1
Q
Replication processes are ________ among different groups of viruses
A
highly variable
2
Q
What are the 6 general steps for replication in all viruses?
A
- Attach to a susceptible host cell
- Enter the cell
- Disassemble the virus particle (uncoating)
- Replicate its own genetic material and express the associated proteins
- Assemble new virus particles
- Escape from the infected cell (release)
3
Q
Viral attachment requires what?
A
Specific interactions between the viral particle and the host cell; viral attachment proteins on the capsid/envelop with host cell attachment receptor.
4
Q
Initial contact between the virus and the host cell involves what?
A
Short distance electrostatic interactions and more specific interactions with other receptor-like molecules.
5
Q
Describe the affinity between binding sites during viral attachment.
A
Usually low; involves many interactions.
6
Q
Host attachment proteins are often specific to what?
A
Certain cells/tissues.
7
Q
The specificity of host attachment proteins determines what?
A
Tissue/organ specificity (tropism) and it affects the pathology/disease caused by the virus.
8
Q
The variation in viral attachment receptors between species is the cause for what?
A
Determination of virus host range.
9
Q
Give 6 examples of host cell attachment receptors.
A
- Ligand-binding receptors
- Signaling molecules (CD4)
- Cell adhesion/signaling receptors (ICAM-1)
- Enzymes
- Integrins
- Glycoconjugates (often w/sialic acid as terminal residue)
10
Q
The identification of host cell factors in viral attachment has practical implications for what?
A
The design of antiviral drugs.
11
Q
Selective pressure on receptor binding/affinity can lead to what?
A
Evolution of new viral strains.
12
Q
Viruses within given families or even different strains of the same virus may use ______ receptors.
A
Different.
13
Q
Changes in receptor affinity can change the ______ of a virus.
A
Pathology.
14
Q
How do viruses with a broad host range (arboviruses) utilize host receptors?
A
- May use several host receptors to allow growth in different cells/hosts
- May utilize a common receptor found in multiple host species.
15
Q
Viral entry and uncoating is necessary for what?
A
Attachment to → release of viral genome within cell and begin viral replication.
16
Q
A bound virus enters a cell through what two general mechanisms?
A
- Entry within a membrane-bound vesicle (enveloped)
- Direct entry across the plasma membrane (non-enveloped)
17
Q
How do receptor molecules assist the entry process?
A
The nature of the receptor determines the mechanism by which the virus enters.
18
Q
Membrane fusion occurs in what type of viruses?
A
Only in enveloped viruses.
19
Q
Describe the mechanism of membrane fusion for enveloped viruses.
A
Binding to host receptor → conformational changes in viral surface proteins → fusion with host plasma membrane.
20
Q
Direct entry across the plasma membrane occurs in what types of viruses?
A
Non-enveloped ones (poliovirus); rare in veterinary viruses.
21
Q
Describe the mechanism of direct entry across the plasma membrane for non-enveloped viruses.
A
- Proteins from the capsid interior are inserted into the plasma membrane of the host which forms a pore.
- Genomic RNA is released through the pore into the cytoplasm of the host cell.
- Change in capsid structure.
22
Q
Internalization of a virus via membrane-bound vesicles occurs through what process?
A
Receptor-mediated endocytosis.
23
Q
Describe the mechanism for internalization of a virus via a membrane-bound vesicle.
A
Receptor/ligand interactions often initiate cellular signaling pathways that lead to internalization; viruses have evolved to exploit these pathways to gain entry.
24
Q
What is endocytosis?
A
Natural cellular mechanism for internalizing extracellular materials.
25
Describe the mechanism for endocytosis.
- Endosomes interiors become progressively acidic.
- pH change → structural changes in the virus that facilitate exit from endosome/uncoating.
- May fuse with lysosomes to become endolysosomes.
26
Why might additional steps be necessary after the initial entry and uncoating of a virus prior to the replication process?
- Genome may not be a form that can initiate replication-related processes.
- Genome may not be in the correct cellular compartment.
27
Define viral transcription.
Viral/cellular genomic code is turned into messenger RNA (mRNA); in the cells this occurs in the nucleus via cellular DNA polymerase.
28
Define viral translation.
mRNA is used to synthesize proteins made of amino acids; normally occurs in the cytoplasm/ER using cellular ribosomes.
29
Define polymerase.
Enzyme that catalyzes the synthesis of DNA/RNA molecules from individual nucleotides.
