial concentration of the salt and the rate of centrifugation the range of densities that can be established may be precisely controlled. In practice, the gradients are generated in small plastic tubes and centrifugation a hole is pierced in the bottom of the tube and the contents are collected is small volumes. The densities of the fractions can be determined by measuring the refractive index with a refractometer. If particles of different densities are suspended initially in the solution they will migrate and concentrate during centrifugation at position where their density equals that of the gradient. Viruses posses characteristic densities and the method is particularly useful for separating them from cellular contaminants. Simple viruses, such as picornaviruses which contain only nucleic acid and protein, generally have a density in the range 1.33-1.45 g/mcm 3 as shown in fig 4.3. viruses which possess an envelope have much lower densities in the region of

1.20g/cm3. For the latter type of viruses, potassium tartrate solution has become a popular medium for centrifugation as well as caesium chloride, since it does not cause breakdown of these more fragile viruses. The lipid- contained viruses can often from visible bands in density gradients even when only very small quantities of material are present, as shown in fig 4.4

Extraction by non-aqueous solvents As mentioned in 4.1, purification methods must not inactivate the virus particles. Consequently, organic solvents are of little value in the purification of viruses containing lipid, but are widely used with simpler viruses, such as poliovirus and foot-and-mouth disease virus. The most popular solvents are butanol and tri-fluoro-tri-chlorethena. The organic solvent not only removes the host-cell lipid components, but also denatures much of the hostcell protein which then collects as an aggregate at the interphase.

Treatment with detergents and enzymes Water- soluble detergents, such as sodium deoxycholate, sodium dodecyl sulphate, and Tween (fig. 4.5) have been used extensively in the purification of many types of viruses (see p.71) when selective removal of the lipid components can be effected, leaving the nucleocapsids intact. It must be established that detergents do not inactivate non enveloped viruses and that associated that labile virus particles which may be of interest are not lost. Purification can often be aided by treatment with deoxyribonuclease, ribunuclease, trypsin, peronase or papain. These enzymes may selectively degrade cellular nucleic acids and proteins without damaging the virus particles. Although the infectivity of some viruses is resistant of such enzyme treatment, it is always difficult to prove that minor structural features have not been altered.

A purification protocol It is seldom, if every, necessary of advisable to make use of all available methods in the purification of virus. The methods chosen will depend greatly on the individual characteristic of the virus being studied and on the particular requirements of the experiment. Although it is impossible to give a standard scheme for the purification of viruses, a few guide-lines can be formulated which typify the step- wise procedures now in common use. Step 1. The virus harvest is centrifuged at low speed to remove macroscopic cellular debris Step 2. The virus and remaining contaminants can be concentrated by precipitation procedures. Step 3. After suspension of the precipitate in a suitably small volume of buffer an impure virus pellet can be obtained by centrifugation at high speeds.

Step 4. Gel filtration can be used to separate the virus particles from both larger and smaller contaminating components. Step 5. Sedimentation on sucrose gradients can separate the virus from particles which may have the same size but possess different shapes and densities. Step 6. Isopycnic centrifugation can be used to separate particles of different densities Sept 7. The final purified virus is generally collected as a pellet by high speed centrifugation. Step 8. Crystallisation of the concentrated virus suspension would be an ideal final stage, but is very seldom carried out in practice except when required for specific purpose such as X-ray diffraction analysis. It is unlikely that many viruses will ever be crystallised owing to their pleomorphic and enveloped nature.

Criteria for purity The purity of a virus preparation is largely defined by empirical criteria such as, for example, the homogeneity of practices as seen in electron micrographs or the presence of single homogenous peaks isolated from velocity or density gradient. The preparation of an absolutely pure virus suspension is probably unattainable for a number of reasons. For example, in any population of virus particles only some are infectious even though they all appear to be physically similar. Again, usually more than one types of particles, such as precursors and by produced during virus multiplication and these impurities must be removed during purification procedures.

Hence purification procedures must perform two main functions. First they must remove the host-cell components which are present in the initial cell lysates; secondly, they must separated the viral components which differ in infectivity or chemical properties.

