Virology Flashcards
(89 cards)
1
Q
How can viruses be grown?
A
Viruses can only be grown in living tissues.
1920 embryonated eggs found that they count be infected with viruses due to many different types of cells and nutritionally rich.
1950s cell structure found cells could be grown under sterile conditions and infected.
2
Q
What is viral cytopathic effect?
A
Infect and kills cells, cells begin to round, have gaps form between them and bleb.
3
Q
Describe scanning electron microscopes.
A
Produces images of a sample by scanning the surface with a focussed beam of electrons.
Example: 70-90nm
4
Q
Describe transmission electron microscopy.
A
Provides very high resolution images. It images thin 100nms samples via the interaction of electrons as they pass through a specimen.
Example: 27-40nm
5
Q
Describe cryo-electron microscopy.
A
Involves flash-freezing solutions of proteins or other biomolecules and then bombarding them with electrons to produce microscope images of individual molecules. These are used to reconstruct the 3D shape, or structure of the molecule.
Example: 18-25nm
6
Q
What are the impacts of molecular biology on our understanding of viruses and virus disease?
A
- Basis for classification
- Understanding replication and pathogenesis
- Development of antiviral drugs
- Production of novel vaccines
- Understanding virus evolution
- New diagnostic tests
- Molecular epidemiology of viruses
7
Q
What do viruses need from the host cell?
A
All viruses are dependent on the host cell as a source of energy and to provide raw materials.
All viruses use host cell machinery to replicate their genome and to synthesise new proteins.
8
Q
What information is enclosed in the viral genome?
A
Pretty small genome and 1.5% of genome will encode for proteins.
9
Q
What are viral gene products and regulatory signals required for?
A
- Replication of the genome
- Efficient expression of the genome
- Assembly and packaging of the genome
- Regulation and timing of the reproductive cycle
- Modulation of host defences
- Spread to other cells and hosts
10
Q
What information is not contained in the viral genome?
A
- Genes encoding a complete protein synthesis machinery (for example, no ribosomal RNA and no ribosomal or translational proteins)
- Genes encoding proteins of energy metabolism or membrane biosynthesis
- Telomeres to maintain genes or centromeres to ensure segregation of genes
11
Q
What are the types of viral DNA genomes?
A
DNA genomes:
- Double stranded DNA genome
- Single stranded DNA genome
RNA genomes:
- Positive sense single stranded RNA – equivalent of mRNA. If it gets into cell, it can directly make proteins.
- Negative sense single stranded RNA
- Double stranded RNA
12
Q
What are positive and negative sense viruses?
A
Positive in order to make mRNA much be replicated via negative
Negative cannot make proteins straight away
13
Q
What are the functions of viral proteins?
A
- Protection of the genome
- Delivery of the genome
- Help with viral replication
14
Q
What are viral nucleic acids?
A
Virus nucleic acid is enclosed in a capsid, made of multiple protein subunits capsomers.
15
Q
What are the functions of structural proteins?
A
- Packaging of genome and essential enzymes
- Protection of nucleic acid from UV light
- Protection form nucleases
- Provides specificity for attachment of virus
16
Q
What is the nucleocapsid?
A
Nucleocapsid = nucleic acid + capsid
17
Q
Describe icosahedrals.
A
- Sometimes capsomeres assemble into pentamers and hexamers.
- When no hexagons are present the resulting shape is called an icosahedron, which can be made of triangles arranged as overlapping pentamers.
- Icosahedral are polygons with 12 vertices, 20 faces and 30 edges.
- Each face is an equilateral triangle.
- Icosahedral capsid is the most stable and found in numerous viruses: adenovirus, picornavirus, papovavirus, herpes virus.
18
Q
What is icosahedral symmetry?
A
Has axes of 2, 3 and 5 fold rotational symmetry.
19
Q
What is the structure of adenovirus?
A
252 capsomeres = 12 pentons (one at each vertex) + 240 hexons (12 on each face)
20
Q
What is the structure of foot and mouth virus?
A
60 trimeric capsomers. Trimeric proteins join groups of 5/pentamers.
21
Q
What is the structure of feline calicivirus?
A
Capsomers are 90 dimers arranged in rings on the faces.
5 fold and 3 fold dimers join together a ring, which come together to form a capsid.
