MI – 308 Virology & Mycology Unit I Viruses: General 1.1 General characteristics and structural organization of virus 1.2 Cultivation of viruses A. Animal cultivation B. Cultivation in embryonated eggs C. In vitro culture: Cell line, primary and secondary cell lines, continuous cell lines, cytopathic effects D. Cultivation of bacteriophage 1.3 Enumeration of viruses: methods of enumeration of viruses 1.4 Classification of viruses: PCNV, ICNV and Cryptogram system of viral classification 1.5 Sub viral entities: viroids, virusoids, prions, introduction to persistent, latent and slow viruses, oncogenic viruses Early Development of Virology Virology has become a basic biological science around the middle of the century. The subject matter of virology, the viruses cannot be defined by the common sense criteria applied to animals or plants. Many definitions have been proposed: 1. Strictly intracellular and potentially pathogenic entities with an infectious phase and possessing only one type of nucleic acid, multiplying in the form of their genetic material, unable to grow and undergo binary fission, and devoid of a Lipmann system (ie. System of enzymes for energy production) – Lwoff (1957) 2. Elements of genetic material that can determine in the cells where they reproduce the biosynthesis of a specific apparatus for their own transfer into other cells – Luria (1959) 3. Virus are entities whose genomes are elements of nucleic acid that replicate inside living cells using the cellular synthetic machinery and causing the synthesis of specialized elements that can transfer the viral genome to other cells – Modified from Luria and Darnell (1967) Viruses Latin word virus, poison or venom Louis Pasteur used the term virus for any living infectious disease agent. Diseases caused by viruses have been recognized for thousands of years. Diseases caused by viruses like smallpox, yellow fever, potato leaf roll and tulip break have been known for centuries. Mayer (1886) demonstrated the transmissibility of mosaic disease of tobacco by mechanical inoculation with sap of infected plants. Dimitri Iwanowsky (1852) reported the transmission of tobacco mosaic by sap filtered through bacteria proof filter. Sheetal Pithva, Dept. of Microbiology, Government Science College, Gandhinagar Page 1 Beijerinck (1899) succeeded in proving the serial transmission of tobacco mosaic by bacteria free filter in which no microscopic organism could be detected. He described this causative agent as “contagiumvivumfluidum” Walter Reed (1900) showed yellow fever diseases virus transmitted by mosquito Beginning of 20th century viruses are different from bacteria, plant and humans. VilhelmEllermann and Oluf Bang reported leukemia could be transmitted between chickens by cell free filtrate and was probably caused by virus. Peyton Rous (1911) reported virus now known as Rous Sarcoma virus was responsible for malignant muscle tumor in chicken. Frederick Twort (1915) reported that bacteria also could be attacked by viruses. Felix d’ Herelle (1917) established decisively the existence of bacterial viruses. Schelsinger (1933) was the first to determine the composition of a virus. He showed that bacteriophage consists of only protein and DNA. Wendell Stanley (1935) crystallized the tobacco mosaic virus (TMV) and found to be largely or completely protein. Later on Frederick Bawden and Norman Pirie managed to separate the TMV virus particles into protein and nucleic acid. Thus by the late 1930s it was becoming clear that viruses are complexes of nucleic acids and proteins able to reproduce only in living cells. General Characteristics of Viruses Viruses are a unique group of infectious agents whose distinctiveness resides in their simple, acellular organization and patternof reproduction. A complete virus particle or virionconsists of one or more molecules of DNA or RNA enclosed in a coat of protein, and sometimes also in other layers. These additional layers may be very complex and contain carbohydrates, lipids, and additional proteins. Viruses can exist in two phases: extracellular and intracellular. Virions, in the extracellular phase, possess few if any enzymes and cannot reproduce independent of living cells. In the intracellular phase, viruses exist primarily as replicating nucleic acids that induce host metabolism to synthesize virion components; eventually complete virus particles or virions are released. In summary, viruses differ from living cells in at least threeways: Sheetal Pithva, Dept. of Microbiology, Government Science College, Gandhinagar Page 2 1. Their simple, acellular organization; 2. The presence of eitherDNA or RNA, but not both, in almost all virions (human cytomegalovirus has a DNA genome and four mRNAs) 3. Theirinability