The successful removal of host-cell components can be monitored by a techniques involving the use of non-infected cells which have been grown in the presence of radioactive precursors such as PO4, [3 H] uridine or [14C]amino acids. In a typical experiment a homogenised suspension of these radioactive cells is added to a non-radioactive virus lysate and the virus is then purified. The presence of host-cell material can be detected readily by measuring the radioacticity and the purification scheme may be considered successful if the final purified virus is not radioactivities. Although this method has been successfully for picornaviruses, it is not satisfactory for enveloped viruses. As these viruse mature by a budding process it is of testing whether this happens is to label cells with [ 36 S] methionine prior to infection and to allow the virus mature by a budding process it is possible that they trap cellular materials into the virus structure. A method of testing whether this happens is to label cell with [ 35 ]

MODULE 2

STRATEGY OF VIRAL INFECTION

Adsorption The first step in infection of a cell is attachment to the cell surface. Attachment is via ionic interactions which are temperature-independent. The viral attachment protein recognizes specific receptors, which may be protein, carbohydrate or lipid, on the outside of the cell. Cells without the appropriate receptors are not susceptible to the virus. Penetration The virus enters the cell in a variety of ways according to the nature of the virus. Enveloped viruses (A) Entry by fusing with the plasma membrane. Some enveloped viruses fuse directly with the plasma membrane. Thus, the internal components of the virion are immediately delivered to the cytoplasm of the cell (figure 1). (B) Entry via endosomes at the cell surface (figure 2) Some enveloped viruses require an acid pH for fusion to occur and are unable to fuse directly with the plasma membrane. These viruses are taken up by invagination of the membrane into endosomes. As the endosomes become acidified, the latent fusion activity of the virus proteins becomes activated by the fall in pH and the virion membrane fuses with the endosome membrane. This results in delivery of the internal components of the virus to the cytoplasm of the cell Non-enveloped viruses

Non-enveloped viruses may cross the plasma membrane directly or may be taken up into endosomes. They then cross (or destroy) the endosomal membrane. Uncoating Nucleic acid has to be sufficiently uncoated that virus replication can begin at this stage. When the nucleic acid is uncoated, infectious virus particles cannot be recovered from the cell - this is the start of the ECLIPSE phase - which lasts until new infectious virions are made. GENOME REPLICATION & GENE EXPRESSION The replication strategy of the virus depends on the nature of its genome. Viruses can be classified into seven (arbitrary) groups: I: Double-stranded DNA (Adenoviruses; Herpesviruses; Poxviruses, etc) II: Single-stranded (+)sense DNA (Parvoviruses) III: Double-stranded RNA (Reoviruses; Birnaviruses) IV: Single-stranded (+)sense RNA (Picornaviruses; Togaviruses, etc) V: Single-stranded (-)sense RNA (Orthomyxoviruses, Rhabdoviruses, etc) VI: Single-stranded (+)sense RNA with DNA intermediate in life-cycle (Retroviruses) VII: Double-stranded DNA with RNA intermediate (Hepadnaviruses) Gene expression Control of viral replication is achieved by tight regulation of gene expression. The methods used depend on nature of the virus genome/replication strategy, e.g: Segmented genomes are usually transcribed to produce monocistronic mRNAs. One advantage of monocistronic mRNAs is that various proteins can be produced in different amounts, rather than in a constant ratio. Non-segmented genomes tend to produce polycistronic mRNA, which is translated to form a polyprotein, processed by proteolytic cleavage to form the mature gene products. To utilize the cellular machinery, viral mRNAs must contain control signals which are recognized by the cell, e.g. ribosome-binding sites, splice signals, polyadenylation signals. Similarly, some DNA viruses, e.g. Papovaviruses, encode a protein which binds to the origin of replication and stimulates cellular DNA polymerase to replicate the virus genome. "Intermediate" viruses, e.g. Adenoviruses, encode their own DNA polymerase, but are still dependent on other cellular factors for replication. "Complex" viruses, e.g. Herpesviruses, encode a large number of proteins involved in DNA synthesis, and are largely independent of the cellular machinery. (Animated replication of Herpex Simplex 5) ASSEMBLY Involves the assembly of all the components necessary for the formation of the mature virion at a particular site in the cell. During this process, the basic structure of the virus is formed. The site of assembly varies for different viruses, e.g: Picornaviruses, Poxviruses, Reoviruses In the cytoplasm. Adenoviruses, Papovaviruses, Parvoviruses - In the nucleus. Retroviruses On the inner surface of the cell membrane. 6) RELEASE For lytic viruses (most non-enveloped viruses), release is a simple process - the cell breaks open and releases the virus. Enveloped viruses acquire the lipid membrane as the virus buds out through the cell membrane. Virion envelope proteins are picked up during this process as the virus is extruded. Budding may or may not kill the cell, but is controlled by the virus - the physical interaction of the capsid proteins on the inner surface of the cell membrane forces the particle out through the membrane:

7) MATURATION The stage of the life-cycle at which the virus becomes infectious. Usually involves structural changes in the particle, often resulting from specific cleavage of capsid proteins to form the mature products, which frequently leads to a conformational change in the capsid, or the condensation of nucleoproteins with the genome. For some viruses, assembly and maturation are inseparable, whereas for others, maturation may occur after the virus particle has left the cell.

