Week 1 & 2 Flashcards
(99 cards)
1
Q
Virus
A
Infectious, obligate intracellular molecular parasite
2
Q
Virus size
A
20-300nm
3
Q
Light microscopy
A
Observe cytopathic effects of virus-infected cells
4
Q
Negative staining
A
Use compound containing heavy metal
Stains appear as dark areas around virions
Advantage: high quality electron micrograph
Disadvantage: Possible structural distortions resulting from drying
5
Q
X-ray crystallography
A
Virion crystals/molecules placed in beam of X-ray
Diffraction pattern allows molecule/atom positions to be determined
6
Q
Cryo-electron microscopy
A
Wet specimen rapidly cooled to <160C
3D image reconstructed using multiple images
Useful for labile outer shells
7
Q
Capsid
A
Protective protein shell surrounding genome and forming core of viral particle
8
Q
Capsomers
A
Clusters of capsid protein subunits
Triangular faces
9
Q
Nucleocapsid
A
Protein assembled onto nucleic acid
10
Q
Envelope
A
Lipoprotein membrane surrounds nucleocapsid or capsid
Phospholipid - host membrane
Glycoprotein - virus encoded
11
Q
Naked virus
A
No envelope layer
12
Q
Matrix
A
Structural proteins linking the viral envelope with the virus core
13
Q
Icosahedral symmetry
A
Shell built from protein molecules
Less contact with virus genome than helical capsids
Can appear spherical
E.g. adenovirus, herpesvirus, papillomavirus
14
Q
Helical capsid
A
Common in ssRNA viruses - RNA forms a helix coated in protein
Helical symmetry allows entry of negative stain
15
Q
Complex symmetry
A
E.g. poxvirus
16
Q
Family classification
A
-viridae
Type of nucleic acid genome and arrangement (+/-)
Strategy of viral replication
Morphology
17
Q
Genus classification
A
-virus Size of genome Number and size of proteins Serological reactivity Host range and disease produced
18
Q
DNA or RNA genome?
A
- Infect cells in presence of 14C thymidine (DNA) and 3H-uracil (RNA)
- Purify virus particles produced in cells
- Use radioactivity detector to determine whether virus contains H or C
19
Q
ss or ds RNA
A
- Label viral RNA during growth
- Extract nucleic acid from purified particles
- Divide into 2 portions, add ribonuclease to one and incubate. RNase converts RNA polymer into free nucleotides (digests ss not ds RNA)
- Use Trichloroacetic acid to precipitate remaining radioactive RNA polymers from each sample
20
Q
Extracting nucleic acids
A
Lipids and proteins solubilized with SDS detergent + proteinase K
Phenol extraction –> centrifuge
Phenol = weak acid, destroys capsid, not nucleic acids
21
Q
Do DNA viruses have helical symmetry category?
A
NO
22
Q
Baltimore classification
A
I: ds DNA II: ss DNA •III: ds RNA •IV: ss RNA +ve •V: ssRNA –ve •VI: +ve ssRNA that replicate through DNA intermediate VII: dsDNA that replicate through ssRNA
23
Q
Koch’s postulates difficult to confirm with viruses
A
- Virus should have a regular association with clinical disease
- Virus characterized – isolated via animal or cell-culture passage and distinguished from other viruses immunologically or genetically
- Clinical syndrome should be experimentally reproducible in volunteers or lab animals
- Virus should be reisolated from experimentally infected animal
24
Q
Non-taxonomic virus groups
A
Enteric: rotavirus, calcivirus, some adeno
Respiratory: orthomyxo, rhino, paramyxo, corona, adeno
Arbo: bunya, flavi, toga
Sexually transmitted: herpes, papilloma, retro, hep
Hepatitis
25
Hepatitis
A & E: enteric
| B,C,D - blood, sexually transmitted
26
Viral cultivation
Gold standard
Useful for public health, rather than individual patient treatment
Very slow
27
Low temp preservation
Day - 4C (Fridge)
Long term - -7C (dry ice, deep freeze)
Permanent - -196 (liquid N)
Avoid repeat freeze thawing - ice crystals can shear envelope layer
Naked viruses: freeze drying - dehydration of a frozen suspension under vacuum, used for some live-viral vaccines
Buffered transport medium - pH change can be a trigger for viral replication
28
Mammalian cell culture
Primary cells: animal tissues e.g. human foreskin fibroblast - directly from host animal, usually derived from monkeys
tumour?