22
Q
What is the structure of canine parvovirus?
A
12 pentamers and 20 hexamers
23
Q
What is helical symmetry?
A
- Protein subunits can interact with each other and with the nucleic acid to form a coiled, ribbon like structure.
- The best studied virus with helical symmetry is the non-enveloped plant virus, tobacco mosaic virus.
- Most of the helical viruses are enveloped and all are RNA viruses.
24
Q
What is the structure of rhabdovirus and orthomyoxovirus?
A
Helical nucleocapsids.
Bullet shaped with icosahedral nuclear capsid.
25
What is complex viral symmetry?
Do not strictly conform to a simple helical or icosahedral shape.
Poxviruses are large, oval or brick-shaped particles, 200-400nm long. Inside the complex virion, a dumbbell-shaped core encloses the viral DNA and is surrounded by 2 lateral bodies, the function if which is currently unknown.
26
What are peplomers/glycoprotein spikes?
- Glycoprotein (sugar proteins), which protrude from the surface of mature virions
- Can have enzymatic functions ]can be used to bind to cell receptors.
27
What are viral lipid enveloped?
- Lipid bi-layers from host cells are derived by budding.
- Many viruses with icosahedral but not symmetry have a membrane envelope.
- Most of the helical viruses are enveloped and all are RNA viruses.
28
What are the functions of viral envelopes?
- Protecting the viral RNA and DNA genomes
- Evading virus recognition by the immune system
- Facilitating virus entry into cells
29
What are the properties of rinderpest virus?
Enveloped, negative sense RNA, helical symmetry
30
What are the properties of equine herpes virus?
Enveloped virus, double stranded DNA genome, icosahedral nucleocapsid, revealed by detergent
31
How do viruses cause disease?
It is important to remember that viruses infect pets, food, animals, plants insects and wildlife throughout the world. The process by which viruses cause disease is called viral pathogenesis.
32
What does the nature of a virus depend on?
- The effects of viral reproduction on host cells
- The responses of the host’s defence systems
- The ability of the virus to spread in and among hosts
33
Why are viral infections not synonymous with disease?
As many viral infections can be subclinical/asymptomatic, whereas others result in disease of varying severity that is typically accompanied by characteristic clinical signs in the affected host.
34
Define pathogenicity.
Refers to the ability of an organisms to cause disease and harm the host. Qualitative term, an ‘all or none’ concept. For example, a virus is said to be either pathogenic or non-pathogenic.
35
Define virulence.
Refers to the degree of pathology caused by the organism. For example, virus A is more virulent in animal species Y than species Z.
36
What are the different routes that a virus can enter the host?
Respiratory route – localised respiratory disease, such as influenza virus.
Oral route – virus infections are the single most common cause of diarrhoea, such as rotavirus, adenovirus, calicivirus, coronavirus
Cutaneous localised infection – papilloma virus
Percutaneous injection – bites from infected animals, such as rabies and FIV, or bites from insect vector, such as arboviruses/Louping ill.
Sexually transmitted infection – localised genital infections or systemic disease: pox virus, herpes viruses and papilloma virus
37
How do viruses target cells?
Specific interaction – can define the host species and cell type a virus may infect.
38
Give an example of specific interaction of HIV.
- HIV Env binds with high affinity to CD4 molecules on human cells.
- There is a conformational change in Env (virus envelope glycoprotein) which allows a low affinity interaction with another receptor called CCR5.
- Binding to CCR5 in turn promotes further conformational changes in Env that enable virus envelope cell membrane fusion.
39
Describe high specific protein-protein interactions.
Highly specific protein-protein interactions depend on closeness of fit between molecules and even single amino acid substitutions can alter the bonding. There are often several amino acids differences between the same molecule in different species, this can restrict virus host range.
40
Why can humans not support FIV infection?
- FIV Env protein binds with high affinity to feline CD134, which is present on CD4+ lymphocytes.
- FIV can only bind to feline CD134, which has aspartic acid residues at positions 60 and 62.
- Human CD134 fails to support FIV infection.
41
What is the consequence of the conformational change in Env?
There is a conformational change in Env which allows a low affinity interaction with a chemokine receptor CXCR4.
