Physical Properties of Viruses PDF

The Physical properties of the viruses Prof. Dr. Sahar Abd El Rahman Head of Virology Department, Faculty of Veterinary Medicine, Mansoura University 08.10.. 2023 The physical properties of the viruses Stanly was the first to crystalize viruses 1- The shape of the virus....

The Physical properties of the viruses Prof. Dr. Sahar Abd El Rahman Head of Virology Department, Faculty of Veterinary Medicine, Mansoura University 08.10.. 2023 The physical properties of the viruses Stanly was the first to crystalize viruses 1- The shape of the virus. * The shape of virus particles is determined by the arrangement of the repeating subunits ,capsomers, that form the protein coat ,capsid, of the virus. 2- The Molecular Weight of the virus. 3- The size of the virus. The shape of the virus could be detected by Electron microscope  The shape of viruses 1- Brick shaped …………….. as Poxviridae. 2- Spherical shaped as ………………... Retroviridae, Paramyxovirdae, Orthomyxoviridae, Arenaviridae and Flaviviridae. 3- Icosahedrons shaped ……………as Adenoviridae, Papovaviridae and Parvoviridae. 4- Sperm (tadpole) shaped ………………… as Bacteriophages. 5- Bullet shaped …………………….. as Rhabdoviridae. 6- Slender rigid (Rod) shaped ………………. as plant viruses (TMV) 7- Polyhedral shaped ……………… as Birnaviridae and Reoviridae. Polymorphism is found between and within the families of enveloped viruses e.g. rhabdoviruses are bullet or cone or bacilli form shaped. Pox viruses are oval or brick shaped and influenza viruses are usually spherical, although filamentous or threads forms exist. This polymorphism may be due to lack of a matrix protein in viral envelope. or due to flexible shape due to weak bonds between proteins molecules of the capsid.  Capsomers Polymorphism The molecular weight of the virus ( mol. Wt.) Dalton is equal to the mass of one hydrogen atom= 1.67X10-24 g It could be detected by analytic ultracentrifuge depending on its sedimentation rate. The relationship between the nucleic acid (mol. Wt.) and the virus (mol. Wt.) The relationship between the DNA viral nucleic acid (mol. Wt.) and the RNA virus (mol. Wt.) DNA ………………… 1.5x106 to 160-200x106 RNA …………………. 1x106 to 15x106 Factors affecting sedimentation rate of the viruses: Centrifugation force Weight, Size, and density of the virus Length of centrifugation tube Time Viscosity  The size of the virus 1m = 103 mm (millimetres) 1mm= 103 µm (micrometres) 1µm = 103 nm (nanometres) 1nm= 10 Ao Sometimes in old texts Angstroms (Å) are used (the diameter of a hydrogen atom) 1nm = 10-6 mm. Viruses vary greatly in size ranging from 20-25nm for the smallest viruses, foot and Mouth disease virus (Picornaviridae), Parvoviridae and poliovirus (28 nm in diameter) to 300 x 200 nm for the large viruses, poxviridae,. The smallest viruses are little larger than ribosomes whereas the poxviruses (largest virus) are about the same size as the smallest bacteria and can be seen in the light microscope.  The size of the virus Methods used for measuring size of the virus: 1- Ultrafiltration. 2- Ultracentrifugation. 3- Electron Microscope 4- X- ray crystallography. 5- Chromatography and electrophoresis.  Ultrafiltration - The virus preparation is passed through series of membranes of known pore size, the approximate size of any virus can be measured by determining which membrane allowed the infective unit to pass and which hold it back. - Through collodion or cellulose acetate membranes of graded porosity (Millipore filters) made from alcohol, ether, acetone and acetic acids. Advantages of membranes: 1- non toxic 2- do not alter pH 3- do not adsorb large quantities of virus particles. APD= the mean of the filters diameters sizes The actual size of the virus= APDx0.64  Calculation of viral size by Ultrafiltration APD= the mean of the filters diameters sizes The actual size of the virus= APDx0.64 - e.g. filter pore size ( n m ) 10 20 30 50 70 - Detection of virus infectivity - - - - + - Average pore diameter ( A P D ) = 50 + 70 = 60 n m 2 ∴ Actual size of the virus = 60 x 0.64 = 38.40 nm  Ultracentrifugation Centrifugation that reach speeds of 40.000 r.p.m. or more are generally referred to as ultracentrifugation. If particles are suspended in a liquid, they will settle to the bottom at a rate that is proportionate to their size. In an ultra centrifugation, forces of more than 100.000 times gravity ,g , (30.000-33.000 r.p.m.) may be used to drive the particles to the bottom of the tube. The rate of sedimentation of virus particles depend upon its size , the density and viscosity of the suspending fluid. The relationship of sedimentation and size of virus particles is governed by Stokes law.  Electron Microscope As compared  Electron Microscope The resolution of the light microscope is only 1/100 that of the electron microscope (because EM magnificent the object from 10.000 to 100.000 times its original size). Also EM has a resolution power better than 5Ao as compared to 0.3µm (3000Ao) for light microscope. So it possible to study the morphology of viruses in details.  THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE FEATURE LIGHT MICROSCOPE ELECTRON MICROSCOPE Electromagnetic Visible light Electrons spectrum used 760nm (red) – 390nm app. 4nm Colours visible Monochrome Maximum app. 200nm 0.2nm resolving power Fine detail Maximum x1000 – x1500 x500 000 magnification Radiation Tungsten or quartz High voltage (50kV) source halogen lamp tungsten lamp Lenses Glass Magnets Interior Air-filled Vacuum Focussing Human eye (retina), fluorescent (TV) screen, screen photographic film photographic film Stains Water Soluble dyes Heavy metals Support Glass Slides Copper Grid  Methods of EM examination 1- Shadow casting method 2- Negative staining method 3- Positive staining method 4- Thin sectioning 5- Freeze drying (etching) techniques 6- Carbon replicas methods  Shadow This method consists casting of permitting method vapor of heavy metals (chromium, gold or platinum ) after heating to projected and directed obliquely at an angle onto a membrane to which derided viral particles are attached in vacuum chamber. The metal vapor coats it with an electron opaque layer of metal as will the film area immediately in front at the particles. The areas behind the particles, however, will be protected from the flow of a vaporized heavy metal, thus when the specimen is exposed to the beam of the electrons of the microscope, the electron flow will be interrupted by the heavy metal coating over the particle and to one side, giving the impression of an opaque body with an adjacent shadow. The latter representing the portion of the film that was not coated with metal i.e. absence of metal deposition behind a particle form a shadow whose features reveal the size and shape of the particle in all electron microscope preparations, it is necessary that the material be fixed and desiccated Shadowing casting method of electron microscope Shadowing casting method of electron microscope: a ) A sample of heavy metal is vaporized in a vacuum chamber. b) any object in the path will cast a shadow on the grid on which it is supported. c) A double-shadow virus in the electron microscope.d) An icosahedral model is placed in two light beams to show the equivalence of the shadows.  Negative staining method of Electron A marked improvement inMicroscope EM technique for the study of virus particle detail is the method of negative staining. The purified viral particles or distilled water lysates of viral infected tissues or cell culture are mixed with a solution of a salt highly opaque to electron usually sodium phosphotungestate. The mixture is then spread in a thin layer on a carbon membrane and dried. In negative staining, the stain do not bind chemically to the virus but merely surrounds it with a dried down, tightly fitting amorphous glass like layer of heavy metal salts. Under electron microscope, the electron beam is scattered by the heavy atoms but passes relatively uninhibited through the lighter atoms of the biological specimen (virus). The specimen appear as a translucent area an opaque background on the screen. The penetration of the salt between protruding parts of the virus reveals details of surface structure. The salt deposit around the virus particles become clearly visible in EM examination not the virus, so it is called Negative staining Advantages of this method are that large numbers of virus preparations may be examined quickly, good preservation is obtained and impure virus preparation may be used. Negative staining method Stain: Sodium Phosphotungestate  Positive staining method of Electron Microscope Certain components of viruses can be stained by salts that become selectively absorbed. Uranyl acetate stains the viral nucleic acid and other components. On the other hands ferritin is specifically adsorbed by viral proteins Positive staining can be combined with negative staining to improve the resolution. Deposition of these stains on the components of virus particles increase their resolution. This is basically achieved by enhancing the contrast of the particles. We can therefore say that these stains are directly staining the virus particles. For this reason, this method Positive staining method Stain: Uranyl acetate Methyl methacrylate Plaster of paris  Thin sectioning is helpful in studying the virus particles within the host cells (studying virus pathogenesis), virus in centrifugal pellets or in crystal structure studying of viruses. The specimens are fixed, dehydrated and embedded in a plastic such as methyl methacrylate. Thin sections are cut with an ultra microtome equipped with diamond knife , mounted ,negatively stained and examined under EM. Freeze drying (etching) techniques are used to prevent any possible distortion that may occurs to the particles while studying them. This method helps in getting a correct idea about the shape and size of the virus particles. The specimen is super cooled in a drop of water to form a solid block of ice and is placed in an evaporating unit under liquid nitrogen. The nitrogen is removed briefly to produce a sublimation or etching of the ice block , there by exposing the surface of the specimen which is then shadowed. Carbon replicas methods are similar to plaster of Paris moulds. These methods are prepared in many cases to bring out the surface characteristics of virus particles. Different methods of E.M and its uses Technique Nature of study Metal shadowing Morphology, surface details Negative staining surface details Positive staining internal structure. Thin sectioning internal structure Freeze drying size and shape ; morphology Carbon replica surface details  X- ray crystallography When x-rays are projected on the crystal from a suitable source, some rays pass through the crystal in straight lines while other diffracted due to the facing of molecules in the crystal and form cones. These cones were studied by crystallographers and by special mathematical formulas, they estimate the structure of the examined crystals. These phenomena were used in studying the morphology and structure of the virus particles, but x-rays diffraction requires pure virus preparations for analysis. The method has yielded important data on structure of the whole viruses, the proteins and nucleic acids of the viruses ( e-g. mosaic virus disease, poliovirus. So plant and animal viruses can be compared and constructed with this method. X- ray crystallography Thank you for your attention