Viruses 2 - replication Flashcards
reminder - viruses 3 in on paper cards (69 cards)
1
Q
What type of mutations do RNA viruses have?
A
- more unstable mutations
2
Q
What happens if a virus is more virulent?
A
- control is more difficult
3
Q
What is the control of non-enveloped viruses like?
A
- more difficult to control and spread more easily
4
Q
What do we use in diagnosis and vaccination?
A
- use structural vs non-structural proteins
- DIVA vaccine/test
5
Q
What is antigenic drift?
A
- small changes eventually lead to changes in surface proteins (HA and NA)
- happening all the time as the virus replicated such as point mutations
6
Q
What is antigenic shift?
A
- abrupt major changes to those surface antigens (HA/NA) - like a host species jump
- can occur if your genome is segmented
7
Q
The first stage of viral replication is attachment and entry.
what are the different ways this can happen?
A
- penetration (injection of genome) = non-enveloped
- Fusion = enveloped
- Endocytosis = enveloped
8
Q
How do viruses bind?
A
- viruses bind to receptor in cell surface
- glycoprotein on virus binds to protein/polysaccharide of receptor
9
Q
What is haemagglutinin?
A
- H-antigen
- H1-18
- essential for attachment
- leads to variation in virulence based in tropism
10
Q
What is neuraminidase?
A
- N-antigen
- N 1 -11
- essential for escape
11
Q
What is tropism?
A
- the ability of specific virus to infect particular cell, based in virus-receptor interaction
12
Q
What are the two types of pathogenesis?
A
- highly pathogenic
- lowly pathogenic
13
Q
What does a change in tropism lead to?
A
- change in pathogenesis, symptoms and virulence
14
Q
What is endocytosis?
A
- part of the cell machinery for moving large-sized materials into cell through engulfing
15
Q
How can viruses exploit endocytosis and what different methods do they use?
A
- to gain gain entry
- they use different methods of endocytosis such as vesicles and pits
16
Q
What might enveloped viruses do during endocytosis?
A
- may fuse with endosomal membrane
17
Q
What happens once a virus has entered a cell through endocytosis?
A
- once inside there is a pH change which releases virus from endosome
18
Q
What is the non-endocytic route of entry?
A
- virus released directly into cytoplasm
- enveloped viruses with fusion at cell surface
19
Q
What is the non-endocytic penetration route?
A
- non-enveloped virus attached to host cell and injects virus into cell
20
Q
Where do DNA viruses replicate?
A
- replication of genome in nucleus
21
Q
Where do RNA viruses replicate?
A
- replication of genome in cytoplasm
22
Q
What is uncoating?
A
- release of the viral genome from capsid so it can replicate inside host
23
Q
How can viruses escape a cell?
A
- pH change
- fusion
- viral envelope with endosomal membrane
24
Q
What varies greatly in viruses?
A
- extent of nucleoprotein complex and capsid disintegration
25
What must viruses do for replication?
- must replicate its genome
- produce proteins e.g., capsid, glycoproteins if enveloped
- assemble genome and capsid (and envelope)
26
What is mRNA used for?
- used for transcription (DNA > mRNA > ribosome > protein)
27
Viruses - different strategies depend on genome:
- DNA v RNA
- RNA > single or double-stranded
- positive or negative sense RNA
28
What is positive RNA?
- works as mRNA - can inject genome directly
29
What is negative sense RNA?
- must create template, like mRNA before its read
30
DNA has 2 strands what are they?
- positive/sense
- negative/antisense
31
RNA has 1 strand - what is it?
- positive or negative sense
32
What does RNA positive sense do?
- injects genome into cell to make proteins
33
What does RNA negative sense do?
- must first produce positive stand to produce proteins
34
DNA viruses mRNA transcription occurs where and what does it utilise?
- in the nucleus
- utilises cellular RNA polymerase
35
Where does DNA viruses mRNA get transported?
- transported to ribosomes in cytoplasm for translation
36
What do RNA viruses use for transcription?
- cells don't possess enzyme for RNA transcription
- therefore, they must use own enzyme
37
Where do RNA viruses travel for transcription?
- most remain in cell cytoplasm (no need to enter nucleus)
38
What are ribosomes?
- cellular organelles composed of RNA protein
- site of protein synthesis
- site of translation of viral mRNA (so are essential to replication)
39
What are early proteins?