- This in turn promotes further conformational changes that enable membrane fusion.
- Humans CD134 cannot replace cat CD134
- Human CXCR4 can replace cat CXCR4
42
Describe how non-specific ligands have wide range potential.
Influenza haemagglutinin binds sialic acid (ubiquitous). Infection of cells is dependent on host protease to cut hinge. A single amino acid change to the cleavage sit can alter host specificity of infection and the severity of the disease, as there are more mature proteins on the surface that can be bound. Mutation can be more easily cleaved and more activated for infection.
43
What are the possible normal function of infected cells?
- Provide structure and support
- Facilitate growth
- Allow passive and active transport
- Produce energy
- Create metabolic reactions
- Aids in reproduction
44
Describe lytic infection.
- Many acute virus infections end in cell death causing complete loss of function accompanied by the release of new virus particles (cytopathic effect).
- Release of virus from one cell can infect many thousands of new cells.
- Which causes extensive loss of cell function, such as loss of mucocilliary clearance and localised respiratory disease.
- Generalised multisystem disease, such as Canine Distemper
45
Describe latent infection.
- Virus infections that persist within a cell.
- The virus is dormant and is not replicating – therefore does not cause cell death.
- Latent virus infections can persist for the lifetime of the host.
- Latently infected animals act as a source of infection for others
46
How does persistent infection alter host cell function?
This is a subtle difference from latent infection where the cells produce new virus proteins and can continually shed virus. This type of infection is often associated with a failure of the immune response to clear the virus infection.
47
How does enhanced cell growth alter host cell function?
Viruses require many different functions of the host cell for propagation. Examples include the machinery for translation of viral mRNAs, sources of energy, and enzymes for genome replication. Therefore, viruses often alter the growth characteristics of host cells to enable optimal viral replication.
48
How does apoptosis alter host cell function?
Can also be induced by the biochemical alterations initiated by virus infection. Cell death is the result of a cascade of reactions that ultimately leads to nuclear membrane breakdown, chromatin condensation, loss of membrane integrity (called “blebbing,” in which bubbles of cytoplasm appear on the cell surface), and eventually DNA degradation.
49
What is the inflammatory response to infected cells?
- The virus genome is detected by specialised receptors called pattern recognition reception.
- Examples are Rig-I, TLR3, TLR7, TLR8 and TLR9
- Activation of PRR induces inflammatory cytokines, such as TNF-a, IL-6 and IL12, and type 1 interferons IFN-a and IFN-b.
- Pattern recognition mechanisms to detect thing that we do not make or possess in the body and so their presence is due to infection.
50
Describe the interferon response against viruses.
- Interferon response factor migrate to nucleus and cause transcription and antiviral immune response
- Includes production of type 1 interferons
- Produced and secreted by cell and alerts neighbouring cells, as they have interferon receptor
- Neighbouring cells produce proteins and antiviral factors that may protect it from also being infected.
51
What are the additional effects of interferon, cytokines and chemokines against viruses?
- Increase MHC expression
- Activates macrophages and dendritic cells
- Recruits and activates NK cells
52
What is the action of macrophages and dendritic cells against viruses?
- Secrete interferon
- Produce cytokines
- Present antigen to T cells
- Activate NK cells
53
How is a fever generated?
1. Production of endogenous pyrogens: Interferon ,TNFa.
2. Binding to opioid receptors on nerve cells in the hypothalamus.
3. Activation of COX-2 in the hypothalamus leading to increased PGE2
4. Altered firing rate of temperature sensitive neurons in the anterior hypothalamus
54
Define sickness behaviour.
Motivational state responsible for re-organizing perceptions and actions to enable ill individuals to cope better with an infection.
55
What are the characteristics of sickness behaviour?
- Decreased motor activity
- Social withdrawal reduced responsiveness
- Reduced food and water intake
- Increased slow-wave sleep
- Altered cognition
- Increased pain sensitivity
56
What is released in response to PAMPs?
Mediated by Effects of pro-inflammatory cytokines (especially IL-1) on the CNS (IL-1, IL-6, TNF, interferon) released in response to Pathogen-associated molecular patterns (PAMPs).
57
How does foot and mouth disease enter the host?