29
Cytopathic effects
Cytoplasmic inclusions (virus replicates)
Nuclear inclusions
Lysis - zone of cell death
Multinucleated syncytium (50-100 nuclei)
Transformation (--> malignancy)
Poliovirus - death in 24 hours
Pyknosis - cells shrink and die
30
Haemagglutination assay
Measures ability of virus to agglutinate RBCs
End point titration = last shield
Shiled - virus cross-linking RBCs
10^4 particles need to agglutinate
Haemagglutination inhibition: diluting antibodies, keep virus constant and serotyping
31
Antigen capture assay
High sensitivity
e.g. HIV p24 assay - capsid protein
Capture antibody in well
Prone to giving false positives
32
Anti-viral antibody assay
Viral antigen in well
High specificity
E.g. HIV western blot
Done as a confirming assay after capture assay
33
DNA detection
Southern blot
| PCR
34
RNA detection
Northern blot
| RT-PCR
35
T/F: Poxvirus has a capsid with helical symmetry
FALSE
36
T/F: For cryo-electron microscopy, purified virus is stained for 24 hours in Fourier reagent
FALSE: Cryo-electron microscopy is performed on UNSTAINED, but frozen virus in order to preserve fragile structural components
37
T/F:
| Viruses in the arbovirus family have a DNA genome in a helical capsid
FALSE: arboviruses are not a taxonomic family, but an epidemiological grouping of viruses that are transmitted by insects. Additionally, NONE of the DNA viruses of medical importance have a helical capsid.
38
T/F:
A virus with an RNA genome has the ability to incorporate tritiated-uracil (3H-U) but not 14C-thymidine during replication
True
39
T/F:Assaying a sample for influenza virus using electron microscopy to count particles will give a lower number of viruses than a plaque assay in MDCK cells
False: assaying virus by EM will count both infectious and non-infectious virus particles. Whereas plaque assays require virus to infect, replicate and kill cells, and therefore the non-infectious virus particles are not counted.
40
T/F:
| PCR detection can more rapidly identify a virus than cultivation techniques.
True: cultivation is slow but useful for diagnosis
41
T/F: During the identification of the SARS coronavirus X-ray crystallography was used to determine the virion morphology
False:
| X-ray crystallography not useful for enveloped viruses
42
T/F: During rate zonal centrifugation virus is separated on a pre-formed density gradient in around 2 hours.
TRUE: separates based on size and density: separation depends on velocity that particles move through a pre-formed gradient
43
T/F:
| An icosahedral capsid with 5:3:2 fold axes of symmetry has 15 vertices.
FALSE: does have a 5-3-2 symmetry but it has 20 faces and 12 VERTICES
44
T/F:The RNA polymerase packaged into measles virus particles, a paramyxoviridae, is a non-structural viral component.
FALSE:
Measles does have RNA pol BUT if protein/nucleic acid is packaged into particles and transferred to newlyinfected cells, it is considered a virion structural component
45
T/F: During a virus infection where the multiplicity of infection equals 1 there are equal numbers of infectious virus and infectible target cells. However, under these conditions only a proportion of these infectible cells will contain virus during the first round of viral replication.
TRUE: Viral infection is a random event that follows a Poisson distribution. Therefore, where the multiplicity of infection = 1, some cells will bind more than one virus and some virus will not collide with a target cell.
46
T/F:
| Small viruses, like the picornaviridae, may passively enter cells using pinocytosis.
FALSE: Viral entry is NOT passive, requires receptors - either endocytosis or fusion
47
T/F:
The haemagglutinin trimers in the envelope of influenza virus bind to the sialic acid residues on cellular glycoproteins to initiate virus entry.
True: influenza enters through glatharin coated pit after haemagglutinin trimers bind to sialic glycoproteins present in PM
48
T/F: During entry of the influenza virus, uncoating of virus in the endosome depends upon activation of neuraminidase at pH 10.
False: Endosome becomes acidified (pH5)
Haemagglutinin molecule undergoes structural change that expose hydrophobic fusion domain that becomes inserted into endosome membrane
49
T/F: Conformational changes in the fusion protein (F) of measles virus drive the fusion of virus at the plasma membrane and may also lead to cells fusing into a multinucleated giant cell, or syncytium.