30
What are the 3 main types of polymerases to know?
- DNA Dependent RNA polymerase (DdRp): DNA → RNA
- RNA Dependent RNA polymerase (RdRp): RNA → RNA
- RNA Dependent DNA polymerase (RdDp): RNA → DNA
31
Define ribosome.
Molecular machine that catalyzes the translation of genetic information into proteins.
32
Describe the variation of viral protein and nucleic acid synthesis between viruses.
The process varies markedly between viruses which is the key in determining taxonomic relationships between viruses.
33
What type of viruses are adenoviruses in general?
Non-enveloped, linear, DsDNA.
34
What type of viruses are retroviruses in general?
Enveloped, single-stranded, positive sense RNA.
35
What type of virus is Rhabdovirus in general?
Enveloped, single stranded, negative sense RNA.
36
What type of virus is Picornavirus in general?
Non-enveloped, single stranded, positive sense RNA.
37
Describe the commonalities seen between positive sense RNA viruses.
- RNA genome capable of producing productive infection upon insertion to host cell.
- + sense RNA able to associate with cellular ribosomes.
- Viral proteins synthesized as larger precursor proteins called polyproteins.
- Often induce remodeling of cellular membrane structures to provide sites for RNA synthesis.
38
Describe the mechanism of the assembly and release of non-enveloped viruses.
- Virtually all are icosahedral capsids of varying complexity.
- For simple icosahedrons, capsid elements associate spontaneously into repeating simple structural subunits called protomers.
- Protomers assemble into mature icosahedral capsid that may occur with or without genomic material.
39
Describe the HPV vaccine.
Consists of virus-like particles with empty capsids that are generated by recombinant yeast S. cerevisiae.
40
What does it mean that the HPV vaccine is multivalent?
Contains the gene of interest coding for L1 (capsid proteins) of multiple HPV types.
41
Describe the process of assembly and release in non-enveloped viruses.
May necessitate host and/or viral accessory proteins such as host chaperones or viral scaffolding proteins.
42
What is the function of host chaperones in the assembly and release of non-enveloped viruses?
- Mediate folding and interaction of viral capsid proteins.
- Mediate capsid protein interaction w/genomic RNA/DNA.
43
How do viral scaffolding proteins assist in the assembly and release of non-enveloped proteins?
- Some viral proteins assemble temporary scaffolds that guide capsid assembly.
- Removed before virion maturation.
44
Most non-enveloped viruses are released through what process?
Host cell lysis; assembly and release are separate steps.
45
How do enveloped viruses acquire their envelope during assembly?
As internal structures bud through membranes.
46
How are enveloped viruses released from the host cell?
- Some bud through the plasma membrane.
- Others bud through the interior secretory compartments and are then carried out of the cell by the excretory pathway (exocytosis).
47
In viruses that bud from the plasma membrane (enveloped), assembly and release occur ______.
Simultaneously.
48
For enveloped viruses with helical nucleocapsids, how does budding occur?
Through interactions with matrix protein and glycoproteins/surface membrane.
49
Under what conditions does a virus enter the secretory pathway (exocytosis) for release from the host cell?
Enveloped viruses in which the envelope is acquired from the internal membrane.
50
Describe the function of viral surface glycoproteins.
Surface glycoproteins in enveloped viruses are produced as precursor proteins that are cleaved which 'primes' the virus for entry/uncoating; this is necessary for the infectious particle as it 'activates' the virus.
51
DNA/RNA packaging in virions is selective for what?
Viral genomes via packaging signals.
52
What are the two major alternative mechanisms for DNA/RNA packaging in virions?
- Co-condensation of protein with DNA/RNA: protein and RNA assembled at the same time.
- DNA/RNA transfer into a preformed, empty capsid: typically involves viral ATPase or helical pump.
53
Describe assembly and release of viruses in epithelial cells.
- Display polarity in that the apical and basal surfaces are chemically unique.
- Viruses bud from one other the other depending on the destination (exterior vs interior).
54
Describe the historic propagation of viruses.
- First in the spp which the infection was identified, then in other animal models.
- Allowed for basic studies and tests of host range; still used to assess viral properties.
55
What are some major drawbacks to the historic propagation of viruses?
- Husbandry/safety in use of larger animals.
- Unknown co-infections.
56
Define in vivo.
- Latin: within the living.
- Procedures performed in a whole, living organism.