By ingestion or inhalation and sets up a primary oro-nasal infection. Attaches to integrins, binding to an RDH motif in the virus capsid protein, which mimics the natural ligand on matrix proteins.
58
What does foot and mouth target?
Integrins are found at high density on basal epithelial cells in the oropharynx, which becomes infected through small abrasions or Tonsil crypt.
- The RGD motif is common to many species. Therefore FMD should have a wide host range: cattle, sheep, goats, pigs, deer, elephants, kangaroo, hedgehog and man.
- The virus does not however replicate equally well in all hosts, as horses, cats and dogs are resistance.
59
What is the normal function of squamous epithelium and basal epithelial cells targeted by foot and mouth disease?
- To protect the sensitive submucosa
- To maintain adhesion of epithelium to basement membrane
- Repair/regenerate damaged epithelium by producing growth factors and chemical mediators.
60
How does foot and mouth disease alter cell function?
Shutdown of host cell protein synthesis:
- Loss of host cell integrity and ultimately cell death with lysis and release of infectious virus.
- A single infected cell releases thousands of virus particles
- Virus spreads locally to basal epithelial cells with high integrin expression
- Several sounds of virus replication are needed before macroscopic lesions appear
- The presence of the virus causes an inflammatory response both in the epithelium and in the adjacent tissues.
61
What are the early clinical signs of foot and mouth disease?
- Prostaglandin synthesis sensitises local nocioreceptors.
- Iocal pain sensations
- Clinical signs are drooling ad lip smacking
62
What are the later clinical signs of foot and mouth disease?
Generalised signs of fever and malaise inappetence are mediated by release of interferon and cytokines from infected cells and inflammatory leukocytes. Later clinical signs are mediated by inflammation at the site of developing infection, such as lameness.
- Integrins are expressed at highest levels where shear forces are high, for example, in the laminae, coronary band of the foot and the lining of the mouth.
- Lesions are non-fatal but painful.
- Basal epithelium is most affected: prolonged healing.
63
How can viruses be grown in living tissue?
Embryonated eggs – viruses can be grown in embryonated eggs as eggs contains lots of different types of living cells. This makes eggs versatile for growing viruses.
Cell culture flask – viruses can be grown in tissue culture using a monolayer of cells attached to the bottom of a flask or dish.
64
Explain what happens when a virus enters a host cell.
1. When a single virus enters a cell, it replicates producing new viral particles.
2. The new virions are then released when the cell dies.
3. The released virus particles then infect neighbouring cells, these cells are in turn lysed and after a few hours/days of this repeating, depending on how fast the virus grows, the presence of the virus can be detected by the appearance of a hole/plaque in the monolayer.
65
What is a plaque assay?
In the plate, there are 5 visible plaques formed after 100 ul sample of virus is diluted 1:1000000 and is incubated on the cells. So, the titre of this virus would be described as 5x10^6pfu/ml (pfu = plaque forming unit).
66
What can plaque assays be used for and how?
To track the progress of a virus infection in vitro by the following experiment.
1. Identical cultures of cells are infected with virus.
2. The culture media and the cells are harvested separately at hourly intervals.
3. The number of infectious extracellular virus particles in the culture media (released) or associated with the cells (intracellular) is then measured by plaque essay.
67
Describe the inoculation phase of virus growth.
At time 0, the small amount of added virus is all detected in the culture medium.
68
Describe the virus attachment stage of virus growth.
The level of virus in the culture medium drops during the first house as the virus particles adhere to the cell surface. Some of the adherent virus can still be detected after it is released by detergent lysis of the cell.
69
Describe the uncoating stage of virus growth.
During the next stage, the virus enters the cells an releases its nucleic acid into the cell. For thus to happen, the virus must be dismantled inside the cell and therefore no infectious particles of virus can be detected by plaque assay at this time.
70
Describe the eclipse phase of viral growth.
During this time, the new virus proteins and nucleic acids are being synthesised. No infectious virus particles can be detected during the lag phase.
71
Describe the virus assembly phase of viral growth.
At the end of the lag phase new infectious virus particles can be detected. At first, this is all cell associated/intracellular as the new nucleocapsids assemble prior to release from the cells.
72
Describe the cell lysis stage of virus growth.