True
50
T/F:
| HIV envelope gp160 glycoprotein is cleaved by a virus-coded protease during translation on cytoplasmic polysomes.
False: HIV envelope glycoproteins translated on RER and cellular enzymes in this compartment cleave gp160 precursor protein into mature gp120 and gp41
51
T/F:
The snRNA components of the spliceosome include ribosomal RNA and can interact with viral RNAs in the endoplasmic reticulum.
False: Spliceosomal snRNA include U1,2,6 and only found in nucleus
viral RNA can only undergo splicing in nucleus
52
Inclusion bodies
```
Accumulated viral proteins at site of viral assembly
Adeno - nuclear inclusions
Reo - cytoplasmic
Herpes - intranuclear inclusion bodies
Measles - syncytia and cell inclusions
HIV - multinucleated syncytia
```
53
Infectivity/serology assay
Slow
High sensitivity - detecting viral antigens
Poor sensitivity, high specificity - detecting host antibodies
54
SARS outbreak
Severe flu symptoms & coughing mucus
Virus cultivation: cytopathic growth on vero cells
Coronavirus : morphology from EM
55
Virion
Infectious viral particle
56
Capsid
Protein shell surrounding genome
57
Nucleocapsid
Nucleic-acid protein assembly within virion
58
Virions are metastable
Stable - protect genome
Unstable - come apart quickly on infection
Potential energy used for disassembly
59
Biologically pure virus
1. plaque purify original - select virus from single plaque to grow into a stock
2. limiting dilution of original - biological cloning
3. generation from molecular clone (plasmid)
Purifying virus requires: cell disruption, centrifugation, density gradient centrifugation (most important)
60
Cell disruption
1. Safest method for dangerous pathogens - freeze and thaw (2,3 cycles) - ice crystals sheer membrane
2. Non-ionic detergents will lyse cytoplasmic but not nuclear membranes e.g. Triton X-100 and Nonidest P40 (useful if cytoplasmic virus)
3. Homogenizers: chop up cells
61
Blender-type homogenizers
Rotating blades - sheer cells and breaks up membranes
Membranes:
RER w/ ribosomes = microsomes
If nuclei are broken --> viscous homogenate --> difficult to work with
62
Centrifugation
Low speed centrifugation - separate nuclei and large cell fragments
Virus will be in supernatant
63
Sedimentation coefficient
Used to distinguish virus from other components
64
Ultracentrifugation
Strong rotors
Sealed - run in a vacuum to reduce friction
Virus will be in a pellet --> resuspended in buffer -> concentrate purified by density gradient centrifugation
65
Rate zonal centrifugation
Density of particles being separated are greater than density of solvent
Separation: size
If centrifuge not turned off - particles will pellet
66
Equilibrium centrifugation
Solvent density encompasses density of particles
Separation: particle density
Carried out until equilibrium
67
SDS electrophoresis
SDS: Anionic detergent
Proteins containing bound SDS gain neg charge
Intensity of protein bands directly correlates with protein mass
68
Virus assembly to form icosahedral capsids
5:3:2 symmetry
| Spherical shell is most economical to enclose genome nucleic acid
69
Triangulation
Describes how limited sets of small proteins make icosahedral particles with different sizes
70
Triangulation number
Description of number of equilateral triangles into which each of the 20 triangular faces of the icosahedron is divided
71
Polio
```
Picornaviridae
Enteric infection
Neurotropic --> paralysis
Icosahedral - not easily seen under EM
EQUIMOLAR
T=3
```
72
Adenovirus
```
Infect conjunctiva and respiratory tract
Highly contagious
Transmitted by droplet & fomite
T=25 (complex structure)
NOT equimolar proteins
```
73
Papilloma virus
Replicates in keratinized tissue (skin)
| All capsomers are pentons
74
Togavridae
e.g. Sindbis - envelope glycoproteins have a symmetrical arrangement following the icosahedron rules T=4
75
Maturation of HIV particles
Icosahedral core of HIV undergoes proteolytic processing after budding from cells to mature into rod shape