EFFECT OF VIRUSES ON CELLS Many viruses inhibit host RNA, DNA or protein synthesis (or any combination of these). The mechanisms by which the virus does this vary widely. Cytopathic effect (CPE) The presence of the virus often gives rise to morphological changes in the host cell. Any detectable changes in the host cell due to infection are known as a cytopathic effect. Cytopathic effects (CPE) may consist of cell rounding, disorientation, swelling or shrinking, death, detachment from the surface, etc. Many viruses induce apoptosis (programmed cell death) in infected cells. This can be an important part of the host cell defense against a virus - cell death before the completion of the viral replication cycle may limit the number of progeny and the spread of infection. (Some viruses delay or prevent apoptosis - thus giving themselves a chance to replicate more virions.) Some viruses affect the regulation of expression of the host cell genes which this can have important results both for the virus's ability to grow, and in terms of the effect on the host cell. The cytopathic effects produced by different viruses depend on the virus and the cells on which it is grown. This can be used in the clinical virology laboratory to aid in identification of a virus isolate.

Assays for plaque-forming units The CPE effect can be used to quantitate infectious virus particles by the plaque-forming unit assay (figure 5). Cells are grown on a flat surface until they form a monolayer of cells covering a plastic bottle or dish. They are then infected with the virus. The liquid growth medium is replaced with a semi-solid one so that any virus particles produced as the result of an infection cannot move far from the site of their production. A plaque is produced when a virus particle infects a cell, replicates, and then kills that cell. Surrounding cells are infected by the newly replicated virus and they too are killed. This process may repeat several times. The cells are then stained with

a dye which stains only living cells. The dead cells in the plaque do not stain and appear as unstained areas on a colored background. Each plaque is the result of infection of one cell by one virus followed by replication and spreading of that virus. However, viruses that do not kill cells may not produce plaques. Assays for viruses Some methods (e.g. electron-microscopy) enable every virion to be counted but are not informative about infectivity. Other methods (e.g. hemagglutination) are a less sensitive measure of how much virus is present, but again are not informative about infectivity. Other methods, e.g. plaque assay, measure the number of infectious virus particles.

NUCLEAR DNA VIRUSES

PARVOVIRUS FAMILY Parvoviruses are very small (18to -25nm diameter), single stranded DNA viruses (parvum=small). They have an icosahedral capsid, and are non-enveloped. DNA replication occurs in the nucleus. Human parvovirus B-19 replicates in dividing cells primarily in erythrocyte progenitors in the bone marrow - and causes fifth disease (erythema infectiosum). This is usually a mild disease but the decreased production of red blood cells can be a problem in people with various types of severe hemolytic anemia

The Baltimore Classification The Baltimore system of virus classification provides a useful guide with regard to the various mechanisms of viral genome replication. The central theme here is that all viruses must generate positive strand mRNAs from their genomes, in order to produce proteins and replicate themselves. The precise mechanisms whereby this is achieved differ for each virus family. These various types of virus genomes can be broken down into seven fundamentally different groups, which obviously require different basic strategies for their replication. David Baltimore, who originated the scheme, has given his name to the so-called "Baltimore Classification" of virus genomes. By convention the top strand of coding DNA written in the 5' - 3' direction is + sense. mRNA sequence is also + sense.The replication strategy of

the virus depends on the nature of its genome. Viruses can be classified into seven (arbitrary) groups:

I: Double-stranded DNA (Adenoviruses; Herpesviruses; Poxviruses, etc)

Some replicate in the nucleus e.g adenoviruses using cellular proteins. Poxviruses replicate in the cytoplasm and make their own enzymes for nucleic acid replication.

II: Single-stranded (+)sense DNA (Parvoviruses)

Replication occurs in the nucleus, involving the formation of a (-)sense strand, which serves as a template for (+)strand RNA and DNA synthesis.

