VIRUS

Infectious go from cell to cell, host to host

Obligate intracellular parasites need to
enter cells and replicate
Nucleic acid genome- DNA/RNA
RNA first nucleic acid on earth (evolution)
Protein coat derived from a host cell
Replicate by assembly of pre-formed
components into many particles
1. Attach to cells nucleic acid into cell
(cytoplasm/nucleus) mRNA
proteins new proteins (+ new
nucleic acids) integrate into viral
proteins
Susceptible cell functional receptor for
given virus; may or may not support viral
replication
Resistant cell no receptor; may or may
not be competent to support viral replication
Permissive cell- has capacity to replicate
virus; may or may not be susceptible
Susceptible + Permissive = REPLICATION OF
VIRUS PARTICLE
*all viruses must make mRNA that can be
translate by host ribosomes: they are
parasites not only in cell but also of the host
protein synthesis machinery
*genes are more diverse
VIRUSES ALIVE? Depends on definition of
virus.
Virus has two phases
1. Virion (extracellular) not alive,
nucleic acid + protein, cant
reproduce
2. Infected cell (intracellular)
alive, exists as replicating
nucleic acids
a) Tobacco mosaic virus 1st virus
discovered
b) Mimivirusc) Megavirus chilensis
d) Pandora virus- biggest virus
e) Yellow fever virus- 1st human virus
f) Rabies virus
g) Variola virus
h) Chicken Leukemia virus cell free
transmission (cancer)
i) Poliovirus
j) Rous sarcoma virus
k) Bacteriophages

l) Influenza virus
Filterable infectious agents (FIV) Contagium
vivum fluidum virus
Small enough to pass through a 0.2-micron
filter- key concept that distinguished virus
from other mcgs
Virus classification
nature and sequence of nucleic acid in
virion
symmetry of protein shell
+/- lipid membrane (envelope)
Dimensions of virion & capsid
Origins of viruses
1. Progressive/escape hypothesis
2. Regressive/reduction hypothesis
3. Virus-first hypothesis
Cell components: PRECURSOR
RNA virus genomes = eukaryotic
mRNA
Plasmids = cccDNA
(prokaryotic/eukaryotic)
Transposons = mobile genetic
elements (prokaryotic/eukaryotic)
Mechanisms of virus evolution
1. Mutations
2. Recombination
3. Reassortment
CULTIVATION
Virus Cultivation in animals
1. Whole animals
2. Embryonated eggs
3. Cell/tissue culture technique
Virus Cultivation of plant viruses
1. Plant tissue culture
2. Cultures of separated plant cells
3. Plant protoplast cultures
4. Whole plants: mechanical inoculation
of leaves
Virus Cultivation of bacterial viruses
1. Broth/agar cultures of young, actively
growing bacterial cells
Embryonated eggs eggs disease free &
flocks not vaccinated
Amniotic cavity inoculation virus
Allantoic cavity inoculation - vaccine
production
Choriallontoic membrane inoculation
lesions, quantitative assay

USES
Primary isolation &
identification

ADVANTAGES
Sterile

Maintenance of stock
cultures

(-) immunologic
factors

Vaccine production

Inexpensive,
availability

2. Cell/tissue culture method

MONOLAYERS
a. Primary cell culture tissue + trypsin
eg. foreskin; vaccine production
a. 20-30 times cell division die
b. more preferred
b. Immortalized cell line
grow forever because of aberrancies in
mitotic regulation
derived from tumors (usually
aneuploid)/
in vitro

PURIFICATION
Virus properties utilized:
Large size (relative to proteins)
Stable
(+) Surface proteins
Methods:
a. Preliminary step: [] of virus particles
Ppt with ammonium sulfate,
EtOH, or PEG (polyethylene
glycol)
b. Succeeding step: separation
Differential centrifugation
Density gradient centrifugation
Chromatography
Electrophoresis
1. Centrifugation
Separate particles
Produce aqueous suspension:
cell components + viruses