- non-structural (enzymes etc.)
40
What are late proteins?
- structural (capsid and envelope)
41
What is assembly/packing?
- packaging of new genomes with viral proteins to form new virus particles
42
What is reassortment?
- segmented genome allows exchange of gene segments in coinfected cells
43
What does reassortments still have the potential to do?
- still has the potential to alter nature of infection
- e.g., may affect resistance to pre-existing immunity
44
What is viral mutation?
- pulls multiple strains and combines into a new one which the host may not have immunity against
- this is why flu jabs are repeated - new mutations/strains
45
What happens during assembly and exit phase of viral replication?
- re-assembly or viral components and egress from cell
46
What are the different methods of viral release and what are each of these?
- budding from plasma membrane (enveloped viruses acquire envelop)
- exocytosis ( cell wall lets virus pass)
- lysis (cell rupture - non enveloped)
47
What is the structure of canine parvovirus and how does this aid spread?
- a small non-enveloped virus, which means it can live in the environment and be difficult to destroy
48
How does canine parvovirus virus enter and exist a cell?
- penetration and lysis
49
What virulence does Canine parvovirus have and how does it damage the body?
- high virulence
- it damages the GI tract cells and slough off gut cells, can also damage spleen and bone cells
50
Why do we culture viruses?
- research
- vaccine production
- as tool - to express viral proteins
- diagnostics
51
We can culture viruses using living cells - what can these be?
- animal host
- fertilised chicken eggs
- cell cultures
52
When can we use animal hosts for culturing and why is there issues using these?
- only where limited success with eggs or cell cultures
- ethical clearance required
53
What age do we use chicken eggs for culturing?
- use at 8-11 days
54
There are 4 main sites of inoculation in chicken eggs that can be used for different viruses what are these?
- chorioallantoic membrane inoculation
- amniotic inoculation
- yolk sac inoculation
- allantoic inoculation
55
What is a must in cell culture?
- must be appropriate for virus (tropism)
56
Cell cultures are usually derived from what? and what is an advantage of this?
- tissue samples such as lung, kidney, liver
- ADVANTAGE - easy to manage and scale up
57
What are the steps required for preparation of cell culture?
1. collect sterile tissue sample and cut very small
2. treat with proteolytic enzymes, obtain cells
3. suspend in growth medium (contains amino acids, vitamins, salt, glucose, buffering solution, foetal calf serum, antibiotics)
4. cells grow - cover flask, attach to slides
5. treat with trypsin - remove cells - transfer to new flasks = passaging
6. once cells growing well - can use for viral culture
58
What are the two culture types?
- primary cell cultures
- continuous cell lines
59
What are primary cell cultures?
- from freshly prepared tissue sample
- can survive approx. 15 passages (differentiation prevents further cell division)
60
What are continuous cell lines?
- immortalised cell - continue to grow
- often derived from tumours
- disadvantage - loss of cell receptors
61
What are the steps involved in viral culture?
- obtain clinical sample, swab, faeces, tissue
- keep cold (ice) or freezing for long-term storage
- inoculate in culture medium with antibiotics
- look for cytopathic effects as a signs of viral infection
62
What are cytopathic effects?
and
When looking for cytopathic effects what do you look for?
- visible changes to host cells observed under light microscope
- holes, cells round up and detach
- syncytia, cells fuse, multi-nuclei
- inclusion bodies (protein masses within cells)
63
What are non-cytopathogenic viruses?
- many viruses, no CPE or signs of replication but will get viral particles
- will have to stain for virus protein - colour change
64
What are infectivity assays used for?
- to quantify number of infectious particles produced
65
-What are plaque assays used for?
- to quantify plaque forming units (pfu/ml)
66
What do serial 10-fold dilutions use?
– use late dilutions to inoculate cell cultures
67
What does adding agarose overlay do?
– prevents virus particles contacting the medium
68
What causes plaque formation?
Virus – infects neighbouring cells cause plaque formation
69
What are the stages to plaque assays?
1. mix virus dilution with cells. plate. Overlayer cells with agarose
2. remove agarose later, stain cells to visualize plaques in the monolayer
3. virus titre is determined by counting plaques and multiplying by the dilution factor. Plaque counts from at least 3 replicates at each dilution should be averaged
