Viruses 2 - replication Flashcards
reminder - viruses 3 in on paper cards
What type of mutations do RNA viruses have?
- more unstable mutations
What happens if a virus is more virulent?
- control is more difficult
What is the control of non-enveloped viruses like?
- more difficult to control and spread more easily
What do we use in diagnosis and vaccination?
- use structural vs non-structural proteins
- DIVA vaccine/test
What is antigenic drift?
- small changes eventually lead to changes in surface proteins (HA and NA)
- happening all the time as the virus replicated such as point mutations
What is antigenic shift?
- abrupt major changes to those surface antigens (HA/NA) - like a host species jump
- can occur if your genome is segmented
The first stage of viral replication is attachment and entry.
what are the different ways this can happen?
- penetration (injection of genome) = non-enveloped
- Fusion = enveloped
- Endocytosis = enveloped
How do viruses bind?
- viruses bind to receptor in cell surface
- glycoprotein on virus binds to protein/polysaccharide of receptor
What is haemagglutinin?
- H-antigen
- H1-18
- essential for attachment
- leads to variation in virulence based in tropism
What is neuraminidase?
- N-antigen
- N 1 -11
- essential for escape
What is tropism?
- the ability of specific virus to infect particular cell, based in virus-receptor interaction
What are the two types of pathogenesis?
- highly pathogenic
- lowly pathogenic
What does a change in tropism lead to?
- change in pathogenesis, symptoms and virulence
What is endocytosis?
- part of the cell machinery for moving large-sized materials into cell through engulfing
How can viruses exploit endocytosis and what different methods do they use?
- to gain gain entry
- they use different methods of endocytosis such as vesicles and pits
What might enveloped viruses do during endocytosis?
- may fuse with endosomal membrane
What happens once a virus has entered a cell through endocytosis?
- once inside there is a pH change which releases virus from endosome
What is the non-endocytic route of entry?
- virus released directly into cytoplasm
- enveloped viruses with fusion at cell surface
What is the non-endocytic penetration route?
- non-enveloped virus attached to host cell and injects virus into cell
Where do DNA viruses replicate?
- replication of genome in nucleus
Where do RNA viruses replicate?
- replication of genome in cytoplasm
What is uncoating?
- release of the viral genome from capsid so it can replicate inside host
How can viruses escape a cell?
- pH change
- fusion
- viral envelope with endosomal membrane
What varies greatly in viruses?
- extent of nucleoprotein complex and capsid disintegration
What must viruses do for replication?
- must replicate its genome
- produce proteins e.g., capsid, glycoproteins if enveloped
- assemble genome and capsid (and envelope)
What is mRNA used for?
- used for transcription (DNA > mRNA > ribosome > protein)
Viruses - different strategies depend on genome:
- DNA v RNA
- RNA > single or double-stranded
- positive or negative sense RNA
What is positive RNA?
- works as mRNA - can inject genome directly
What is negative sense RNA?
- must create template, like mRNA before its read
DNA has 2 strands what are they?
- positive/sense
- negative/antisense
RNA has 1 strand - what is it?
- positive or negative sense
What does RNA positive sense do?
- injects genome into cell to make proteins
What does RNA negative sense do?
- must first produce positive stand to produce proteins
DNA viruses mRNA transcription occurs where and what does it utilise?
- in the nucleus
- utilises cellular RNA polymerase
Where does DNA viruses mRNA get transported?
- transported to ribosomes in cytoplasm for translation
What do RNA viruses use for transcription?
- cells don’t possess enzyme for RNA transcription
- therefore, they must use own enzyme
Where do RNA viruses travel for transcription?
- most remain in cell cytoplasm (no need to enter nucleus)
What are ribosomes?
- cellular organelles composed of RNA protein
- site of protein synthesis
- site of translation of viral mRNA (so are essential to replication)
What are early proteins?
- non-structural (enzymes etc.)
What are late proteins?
- structural (capsid and envelope)
What is assembly/packing?
- packaging of new genomes with viral proteins to form new virus particles
What is reassortment?
- segmented genome allows exchange of gene segments in coinfected cells
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
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
What happens during assembly and exit phase of viral replication?
- re-assembly or viral components and egress from cell
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)
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
How does canine parvovirus virus enter and exist a cell?
- penetration and lysis
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
Why do we culture viruses?
- research
- vaccine production
- as tool - to express viral proteins
- diagnostics
We can culture viruses using living cells - what can these be?
- animal host
- fertilised chicken eggs
- cell cultures
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
What age do we use chicken eggs for culturing?
- use at 8-11 days
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
What is a must in cell culture?
- must be appropriate for virus (tropism)
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
What are the steps required for preparation of cell culture?
- collect sterile tissue sample and cut very small
- treat with proteolytic enzymes, obtain cells
- suspend in growth medium (contains amino acids, vitamins, salt, glucose, buffering solution, foetal calf serum, antibiotics)
- cells grow - cover flask, attach to slides
- treat with trypsin - remove cells - transfer to new flasks = passaging
- once cells growing well - can use for viral culture
What are the two culture types?
- primary cell cultures
- continuous cell lines
What are primary cell cultures?
- from freshly prepared tissue sample
- can survive approx. 15 passages (differentiation prevents further cell division)
What are continuous cell lines?
- immortalised cell - continue to grow
- often derived from tumours
- disadvantage - loss of cell receptors
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
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)
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
What are infectivity assays used for?
- to quantify number of infectious particles produced
-What are plaque assays used for?
- to quantify plaque forming units (pfu/ml)
What do serial 10-fold dilutions use?
– use late dilutions to inoculate cell cultures
What does adding agarose overlay do?
– prevents virus particles contacting the medium
What causes plaque formation?
Virus – infects neighbouring cells cause plaque formation
What are the stages to plaque assays?
- mix virus dilution with cells. plate. Overlayer cells with agarose
- remove agarose later, stain cells to visualize plaques in the monolayer
- 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