1. cytopathic effects (CPEs) looking at

infected cells, seeing them die;
morphological characteristics
necrosis of target cells
premature death of cells
syncytia formation of large
cell; multinucleate
inclusion bodies
transformation
lysis

a) Differential
- Separates particles different in
sedimentation constants
- Dependent on: particle size,
shape & density
b) Density gradient equilibrium
isopynic
- Separates particles different in
sedimentation rate (size) or
density
* small differences between
viruses
* particles float when
gradient density = buoyant
density
2. Precipitation
- ammonium sulfate purified by
precipitation w/ PEG
- collected by centrifugation

2. plaque formation dilutions; cell

destruction; trypan blue stains dead
cells; agar overlay on monolayer of
infected cells restrict viral diffusion
after lysis of infected cells

3. Removal of contaminants by denaturation

*stability: viruses > cell components
- heat/ pH change
- -denature contaminants + extract
lipids in prep = solvent treatment

3. immunofluorescence

4. Enzymatic digestion of cell constituents

- Resistance to enzyme attack: virions >
free nucleic acid & proteins
- Cellular proteins & nucleic acid
removed from virus prep (ribonuclease
& trypsin)

HeLa cell- Henrietta Lax

1st human cell line
PRESENCE OF VIRUS IN CELL CULTURE

Recombinant DNA technology

Vector: biological vehicle (plasmid/phage)
- transfect a bacterium

5. Chromatography
Separation of molecules based on diff in
STRUCTURE and COMPOSITION
Separation: interaction of 1+ solutes & 2
phases
PHASES:
Mobile phase: gas/liquid passes through a
column
Stationary phase: solid/liquid does not
move
- Surface adsorption, relative solubility,
charge
Types:
a. Gel filtration
- Separates protein based on size
- Small particles slower
(trapped in pores of beads)
- Larger particles faster (cant
penetrate pores, relegated only
to the interstices)
b. Ion exchange
- Purify viral proteins based on
overall charge
- Matrix/support: cellulose,
dextran, resin
- Addtn of salt + changes in pH
c. Affinity
- Differential affinity of solutes in
soln to stationary phase
- 1-step purification of target
molecule
- Specific ligand- immobilized on
a support, bind to target
molecule
6. Electrophoresis
Proteins + NA
Agarose/polyacrylamide
MW rate of movement
SDS-PAGE: estimating protein MW
PARTICLE COUNT
1. Direct count
- EM
- Non-culturable viruses
Virions of single type
[] prep
Known morphology of
virus
- Virus morphology

Unknown virus [] + known small

particles []
DISADVANTAGES: expensive
equipment & maintenance, low
sensitivity, requires experienced
observer

2. Indirect count
a. Hemagglutination assay
-aggregation of RBCs w/
hemagglutinating viruses
-measures HEMAGLUTTINATION
units
- 1 HA unit = min # of viruses
required to agglutinate RBCs
- relative estimates of titers
b. Measurement of enzyme activity
- Reverse transcriptase activity in
retroviruses
3. Serological assays
-antibody detect viral proteins
-viral proteins detect immune
responses
Antigen foreign; immune response
(immunogen)
Antibody- glycoprotein; bind to an
antigen (immunoglobulin)
a. Complement fixation test
COMPLEMENT: In normal serum,
enzymatic proteins interact to
combine Ag-Ab = lysis (when Ag is
intact w cell)
2 Ag-Ab pairs compete for limited
amt. of complement
1st pair = test system test
Purified virus (test Ag) +serum
(test Ab)
2nd pair = indicator system
sheep RBC(Ag) + antisheep
hemolysin (Ab)
b. Immunoblot
- Viral proteins using Ab +
indicator molecule
c. Immunofluorescence
- Viral proteins using Ab
(fluorescein isothiocyanate,
FITC)
- Direct, indirect, sandwich
d. Immunoprecipitation
- Specific Ag with +
contaminating proteins prep
e. ELISA

f.

