Introduction to Virology

Bio 695

Spring 2007

Ashim Malhotra, PhD

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

Introduction

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 (1898-1900) 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

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

The Cultivation of Viruses

Inoculation of a living hostA. Animal viruses1. Suitable host animals2. Embryonated eggs3. 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 plaquesB. Bacteriophages (viruses that infect bacteria)Cultivated in broth or agar culturesBroth cultures- clear, while plaques form in agar culturesC. Plant viruses can be cultivated in1. Plant tissue cultures2. Cultures of separated plant cells3. Whole plants-may cause localized necrotic lesions or generalized symptoms of infection4. Plant protoplast cultures

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

Viral Symmetry Patterns

Viral Symmetry Patterns

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.

Tobacco Mosaic Virus

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

TEM of Plant Viruses

Adenovirus

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

Viral assays

1. Particle counta. Direct counts - EMb. 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)

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

Principles of Virus TaxonomyGrouped 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

Viral envelopes and enzymes

Envelopes are membrane structures surrounding some (but not all) virusesa. Lipids and carbohydrates are usually derived from the host membranesb. Proteins are virus specificc. 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 replication1. Poxviruses are large (200 to 400 nm) with an ovoid exterior shape2. 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

Virological Methods

• EM

• Complement Fixation assay

• Haemagglutination inhibition test

• ELISA

• Single Radial Haemolysis

• Immunofluorescense

• Neutralization

• Molecular methods