III: Double-stranded RNA (Reoviruses; Birnaviruses)

These viruses have segmented genomes. Each genome segment is transcribed separately to produce monocistronic mRNAs. IV: Single-stranded (+)sense RNA (Picornaviruses; Togaviruses, etc) a) Polycistronic mRNA e.g. Picornaviruses; Hepatitis A. Genome RNA = mRNA. Means naked RNA is infectious, no virion particle associated polymerase. Translation results in the formation of a polyprotein product, which is subsequently cleaved to form the mature proteins. b) Complex Transcription e.g. Togaviruses. Two or more rounds of translation are necessary to produce the genomic RNA.

V: Single-stranded (-)sense RNA (Orthomyxoviruses, Rhabdoviruses, etc)

Must have a virion particle RNA directed RNA polymerase. a) Segmented e.g. Orthomyxoviruses. First step in replication is transcription of the (-)sense RNA genome by the virion RNA-dependent RNA polymerase to produce monocistronic mRNAs, which also serve as the template for genome replication. b) Non-segmented e.g. Rhabdoviruses. Replication occurs as above and monocistronic mRNAs are produced.

VI: Single-stranded (+)sense RNA with DNA intermediate in life-cycle

(Retroviruses) Genome is (+)sense but unique among viruses in that it is DIPLOID, and does not serve as mRNA, but as a template for reverse transcription.

VII: Double-stranded DNA with RNA intermediate (Hepadnaviruses)

This group of viruses also relies on reverse transcription, but unlike the Retroviruses, this occurs inside the virus particle on maturation. On infection of a new cell, the first event to occur is repair of the gapped genome, followed by transcription.

MODULE 3

BACTERIOPHAGE

Bacterial viruses contain either a ssRNA, ssDNA, or a dsDNA genome (rarely a dsRNA genome). Among the ssRNA and ssDNA phage, the known examples all are positive stranded. The terminology "positive" and "negative" stranded viral genomes relates to whether the nucleic acid referred to can form base pair interactions with viral mRNA (which is defined as the positive strand). A positive strand ssRNA virus has a genome that acts as mRNA immediately after entering the cytoplasm of the infected host cell.

The morphology of the viral capsids can be filamentous, icosahedral, or prolate icosahedral with helical tails (Figs. 10.2, 10.4, 10.5). While viruses of eukaryotes can be either naked or enveloped (Fig. 10.3), bacteriophages are only naked (without membrane envelopes).

A typical lytic phage goes through a replication cycle generally described by Fig. 10.8 of the textbook, starting with the specific attachment of the virus to a distinct cell surface receptor. There is a tremendous specificity in the interaction between the virus and the host.

The example described in class of a ssRNA phage is MS2

MS2 is a small, icosahedral phage with a genome size of 3569 nucleotides, with 4 genes: A (maturation) protein, coat protein, replicase protein, and lysis protein.

Attachment of the phage during infection is at the sides of the F pilus of E. coli.

As the phage genome is a polycistronic mRNA molecule, the relative synthesis of the 4 phage proteins during infection is altered by elaborate translational control, via differential access of ribosome binding sites (RBS) to the host ribosome. The coat gene has the most readily accessible RBS on the phage RNA molecule, which acquires complex secondary structure in the cell. In contrast, the A (maturation) gene can only be translated during the replication of viral + strands (that is, the ribosome begins translation of the A gene while the + strand is in the process of being polymerized; see Fig. 19.2 and Slide 8 from handout). (I will not ask you about translation of viral lysis protein).

The MS2 replicase protein associates with 3 other host proteins to form a viral RNA-specific RNA polymerase. There is no DNA intermediate during MS2 replication. The MS2 replicase is an RNA-templated RNA polymerase and makes both positive and negative strands of viral RNA. The only role for viral RNA negative strands is to serve as template for synthesis of viral positive strands.

The example of a filamentous ssDNA phage is M13.

M13 binds to the tip of the F pilus on E. coli cells containing the F plasmid.

Unlike other phages described in class, progeny M13 phages are released from an infected cell without lyzing/killing the host cell. The phage capsids are assembled as the virus "buds" from the cell, allowing continued growth of the host (Fig. 19.5).

Since M13 is a positive strand ssDNA phage, there is no synthesis of viral proteins until after the synthesis of the viral negative strand. The replication of M13 is very similar to that of the phage X174.

The example of an iscosahedral ssDNA phage is X174.

circular, + ssDNA, 5368 nucleotides in length, encoding 10 genes (Fig. 19.3).

The virus attaches to LPS of the outer cell wall membrane of E. coli and closely related enteric bacteria.

The viral DNA enters the cell and is converted into a ds circular molecule by the host DNA synthesis machinery. Host topoisomerases supercoil the dsDNA, creating the replicative form I (RFI) molecule that is active for replication of viral + strands.