Direct: Ag; Indirect: Ab

Enzyme: alkaline phosphatase,
horse radish peroxidase
- Substrate: p- nitrophenyl
phosphate
- Product- p-nitrophenol
Radioimmunoassay
- Competitive binding of
radioactively tagged Ag and
untagged Ag to an Ab
- Tag: isotope of iodine

4. Molecular techniques
PROBEa. Southern blotting - DNA
b. Northern blotting- RNA
c. Polymerase chain reaction (PCR)
- in vitro amplification of DNA
- synthesis of specific nucleotide
sequences from small amt. of DNA
- large amt of specific NA sequences
INFECTIOUS UNIT COUNT
1. FOCI assays
a. Plaque assay bacteriophages
Detection:
Staining
- trypan blue: dead; neutral red:
alive
Hemadsorption cells alive,
adsorb RBC
CPEs- disrupt monolayer
(syncytia/transformation)
Immunofluorescence- Ab labeled w/ fluorescent dye
- Foci of stained cells
b. Transformation assay infectious, noncytocidal transforming viruses
c. Local lesion assay plant viruses
d. Pock method
POCK: lesion on CAM of embryo after
virus infection
- Virus that are released from the
infected cells too slow to give
rise to secondary infectious
centers
2. ENDPOINT/QUANTAL assays
- # of infectious particles not
counted but measure the value
(ALL/NONE)
- ADVANTAGE: more accurate
than direct count, gives
infectious number
- DISADVANTAGE: costly,
variability with animals, difficult

to define endpoint, labor

intensive, not for routine use
CHARACTERIZATION OF VIRUSES
A. PHYSICOCHEMICAL PROPERTIES
1. Density proportion of NA in virus
2. Sedimentation/diffusion coefficient- mass,
density, shape of virus particle
3. UV absorption- purity
4. Electrophoretic mobility affected by size and
net charge
5. Stability to heat response to heat
6. Stability to lipid solvents- enveloped viruses
susceptible; decrease in virus infectivity
7. Stability to low pH- enteric virus stable at low
pH
B. VIRUS ULTRASTRUCTURE
1. Physical methods:
a) Ultrafiltration virus size
Molecular sieve, materials of known pore
size
b) Ultracentrifugation
Analytical
- sedimentation properties
- size
- shape
- density of specimen
- accurate determination of native MW
protein

Preparative
relative density
proportion of NA and proteins
derivation of approx. size of viruses
sucrose or CsCl gradient

c) Spectroscopy
- UV (NA content)
- Visible light (light scattering
properties)
d) Electrophoresis
-DNA, RNA, proteins (size/sequence
similarities)
e) X-ray diffraction
-virus structure at atomic level
f) Nuclear Magnetic Resonance
-atomic structure of all kinds of molecules
incl. proteins & NA
- small molecules (30-40,000 Da)
2. Chemical Methods
Over all composition of virus
Construction of particle
Show nature of NA
- structural analogs of
pyrimidines that interfere with
DNA synthesis

acridine orange staining &

Fuelgen rxn
- mild alkali treatment; cleaves
phosphodiester bonds in RNA
Type of capsid protein interaction:
-used to denature virus capsid: interaction
between components
a. electrostatic (+) ionic salt, alteration
of pH
b. hydrophobic- (+) reagents: urea
c. lipid components non-ionic
detergents/ organic solvents
3. Electron Microscopy detail
Transmission Electron Microscope (TEM)morphology & ultrastructure; estimation of
particle size

a. Negative staining virus (bright)

b. Shadow casting/Metal shadowing
opaque material vaporized; metal
scatters; shadow shape & height
c. Freeze etching
Scanning Electron Microscope (SEM)
surfaces of mcgs in detail
*Cryoelectron microscopy computer
tech
C. MOLECULAR BIOLOGY OF VIRUSES
1. Sanger dideoxy method: NA sequencing
2. Edman degradation method: Protein
sequencing