57
Define in vitro.
- Latin: within the glass.
- Procedures performed outside of a living organism.
58
Define in silico.
Procedures performed on a computer or via computer simulation.
59
Many viruses are initially propagated where and why?
In embryonated eggs because there is a wide variety of cell and tissue types present in the developing embryo and its environment.
60
Development of in-vitro techniques allowed for what?
Easier/more controlled propagation and more intricate studies.
61
Describe the importance of cell culture as an in vitro technique.
Allows for maintenance of cell lines outside of a host animal (primary cell lines).
62
The generation of immortalized cell lines eliminated the need for what?
The need to continually produce cells from new tissue sources; less variation between batches and more reliable studies.
63
What are the pros of in vitro techniques in virology?
- Enhance quality and reliability of diagnostic testing.
- Allow ID/isolation of viruses as etiologic agents of specific diseases.
- Reduced concern for co-infection with adventitious viruses.
64
What is the cytopathic effect?
Observable change in cells infected with a virus that is absent in uninfected cells: lysis, changes in morphology, syncytia, changes in binding capacity, and inclusion bodies.
65
What happens if a virus does not induce an appreciable cytopathic effect?
Virus-specific tests are needed; often in the form of immunologic-based assays.
66
What discoveries can be made due to in vitro techniques that allow for the recognition of viral growth in culture?
- Cytopathic effect.
- Quantification of virus in a sample.
- Isolation of individual plaque forming units.
- Can identify mutations due to phenotypic changes.
67
Studies of viruses in vivo and in vitro often require what?
Specific, reproducible quantities of virus.
68
How is the quantification of a virus useful in assessing clinical cases?
- Various tissues; determining pathogenicity, selecting correct specimens.
- Determining 'viral load' (burden) over time when assessing treatment.
69
Different types of quantitative viral assays often yield ______ results.
Different.
70
What are the 2 general types of viral quantification?
- Physical assays: don't depend on biological activity of virus.
- Biological assays: depend on viral replication cycle.
71
Give examples of physical quantitative viral assays.
- EM particle counts.
- Hemagglutination assays.
- Immunological assays (ELISA).
- Quantitative PCR.
72
Give examples of biological quantitative viral assays.
- Plaque assays.
- Endpoint titration methods.
73
Describe hemagglutination.
- Some virus-infected cells acquire the ability to bind blood cells on their surface (aggregation).
- Does not accurately count viral particles but is useful in comparing relative concentrations between samples.
74
Describe direct particle counts by electron microscopy.
- Requires expensive machinery/highly trained techs.
- Not very sensitive/specific.
- Most useful with non-enveloped viruses that produce stable particles with unique shapes.
75
Describe quantitative PCRs.
- Determines concentration of virus-specific nucleic acid in a test sample.
- Does not necessitate viral particles.
- Uses copy-number controls to determine the concentration of the target nucleic acid.
76
What is the most common biological assay in virology?
Plaque assays.
77
Who is responsible for the development of plaque assays?
Felix d'Herelle via bacteriophage studies.
78
What are the characteristics of plaque assays?
- Each plaque originates from a 'single' virus/phage, although many are present in the plaques.
- Allows for counting of infectious particles in a sample and determination of concentration (titer) based on dilution.
79
When performing plaque assays, what is necessary for noncytopathic viruses?
Immunofluorescent and immunohistochemical staining procedures.
80
Variations in plaque assays/CPE may identify what?
Mutants in a population.
81
What is an endpoint titration assay?
Inoculation of test animals or embryonated eggs in which successful infections are scored through disease/death of test animal/egg or by confirming immune response.
82
Endpoint titration assays are used to calculate what?
- ID50: dilution of virus that would infect 50% of test animals.
- LD50: dilution of virus that kills 50% of test animals.
- Tissue culture infectious dose 50.
83
Define particle to PFU ratio.
Difference in number of virus detected via physical and biological assays: 100's to 1000's more detected via physical assays.
84
Why are so many more viruses detected via physical assays than biological assays?
- Virion assembly is inefficient and error-prone.
- Not all viruses that bind/enter are capable of establishing productive infection.
- Replication process is error-prone.
- Virus particles can be inactivated when stored at suboptimal conditions.
- Host cell defenses prevent a productive infection.
- Infectivity tests may be performed on animals/cells not optimum for detecting infectious particles.
Veterinary Virology
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