During the final stage of the replication cycle, the cells begin to be lysed and to release high levels of new infectious particles into the media. At this time, the level of virus in the media may become thousands of times higher than that of the original input virus.
73
Summarise the process of virus replication.
1. Attachment to plasma membrane
2. Entry into cytoplasm/nucleus
3. Uncoating of virus particle
4. Synthesis of mRNA and protein
5. Replication of nucleic acid
6. Virus assembly
7. Exit of virus from cell
74
Describe RNA synthesis.
- Many different viruses and the replication cycle of each has its own special features or variation.
- 1 important common feature in replication is that all viruses have to make mRNA that can be transplanted into protein by host cell ribosomes.
- RNA is said to be positive sense as it can be read by ribosomes and translated into a functional protein.
75
What is the role of sense and anti-sense in virus translation?
- mRNA is a positive sense RNA
- The +ve sense strand is complementary to the –ve sense RNA
- This is like a mirror image copy of a sense RNA and it cannot be read by a ribosome or translated directly into a functional protein.
- The bases in RNA pair up like those in DNA
- And thus, the –ve sense RNA has the opposite base in each position
76
What is the purpose of the 5' cap?
- Recognised by the translational machinery of the cell – aid in protein production.
- Caps protect the growing RNA chain from degradation by nucleases – aid in stability.
77
What is the purpose of polyadenylated tails?
Newly synthesized RNA has a poly(A) tail added at the end.
- Polyadenylation increases the stability of mRNAs - protects against degradation.
- Plays a role in the initiation of translation – promotes export from the nucleus.
78
What must all viruses produce to make proteins?
Positive sense mRNA
79
Distinguish dsDNA and ssDNA.
dsDNA (papovaviridae, adenoviridae, Herpesviridae, poxviridae) form DNA directly
ssDNA (parvoviridae, circoviridae) must form dsDNA and then mRNA.
80
What are the different ways mRNA can be made in RNA viruses?
+ve ssRNA (piconaviridae, togaviridae, Flaviviridae, caliciviridae, coronaviridae, astroviridae, arteroviridae) form mRNA+ directly
dsRNA (reoviridae) form virus RNA polymerase and then mRNA+
-ve ssRNA (bunyaviridae, arenaviridae, rhabdoviridae, paramyxoviridae, Orthomyxoviridae) form virus RNA polymerase form mRNA+
81
How do retroviridae produce mRNA?
- Have 2 copies of a positive sense single stranded RNA genome
- Carry reverse transcription
- This copies the +vessRNA into double stranded DNA
- Integrase integrates this into the host genome DNA
- This can be transcribed to form mRNA using host polymerases
82
What are the characteristics of foot and mouth disease?
Fever
Vesicles in mouth
Lameness
Inappetence
Prolonged recovery
83
What is the structure of food and mouth disease virus?
Non enveloped capsid virus wit 60 trimeric capsomers and icosahedral symmetry. Positive sense RNA genome.
84
How do FMD viruses attach to integrins?
- Integrins bind to an amino acid RGD motif on protein molecules found in the basement membrane.
- There is an RGD motif in the virus capsid protein mimicking the ligand of the matrix protein.
- Found at high density on basal epithelial cells in the oropharynx, which become infected through small abrasions.
85
How do FMD viruses enter the cytoplasm?
- Receptor mediated endocytosis via clathrin coated pits
- After binding to integrins, the virus is transported to clathrin coated pits and is endocytosed.
- H+ ions are pumped into the endosome.
- pH drops and signals for virus to get out before damage.
86
How are FMD viruses upcoated?
- The acidification in the endosome causes a conformational change in the virus capsid.
- A pore is formed in the endosome membrane through which the genome escapes into the cytoplasm.
87
How do FMD viruses replicate their genomes?
- FMD already has a positive sense RNA genome complete with a poly A tail.
- FMD makes 1 big polyprotein (polycistronic RNA):
88
How are new FDM viruses assembled?
- FMD has to make 60 copies of its polyprotein to make 1 new virus
- Capsomeres, VP1, VP2 and VP3, self assemble to form the viral capsid with icosahedral symmetry.
- The capsid will package the viral RNA.
89
What are inclusion bodies?
Thousands of new virus particles self-assemble into procrystalline arrays within the cell. These aggregates are called inclusion bodies.
