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General Characteristics
of Viruses
Michael Piatak, Jr, PhD
AIDS Vaccine Program
SAIC Frederick, Inc.
National Cancer Institute, Frederick
HIV !
Avian Flu !
SARS ! Ebola !
West Nile ! Smallpox !
Hepatitis A,B,C,D,E !
Polio ! HPV !
Viruses infect all living things
• Animal kingdom
• Plant kingdom
• Microbes
•Specificity/selectivity for host
Manifestations of Viral Infections
•
•
•
•
Asymptomatic
Acute viral syndrome (influenza, rhinovirus, etc)
Persistent viral syndrome (EBV)
Chronic infection
– Reactivating (HSV-1/2, VZV (chicken pox/shingles))
– Progressive (HBV, HCV, HIV)
• Cancer (EBV, HPV-16, HBV, HCV, KSHV)
• Death (HIV, et al)
• Acute death (smallpox, Ebola, SARS)
Viral pathogenesis --- disease
• Virus replication
• Targeted cells
• Host response
What is a virus?
What are the essential characteristics
of a virus?
• Physical
• Biological
What is a virus?
• Physical Characteristics
– Genetic Material
• Nucleic acid
– RNA (ssRNA, dsRNA, segmented)
– DNA (ssDNA, dsDNA)
– Protein coat (subunit structure)
• Nucleoprotein
• Capsid
–Capsomeres, Geometrical constraints
– Envelope (some)
HIV-1, SIV and Other Retroviruses
Rhabdoviruses (rabies)
Adenoviruses
Size of Viruses
What is a virus?
• Genetic material limited
• 10^3 to 10^5 bases long
– Codes for unique proteins
•
•
•
•
•
Polymerases – RNA replication
Kinases
Proteases
Structural proteins
Accessory proteins
Targets for antiviral agents - limited
What is a virus?
Biological Charateristics
• Viruses as parasites
• Parasites
– Not capable of independently
completing a full replication cycle
– Cellular parasites
– Subcellular “parasites”
• Viruses
• Viroids
• Prions
What is a virus?
• Are viruses alive?
• Characteristics of living organisms?
What is a virus?
Biological Characteristics
• Viral replication strategies
• Viral replication cycles
– Binding/entry
– Uncoating
– Genome replication (w/wo integration)
– Protein production
– Assembly
– Budding
• Host cell tropism (receptors)
• Lytic vs. non-lytic infection
Vaccinia
Poliovirus
Adenovirus
Herpesvirus
How to characterize and study
viruses
• Structural & Morphological: X-ray crystallography,
Electron microscopy
• Biochemical: Gel electrophoresis, filtration, metabolic
labeling, Enzymatic assays (Reverse Transcriptase,
Neuraminidase)
• Cell/Virus interaction: Hemagglutination, Titration, Focus
forming, Plaque assay, Syncytia, In vivo labeling
• Immunological: ELISA, Western blot, IFA, Plaquereduction neutralization
• Physical: Differential centrifugation, Solvent resistance
• Nucleic acid based: PCR, Sequencing, Hybridization, In
vivo labeling, etc…
• Host/Virus interaction: Host range, LD50, Vaccinationchallenge, Suckling mice, Epidemiology studies
Morphological Assays
• Electron microscopy
• Isolated virus or
infected cells
• Nonenveloped or
enveloped (alternate
method to determine if
an envelope is
present?)
• Core structure
Biochemical - Density Gradient Separation
Cell/virus –titrate infectious units
Uninfected
Infected
Plaque Assay
Plaque assays are usually over layered
with agarose
Syncytia Assay
Titration: TCID50 Assay
1
8/8 = 100%
10
8/8 = 100%
103
8/8 = 100%
104
8/8 = 100%
105
8/8 = 100%
106
8/8 = 100%
107
6/8 = 63% TCID50
108
2/8 = 25%
109
0/10 = 0%
1010
0/10 = 0%
1011
0/10 = 0%
1012
0/10 = 0%
Host/virus interactions – LD50
Metabolic Labeling
• Proteins: Radioactive amino acids,
[35S]Methionine
• Nucleic Acids: Radioactive
nucleotides, [32P]ATP
Immunological
• ELISA
• Western Blot
• Antibody Neutralization
ELISA – antigen capture of HIV-1 capsid protein
Color
light
p24
Western Blot Assays
SIV (gp32+gp41)
SIV (gp32)
HIV-1
Experimental
Mol. Wt. Std.s
SIV (gp32+gp41)
SIV (gp32)
HIV-1
Experimental
Mol. Wt. Std.s
HIV/SIV Example
gp120
gp41
< SIV anti-gp41
membrane
< HIV anti-gp41
SIV, anti-gp41
HIV-1, anti-gp41
Antibody recognizes
extracellular domain
Antibody recognizes
intracellular domain
Molecular Assays
• Is the genetic material isolated
from the virus infectious?
• Assay for viral genome
– RNA verses DNA
– Northern blot & RT/PCR, Southern
blot and PCR
• Sequence analysis
– Mutation analysis
Molecular Assays – Quantitative PCR
Plasma SIV RNA (Copy/Eq/mL)
SIV infection in Rhesus macaques
PMPA + FTC
Weeks
SIVsmE660
CD8 SIVsmE660
SIVmac239
Epidemiology
• Tracking prevalence of viruses in host
populations
Blood components and tissue samples
– Virus culture
• In vitro (tissue culture)
• In vivo (susceptible animal model)
– Molecular testing
• PCR methods
• Southern blot
– ELISA (enzyme linked immunosorbent assay)
• Assay for antigen (viral protein)
• Assay for antibody directed against virus
Acknowledgements
Jeff Lifson, MD
Director, AIDS Vaccine Program
Bruce Crise, PhD
AIDS Vaccine Program
SAIC/NCI-Frederick