HIV1 p24 capsid proteins form fullerene cone structure from hexamer and pentamer interactions
Pentamers concentrate at narrow end to close cone
No tethering of GP onto core
76
Measles - paramyxoviridae
Haemagglutinin and fusion protein
Ribonucleoprotein: nucleoprotein, phosphoprotein, RNA polymerase large protein
Matrix protein
Nucleoproteins:
nuclear and cytosolic localization
Most abundant protein in infected cells
Self assembles to form nucleocapsid
77
Multiplicity of infection
Number of virus infecting a cell
| Calculated by Poisson distribution
78
Attachment/adsorption
Virus binds specifically to receptor on cell plasma membrane
Receptors: protein (Rhinovirus), carbohydrate (sialic acid - influenza) OR 2 different receptors - initial attachment & coreceptor for closer attachment and entry
79
Virus entry
By endocytosis or fusion
Phagocytosis not used - engulfs larger particles
ONLY by RECEPTOR-MEDIATED endocytosis or fusion, NOT passive pinocytosis
80
Endocytosis
Invagination of clathrin-coated pit forming an endosome
Endosome becomes acidic --> conformational change in virus capsid or envelope proteins --> uncoating or fusion and release of viral genome into cytoplasm
81
Adeno entry and uncoating
Penton fibre engages cell adenovirus receptor (CAR)
Acidification --> uncoating
Capsid transported to nuclear pore along microtubule network
82
Polio receptor mediated entry
Interaction between receptor and capsid --> conformational change in VP1 capsid protein --> pore formation
83
Reovirus entry and uncoating strategies
Outer capsid proteins bind to cell receptors
Enters by receptor-mediated endocytosis and undergoes proteolysis in late endosome --> Infectious subviral particle (ISVP)
Modified capsid proteins of ISVP mediate membrane penetration
RNA comes out of reovirus pore
84
Direct fusion
E.g. paramyxovirus - fusion glycoprotein on envelope
| Rotavirus VP4 - cleaved by trypsin
85
Endocytosis or fusion?
Expose cells to weak base during infectione.g. chloroquine or methylanine
Block infection via endocytosis (prevent acidification of endosomes) but won't affect infection by direct fusion
86
dsDNA replication
All EXCEPT pox replicate in nucleus
| Use cellular enzymes
87
Papova SV40 structure
SV40 viral DNA organises with histones into a microchromosome
Anticlockwise genes expressed first
88
Fork replication dsDNA
RNA primer
| e.g. papova and herpes
89
Displacement
Adeno - protein
Parvo - DNA hairpin
No lagging strand, often uses virus encoded DNA pol
90
Poxvirus genome
Has it's own RNA pol, capping enzymes, poly A polymerase, and DNA pol --> replicate in cytoplasm
91
Clinical samples
```
Respiratory - throat washing
Enteric - faecal sample
Meningitis - CSF
Vesicular rash - vesicle fluid
Systemic fever - blood
```
92
PC4 isolation
Highly pathogenic airborne virus
| e.g. Bird flu or sars
93
Direct visualisation by EM
Crude purification of virus from heavily contaminated samples e.g. faeces - Rotavirus, Noward virus
94
Virus cultivation in chicken eggs
Chorioallantoic membrane - Herpes simplex, pox
Amniotic - influenza, mumps
Yolk sac - herpes simplex
Allantoic -influenza, mumps, newcastle disease virus
95
Inclusion bodies
Adenovirus - nuclear inclusions
Reovirus - cytoplasmic inclusions
Hint to where virus is DNA or RNA virus
96
Enumeration by cell transformation
Cell culture - transformed cells grow together in 'foci'
Count the foci
97
Haemadsorption
viral envelope proteins bind red blood cells e.g. influenza, parainfluenza
98
Comparisons of assays
Electron microscopy - 10^10 particles, but not all infectious
Quantal assay in egg: 10^9, ID50
Cell culture/plaque: efficiency of replication is 10x less= 10^8 pfu
Haemagglutination assay: 10^3 assays, need a lot of particles to agglutinate
99
Western blot assay
1. Detergents used to solubilize proteins in cells that are infected with HIV or expressing HIV DNA vaccine
2. Proteins separated by SDS-PAGE and transfererd to filters
3. Anti-viral antibody in patient serum binds to protein bands - high specificity
