1. Introduction to Virology
Bio 695
Spring 2007
Ashim Malhotra, PhD

2. Introduction
Viruses are generally small, acellular entities
Possess only a single type of nucleic acid enclosed in a coat of protein, and sometimes in more complex layers
Must use the metabolic machinery of a living host in order to reproduce [obligate intracellular parasites ]
Many human diseases have a viral etiology
The study of viruses has contributed greatly to our knowledge of molecular biology

3. Brief History of Virology
Edward Jenner (1798)- smallpox vaccination
Louis Pasteur (1881) - first attenuated viral vaccine
C. The word virus, which is Latin for poison
D. Dimitri Ivanowski (1892) demonstrated that the causative agent of tobacco mosaic disease would pass through filters designed to remove bacteria
Martinus Beijerinck ( ) showed that the causative agent of tobacco mosaic disease was still infectious after filtration (i.e., capable of reproduction); he referred to it as a filterable virus
G. Walter Reed (1900) showed that yellow fever in humans was caused by a filterable virus and could be transmitted by a mosquito

4. Brief History of Virology
J. Frederick Twort (1915) first isolated viruses that would infect bacteria, but did not follow up on these observations
K. Felix d'Herelle (1917) firmly established the existence of viruses that infect bacteria, and devised a method for enumerating them; he also demonstrated that these viruses could reproduce only in live bacteria
L. W. M. Stanley (1935) crystallized the tobacco mosaic virus and showed that it was mostly (or completely) composed of protein
M. F. C. Bawden and N. W. Pirie (1935) separated the tobacco mosaic virus particles into protein and nucleic acid components

5. The Cultivation of Viruses
Inoculation of a living host
A. Animal viruses
1. Suitable host animals
2. Embryonated eggs
3. Tissue (cell) cultures-monolayers of animal cells
. Cell destruction can be localized if infected cells are covered with a layer of agar; the areas of localized cell destruction are called plaques
B. Bacteriophages (viruses that infect bacteria)
Cultivated in broth or agar cultures
Broth cultures- clear, while plaques form in agar cultures
C. Plant viruses can be cultivated in
1. Plant tissue cultures
2. Cultures of separated plant cells
3. Whole plants-may cause localized necrotic lesions or generalized symptoms of infection
4. Plant protoplast cultures

6. The Structure of Viruses
Virion size ranges from 10 nm to 400 nm
Nucleocapsid - the nucleic acid plus the surrounding capsid
Viral nucleocapsids are usually constructed without outside aid in a process called self-assembly
Capsid-protein coat that surrounds the genome, protects the viral genetic material, and aids in transfer between host cellsHelical-hollow tube with a protein wall shaped as a helix or spiral; may be either rigid or flexible
Icosahedral-regular polyhedron with 20 equilateral triangular faces and 12 vertices; appears spherical

7. Viral Symmetry Patterns

8. Viral Symmetry Patterns

9. Tobacco Mosaic Virus
TEM: The image was taken with a Philips CM12. The sample was prepared on a holey film (Quantifoil) and rotary shadowed with platinum / carbon (95/5 w%) under a low angle. The dark spot has its origin in imperfections of the foil.

10. TEM of Plant Viruses
Plant Viruses, spherical Bean Mosaic (top) and rod-shaped Tobacco Mosaic (bottom).

11. Adenovirus

12. Virus Purification
1. Differential centrifugation- separates according to size
2. Gradient centrifugation- separates according to density or to sedimentation rate (size and density), and is more sensitive to small differences between various viruses
3. Differential precipitation with ammonium sulfate or polyethylene glycol separates viruses from other components of the mixture
4. Denaturation and precipitation of contaminants with heat, pH, or even organic solvents can sometimes be used
5. Enzymatic degradation of cellular proteins and/or nucleic acids can sometimes be used because viruses tend to be more resistant to these types of treatment

13. Viral assays 1. Particle count a. Direct counts - EM
b. Indirect counts - hemagglutination (virus particles can cause red blood cells to clump together or agglutinate)
2. Infectious unit counts
a. Plaque assays - plating dilutions of virus particles on a lawn of host cells; clear zones result from viral damage to the cells; results are expressed as plaque-forming units (PFU)
b. Infectious dose assays - determining the smallest amount of virus needed to cause a measurable effect, usually on 50% of the exposed target units; results are expressed as infectious dose (ID50) or lethal dose (LD50)

14. Nucleic acids-genome
May be either RNA or DNA, single- or double-stranded, linear or circular
May have the common bases that occur in RNA or DNA, or genome may have one or more unusual bases (e.g., hydroxymethylcytosine instead of cytosine)
Viruses with single-stranded RNA (ssRNA) come in several arrangements:
Plus strand viruses- genomic RNA with the same sequence as the viral mRNA; the genomic RNAs may have other features (5’ cap, poly-A tail, etc.) common to mRNA, and may direct the synthesis of proteins immediately after entering the cell
Negative strand viruses have a genomic RNA complementary to the viral mRNA
Segmented genomes are those in which the virion contains more than one RNA molecule; each segment is unique and frequently encodes a single protein; in some viruses segments may be packaged into more than one virion structure

15. Principles of Virus Taxonomy
Grouped according to:
Nature of the host-animal, plant, bacterial, insect, fungal
Nucleic acid type
DNA or RNA
Single or double stranded
Molecular weight
Segmentation and the number of pieces of RNA
Capsid symmetry
Presence or absence of an envelope and ether sensitivity
Diameter of capsid (or nucleocapsid)
Number of capsomeres in icosahedral viruses
Immunological properties
Gene number and genomic map
Intracellular location of virus replication
Presence or absence of a DNA intermediate (ssRNA viruses)
Type of virus release
Disease caused by the virus, its special clinical features, or its mode of transmission

16. Viral envelopes and enzymes
Envelopes are membrane structures surrounding some (but not all) viruses
a. Lipids and carbohydrates are usually derived from the host membranes
b. Proteins are virus specific
c. Many have protruding glycoprotein spikes (peplomeres)
Enzymes-some viruses have capsid-specific enzymes; these may be required for virus attachment or entry into the host cell; many, however, are involved in viral nucleic acid replication
1. Poxviruses are large (200 to 400 nm) with an ovoid exterior shape
2. Some bacteriophages have complex, elaborate shapes composed of heads (icosahedral symmetry) coupled to tails (helical symmetry); the structure of the tail regions are particularly variable; such viruses are said to have binal symmetry

17. Virological Methods EM Complement Fixation assay
Haemagglutination inhibition test
ELISA
Single Radial Haemolysis
Immunofluorescense
Neutralization
Molecular methods