to reproduce independent of cells and carry out cell divisionas procaryotes and eucaryotes do. Although bacteria such as Chlamydia and rickettsia are obligatory intracellularparasites like viruses, they do not meet the first two criteria. The Structure of Viruses Virus morphology has been intensely studied over the pastdecades because of the importance of viruses and the realizationthat virus structure was simple enough to be understood. Progresshas come from the use of several different techniques: electron microscopy,X-ray diffraction, biochemical analysis, and immunology. Although our knowledge is incomplete due to the large numberof different viruses, the general nature of virus structure isbecoming clear. Virion Size Virions range in size from about 10 to 300 or 400 nm in diameter. The smallest viruses are a little larger than ribosomes,whereas the poxviruses, like vaccinia, are about the samesize as the smallest bacteria and can be seen in the light microscope. Most viruses, however, are too small to be visible in thelight microscope and must be viewed with the scanning and transmissionelectron microscopes. General Structural Properties All virions, even if they possess other constituents, are constructedaround a nucleocapsidcore (indeed, some viruses consistonly of a nucleocapsid). The nucleocapsid is composed of anucleic acid, either DNA or RNA, held within a protein coatcalled the capsid, which protects viral genetic material and aidsin its transfer between host cells. There are four general morphological types of capsids andvirion structure. Some capsids are icosahedral in shape. An icosahedron is aregular polyhedron with 20 equilateral triangular faces and12 vertices. These capsids appear sphericalwhen viewed at low power in the electron microscope. Other capsids are helical and shaped like hollow proteincylinders, which may be either rigid or flexible. Complex viruses have capsid symmetry that is neither purelyicosahedral nor helical. They maypossess tails and other structures (e.g., many bacteriophages)or have complex, multilayered walls surrounding the nucleicacid (e.g., poxviruses such as vaccinia). Sheetal Pithva, Dept. of Microbiology, Government Science College, Gandhinagar Page 3 Both helical and icosahedral capsids are large macromolecularstructures constructed from many copies of one or a fewtypes of protein subunits orprotomers. Probably the most importantadvantage of this design strategy is that the informationstored in viral genetic material is used with maximum efficiency. Many viruses have an envelope, an outer membranous layer surrounding the nucleocapsid. Enveloped viruses have a roughly spherical but somewhat variable shape even though their nucleocapsid can be either icosahedral or helical. 1. Helical Capsids Helical capsids are shaped much like hollow tubes with proteinwalls. The tobacco mosaic virus provides a well-studied exampleof helical capsid structure. A single type of protomerassociates together in a helical or spiral arrangement to produce along, rigid tube, 15 to 18 nm in diameter by 300 nm long. The RNAgenetic material is wound in a spiral and positioned toward the insideof the capsid where it lies within a groove formed by the proteinsubunits. Not all helical capsids are as rigid as the TMV capsid. Influenza virus RNAs are enclosed in thin, flexible helicalcapsids folded within an envelope. The size of a helical capsid is influenced by both its protomersand the nucleic acid enclosed within the capsid. The diameterof the capsid is a function of the size, shape, and interactionsof the protomers. The nucleic acid determines helical capsidlength because the capsid does not seem to extend much beyondthe end of the DNA or RNA. 2. Icosahedral Capsids The icosahedron is one of nature’s favorite shapes (the helix isprobably most popular). Viruses employ the icosahedral shapebecause it is the most efficient way to enclose a space. A fewgenes, sometimes only one, can code for proteins that self-assembleto form the capsid. In this way a small number of lineargenes can specify a large three-dimensional structure. Certain requirementsmust be met to construct an icosahedron. Hexagonspack together in planes and cannot enclose a space, and thereforepentagons must also be used. When icosahedral viruses are negatively stained and viewedin the transmission electron microscope, a complex icosahedralcapsid structure is revealed. Sheetal Pithva, Dept. of Microbiology, Government Science College, Gandhinagar Page 4 The capsids are constructedfrom ring- or knob-shaped units called capsomers, eachusually made of five or six protomers. Pentamers (pentons) havebfive subunits; hexamers (hexons) possess six. Pentamers are atthe vertices of the icosahedron, whereas