Transcript Intro to Virology

Introduction to Virology
Bio 695
Spring 2007
Ashim Malhotra, PhD
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
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 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
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 cellsHelicalhollow 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
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.
TEM of Plant Viruses
Plant Viruses, spherical Bean Mosaic (top) and rod-shaped Tobacco
Mosaic (bottom).
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 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)
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 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
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
Virological Methods
•
•
•
•
•
•
•
•
EM
Complement Fixation assay
Haemagglutination inhibition test
ELISA
Single Radial Haemolysis
Immunofluorescense
Neutralization
Molecular methods