L1 Flashcards
1
Q
Dimitri Ivanofsky showed that Tobacco Mosaic Virus (TMV) was able to
A
pass through a filter while bacteria could not (1892)
First electron micrograph of TMV in 1939
2
Q
Martinus Beijerinck showed that the titer of TMV
A
increased after infecting a plant, proving TMV was not a toxin (1898)
3
Q
Bacteria viruses (bacteriophage) discovered by
A
Frederick W. Twort (1915) while trying to grow vaccinia virus
4
Q
Bacteriophage were instrumental in developing the field of
A
virology and expanding the field of biology
5
Q
Foot and mouth disease
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first animal virus) discovered (1898)
6
Q
Yellow fever virus
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(first human virus) discovered in 1901
7
Q
Viruses are
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obligate intracellular parasites
8
Q
Viruses are not
A
autopoietic
9
Q
Cellular origin
A
Proposes that viruses were once cellular components but over time they evolved separately.
10
Q
Autopoietic origin
A
Proposes that viruses, once autopoietic entities, became dependent on cells for replication.
11
Q
Attributes for Virus Classification
A
Virus particle structure Genome Replication features Serology Stability
12
Q
Nucleocapsid
A
RNA or DNA in a core that is protected by a protein coat (capsid)
Virus is defined by the nucleocapsid structure
13
Q
Nucleocapsid structural symmetry
A
helical
pelomorphic
Icosahedral
14
Q
Nucleocapsid is comprised of repeating
A
protein subunits called capsomeres
15
Q
Envelopes:
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virus-modified cellular membranes acquired upon exit from host
16
Q
Exposure to lipid solvents in the laboratory (e.g., alcohol, ether, acetone, Freon, etc.) renders enveloped viruses
A
noninfectious
17
Q
Enveloped viruses may have nucleocapsids with
A
different structures
18
Q
Smallest virus
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18 nm
19
Q
Largest
A
300 nm
20
Q
Virus Genome DNA
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double or single stranded
21
Q
Virus genome RNA
A
RNA Double stranded Single stranded Plus sense (+)ssRNA Minus sense (-)ssRNA (polarity) Ambisense
22
Q
Ambisense
A
Among the negative RNA viruses, ambisense RNA viruses or ‘ambisense viruses’ occupy a distinct niche. Ambisense viruses contain at least one ambisense RNA segment, i.e. an RNA that is in part of positive and in part of negative polarity.
23
Q
Virus Genome Structure
A
Linear Circular Segmented Diploid Gene arrangement can change this
24
Q
Virus Replication steps
A
Attachment Entry Transcription Translation Replication Assembly Release
25
Viral attachment is the
Binding of a virus receptor to a cellular receptor
26
Cellular receptors
Signaling molecules – induced cellular response to binding
Cell adhesion
Transport
27
Viral receptors usually do not
mimic cell receptor’s normal ligands
28
viral receptors typically are
Typically are spike like projections on particle surface
29
Viral creceptor smay require
a co-receptor- e.g., HIV (CD4, CXCR4)
30
Genetic engineering - can change receptor recognition
Integrin RGD sequence – used by Ad, engineered into lambdaphage
Pseudotyping particles – improve retroviral entry, VSV, Ebola, LCMV
31
Attachment is a major determinant of
virus tropism (host range)
32
Viruses infect essentially all known
forms of life
Specific host ranges, some can infect humans and animals (zoonosis)
Not shared across more divergent hosts (plants, bacteriae
33
Virus Replication – Entry - Pathways
Receptor rrrr3 mediated endocytosis
| Direct penetration of plasma membrane
34
Enveloped virus
| - viral entry
Membrane fusion
Best understood for influenza
HA protein – attachment & fusion
35
Receptor conformational change
| Receptor conformational change
Low pH
| Receptor induced
36
3Uncoating
involves nuclear replication nd cytoplasmic replication after uncoating
37
Nuclear replication
Genome and remaining protein coat (nucleocapsid) transported to the nuclear membrane
Delivery of genome to nucleus
38
Cytoplasmic replication
Release of the genome in cytoplasm
Transportation of the genome to intracellular site of replication
Many RNA viruses replicate in membrane associated complexes
39
dsRNA viruses never release their
genomic material from the entering particle
40
DNA viruses usually rely upon cellular
RNA polymerases
41
The genome of (+)ssRNA viruses can
serve as mRNA
| Production of new transcripts can occur later using a (-)ssRNA template
42
(-)ssRNA, dsRNA viruses must bring their own
polymerase into the cell
43
All viruses need the cell’s
ribosomes to produce protein – no exceptions
44
Viral protein production can be regulated at the
transcript (mRNA) level or translation level
45