An early phage protein is CisA, which nicks the + strand of the viral RFI (at the origin of viral replication) and attaches to the 5' end of the viral DNA (forming an RFII). This leaves the 3'OH group of the + strand accessible to prime + strand DNA synthesis by rolling circle

replication (see textbook, Fig 19.4). After a complete + strand has been copied, CisA religates the DNA, creating a circular ssDNA + strand. Early in infection, the replication cycle produces several RFs; late in infection, the only product of replication is progeny + strands that are packaged into viral capsids.

X174 is also notable for containing overlapping genes, a strategy commonly used in viruses to maximize use of limited genome space. One section of genomic material can specify the production of multiple translational products by using different translational start sites within the same mRNA with different reading frames.

An example of a lytic dsDNA virus is T4. T4 contains linear dsDNA, approximately 170 kb in length. T4 genome is about 85% identical to T2 and T4 phage, with differences relating to receptor binding sites (T4 binds to LPS)

T4 overwhelms the host cell synthesis to enable its own proliferation (Fig. 10.15/slide 16): 1. inhibits host RNA synthesis via ADP ribosylation of host RNA pol sigma factor (inactivating the sigma factor); T4 makes its own sigma factor that associates with core RNA pol for T4-specific transcription. 2. T4 encodes several genes for nucleases that degrade the host cell DNA. T4 makes its own DNA pol, DNA ligase, etc. In addition, T4 DNA is modified so that the T4 DNA is resistant to the nucleases.

During T4 DNA replication, the newly synthesized phage DNA undergoes recombination, forming long concatamers (linear molecules of several genomes attached to one another; Fig. 10.13). The concatamers are processed into pieces each about 170 kb in length, representing one "headful" of DNA. This length is one genome plus about 5000 bp present at both ends. Individual pieces that are packaged into phage heads have different terminal repeats, but every piece has a complete genome plus the duplicated sequence at the ends. This arrangement is said to represent a "circular permutation" of the T4 genome.

In contrast, other dsDNA phages like T7 and lambda DNAs (which also form concatamers during replication) are always cleaved at a particular sequence in the genome, so that all the capsid-packaged linear dsDNA molecules are the same (see Fig 19.7).

A different example of a dsDNA virus is Lambda ( ).

While we could spend many days talking about lambda, our focus in M410 on lambda is on its ability to have two different sorts of interactions with a host cell: it can infect and carry out a lytic replication cycle or the lambda DNA can integrate into the host chromosome and form a lysogen (Fig. 10.16 and Fig. 10.18). Lysogens are reasonably stable associations between the integrated prophage (phage DNA) and the host bacterial chromosome; however, the lysogen can be induced, resulting in the lytic replication of more phage particles. Because of this capacity to make the lysis/lysogeny choice, lambda is a temperate phage (many known examples of temperate phages). In its simplest terms, the lysis or lysogeny choice is determined by the relative abundance of two lambda-encoded proteins in the cell: cI repressor and the Cro activator. If Cro predominates, there will be expression of the lytic lambda proteins (enabling phage DNA replication, expression of capsid components, etc); if cI predominates, a lysogen will form (and the expression of lytic phage replication genes will be repressed in the lysogen).

MODULE 4

Virus Immunology

Types of virus-specific antibodies Different types of viral preparations elicit the formation of different Abs;

Killed virus preparations elicit Abs predominantly directed against the surface of the virions. These Abs have neutralizing and HI activities against the virions as well as CF and precipitating activities against the Ags of the viral coat. Live virus preparations elicit antibodies against all the viral antigens, including both external and internal antigens. Immunization with internal components of the virions produces CF and precipitating Abs active only toward the Ags of these components. Immunization with peptides reproducing segments of virion proteins elicit Abs, the properties of which depend both on the protein and the specific sequences reproduced. Specificity of test methods

The Abs that react in the different tests may overlap though they may not be altogether identical. Neutralization is primarily caused by Ab molecules specific for the sites of the virion that are involved in the release of viral nucleic acid into the cell. CF usually involves additional surface or internal Ags. Neutralization probably requires molecules with a higher affinity for virions than do HI and CF. After viral infection, the titres of Abs to different components rise and fall with quite different time courses. Because of their high specificity, immunological methods can differentiate not only between viruses of different families but also between closely related viruses of the same family or subfamily. By these means, family Ags may be identified. Usually, antibodies detected by neutralization tend to be less cross-reactive and thus are useful in defining the immunological type. Whereas those detected by CF tend to be more cross-reactive and the useful in defining the family. By proper procedures, however, such as immunization with purified Ags, highly specific CF Abs can be prepared. The resolving power of Abs is maximized by the use of monoclonal Abs. Whereas all the methods for measuring viral antigens are needed for classifying a new isolate, the method of choice for diagnostic purposes is ELISA (high sensitivity and low cost.)