Structural proteins are produced in
high quantities
46
Non-structural proteins are only seen inside the
infected cell
47
(+)ssRNA Genome serves as template for
translation
48
(+)ssRNA
| -Polymerase makes (-)ssRNA copy as
template for new genomes
49
(-)ssRNA
| Virus particle must include the
viral polymerase
50
-ssrna Polymerase makes
messenger RNA for translation
51
Viral Genome replicates through full-length
(+)ssRNA intermediate
52
dsRNA
Virus particle includes viral polymerase
53
dsRNA induces innate
immune response so genome stays inside particle
54
mRNA synthesized in particle and exported to
cytoplasm
55
mRNA serves as
(+) strand in virus genome, (-) strand synthesized during assembly
56
ssDNA and dsDNA must gain access to
nucleus
57
Poxviruses are an exception, virion contains the necessary
RNA polymerase and the genome encodes the DNA polymerase for replication
58
ssDNA and dsDNA Prepare the cell for
DNA replication
Growth phase, dNTP production, replication machinery
Ensure genome ends are copied
59
ssDNA and dsDNA Prepare the cell for
DNA replication
Growth phase, dNTP production, replication machinery
Ensure genome ends are copied
60
Virus assembly
Package new genomes into functional particles
Localize structural proteins to aid assembly
Cellular viral “factories”
61
Virus Genome contains
packaging signals
62
Adenovirus –
empty protein coat imports genome
63
Reovirus –
RNA packaged during capsid assembly
64
Retrovirus –
preassembly on a membrane
65
Lysis
Best known for bacteriophage
| Viral molecules that rupture cellular membrane
66
Weak Lysis
Depends on membrane breakdown after cell death
67
Budding (enveloped only)
Enveloped viruses use cell membrane as the outer coat of the virus particle
68
One step growth curve
Infect every cell at same time (MOI > 5)
| Every cell dies at end of infection
69
Phases
Eclipse
Exponential growth
Plateau
70
Eclipse:
attachment and uptake
71
Exponential growth:
replication and assembly
72
Plateau:
cell death
73
Virus Replication - Kinetics Useful to assess:
Mutations
Cell entry
Process design
74
Time for one step growth
Start of infection to beginning of plateau
Bacteriophage – 30 min
Vesicular stomatitis virus (VSV): 6 hours
Vaccinia: 24 hours
75
Productivity
Measure amplification
VSV – 1:1000
Vaccinia – 1:100
76
Initial discovery
Disease in a host
| Contaminant in cell culture
77
Confirmation
Purification of virus
Confirmation of disease
Animals, eggs or cell culture
Cell culture is preferred
78
Principle of Detection and Quantification Methods
Infectivity
Physical
Genome
Serological
79
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Cytopathic Effect (CPE)
infection assay
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Cell Rounding
Syncytia Formation
Inclusion bodies
80
Fluorescent Focus Assay
| infection assay
Infect cells
Expose virus antigen
Stain with labeled antibody
Count areas that fluoresce
81
Plaque Assay
| infection assay
Cell monolayer
Inoculate with dilute virus
Infected cells die leaving a clear area – plaque
82
Infectious Dose
| infection assay
ID50, IU50, TCID50
Systems: Tissue Culture, Eggs, Animals
Inoculate with different dilutions of virus
Calculate concentration based on number infected
83
Particle Assays
Electron microscopy
| Hemagglutinin assay
84
Electron microscopy
Direct image of virus particles
| Calibrate with latex bead standard
85
Hemagglutinin assay
Viruses that bind red blood cells (RBCs)
Mix constant number of RBCs with various virus dilutions
If virus concentration is sufficient, a matrix of RBCs and virus is formed
Matrix does not allow RBCs to pellet
86
Polymerase Chain Reaction
| genome assay
DNA primer specific to virus
Amplify the gene
Very sensitive
87
Southern (DNA) & Northern (RNA) Blots
| genome assay
Isolate DNA or RNA
Separate by electrophoresis
Use labeled DNA probe to detect
88
Serological Assays
Virus neutralization
Enzyme Link Immunosorbant Assay (ELISA)
Western (Protein) Blot
89
Virus neutralization
Antibody binding to virus can block infection
| Virus concentration determined by amount of antibody needed
90
Enzyme Link Immunosorbant Assay (ELISA)
Antibody recognition of virus
| Amplification by enzyme linked to antibody
91
Western (Protein) Blot
Separate proteins by electrophoresis
| Probe proteins using an antibody
92
Viruses are obligate
intracellular parasites; use host cell's replication processes to duplicate themselves
93
Every virus has a
nucleocapsid consisting of genetic material (RNA or DNA) and protein; some viruses are enveloped (coated with a host cell membrane)
94
Human viruses have diameters of
30-300 nm
95
Viruses are classified by the
genome, virus particle structure, and replication strategy
96
RNA viruses replicate in
cytoplasm
97
DNA viruses replicate in
nucleus