Cell-Mediated Immunity
Cytotoxic T lymphocytes: CMI is very important in localizing viral infections, in recovery, and in the pathogenesis of viral diseases. In experimental animals, primary CTLs reach maximal abundance about 6 days after a viral infection and then disappears as infection subsides. However, memory T cells persists and can be recognized by culturing spleen cells with virus-infected cells where within a few days, secondary CTLs appear in culture with much greater activity than in the initial response. Formation of CTLs is elicited by cellassociated Ags present at the cell surface, not only for enveloped viruses, but also for other viruses whose core or nonvirion proteins reach the cell surface. As in humoral immunity, type specific and group specific responses can be seen. Even noninfectious or inactivated viruses can elicit a cellular response because their envelopes fuse with the cell plasma membrane in the initial stage of viral penetration. Moreover, the virions themselves may also be able to elicit the response after absorbing to the macrophages. Both internal virion proteins and nonvirion proteins are often recognized by CTLs. An example is the nucleocapsid proteins of

enveloped viruses, fragments of which reach the cell surface by an unknown route and are recognized very efficiently, giving rise mainly to cross-reactive CTLs. Often, Abs to viral surface proteins do not block their interaction with CTLs, because the humoral and cellular responses recognize different epitopes. Antibody-dependent cell-mediated cytotoxicity: The K cells are the effector cells in ADCC. In vitro, these cells kill virus-infected cells sensitized by IgG from immune donors but not unsensitized targets. ADCC is very efficient in vitro against HSV or VZV infected cells, preventing the usual spread of the virus from infected to neighboring uninfected cells. Therefore, it may play a role in the defense against human infection with these viruses. K cells had been shown to mediate immunity to vaccinia infection rather than Tc cells. Natural Killer (NK) cells: In man, the principal NK cell is the large granular lymphocyte (LGL) which comprise 2-5% of peripheral blood lymphocytes. However, not all lytic cells are LGLs and not all LGLs are NK cells. There is overlap of the NK population with K cells. The Fc receptor of the NK cell is however, not involved in the lytic process. There are also mechanistic differences and K cell activity is less consistently augmented by interferon and other immune modulators. NK activity is subject to both positive and negative regulation in vivo and in vitro. Interferon gamma and IL-2 are potent inducers. Besides producing lysis, NK cells can produce alpha-interferon. The target molecules recognized but NK cells have not been defined but it appears that some determinants are ubiquitous whilst others have a more restricted distribution. An alternative suggestion is that NK cell susceptibility depends on the absence of normal cell surface antigens such as MHC molecules. The importance of NK cells in viral infection is partially understood. It had been shown that mice depleted of NK cells by treatment with Ab against asialo GM1 show an increased susceptibility to CMV.

Immune response of host to virus infections, emphasizing cellular responses

Francis A. Ennis, M.D. Director, Center for Infectious Disease and Vaccine Research A. T cell triggered and cytokine mediated immunopathology. We hypothesize that certain virus infections e.g. Dengue Hemorrhagic Fever (DHF: Immunopathogenesis, click icon), or the Hantavirus Pulmonary Syndrome (HPS) may be due to "over"responses of dengue or hantavirus specific T cells in certain high-responders, due to immune responses genes. These T cell responses are needed for clearance of virus-infected cells but marked inflammatory responses probably mediated by cytokines, cause endothelial cell leakness and shock (DHF) or pulmonary edema (HPS). We are defining CD4+ and CD8+ T cell epitopes, determining the Th1 and Th2 cytokine responses at the protein and m-RNA levels and by immunocytochemistry. TCR usage is determined, and correlations with disease phenotypes are studied. B. Failure to Eliminate Virus in HCV Infection Failure of immune response to eliminate HCV infection is very common and chronic progression liver disease frequently results. Is this due to a poor CD8+ T cell response in those individuals? We are also developing CD8+ T cell clones from the liver tissues and blood cells of patients with HCV infections. We will determine whether interferon therapy

which is the only known treatment, alters the HCV+ CD8+ CTL responses in the patients who respond to therapy or those that do not. We are also defining Th1, Th2 cytokine patterns in liver tissues by immunocytochemistry