Viruses 2 - Replication Flashcards
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 - 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?
1) Penetration (injection of genome) = non enveloped
2) Fusion = enveloped
3) Endocytosis = enveloped
How do viruses bind?
Viruses bind to receptor in cell surface
Glycoprotein on virus bind to protein/ polysaccharide of receptor
What is haemagglutinin?
- H-antigen
- H1-18
- Essential for attachment
- Leads to variation in virulence based on 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 on virus-receptor interaction
What are the two types of pathogenesis?
1) Highly pathogenic
2) 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 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)
What is +ve RNA?
Works as mRNA - can inject genome directly
What is -ve sense RNA?
Must create template, like mRNA before it is read
DNA has 2 strands - what are they?
+ve = sense
-ve = antisense
RNA has 1 strand - what is it?
+ve or -ve sense
What does RNA +ve sense do?
Injects genome into cell to make proteins
What does RNA -ve sense do?
Must first produce +ve strand to produce proteins
DNA viruses mRNA transcription occurs where and what does it utilise?
In the nucleus
Utilises cellular RNA polymerase
Where do 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 their own enzyme.
Where do RNA viruses travel for transcription?
Most remain in cell cytoplasm as no need to enter nucleus
What are ribosomes?
- Cellular organelles composed of RNA protein
- Site of protein synthesis
- Site of translation of viral mRNA - 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 reassortment 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/ shifts
What happens during assembly and exit phase of viral replication?
Re-assembly of viral components and egress from cell
What are different methods of viral release and what are each of these?
- Budding from plasma membrane (enveloped viruses acquire envelope)
- 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 that it can live in the environment and be difficult to destroy.
How does canine parvovirus enter and exit a cell?
Penetration and lysis
What virulence does canine parvovirus have and how does it damage the body?
- High virulence
- It damages the GIT cell and slough off gut cells, can also damage spleen and bone cells.
Why do we culture viruses?
1) Research
2) Vaccine production
3) To express viral proteins
4) 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 an issue using these?
- Only where limited success with eggs or cell cultures
- Ethical clearance required
What age do we use chicken eggs for culturing?
8-11 days
There are 4 main sites of inoculation in chicken eggs that can be used for different viruses - what are these?
1) Chorioallatonic membrane inoculation
2) Amniotic inoculation
3) Yolk sac inoculation
4) Allatonic inoculation
What is a must in cell culture?
Must be appropriate for virus (tropism)
Cell cultures are usually derived from what? What is the 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?
1) Collect sterile tissue sample and cut very small
2) Treat with proteolytic enzymes, obtain cells
3) Suspend in growth medium
4) Cells grow and cover flask by attaching to sides
5) Treat with trypsin - remove cells, transfer to new flasks = passaging
6) Once cells growing cell, can use for viral culture.
What are 2 culture types?
Primary cell cultures
Continuous cell cultures
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?
1) Obtain clinical sample, swab, faeces, tissue
2) Keep cold (ice) or freezing for long-term storage
3) Inoculate in culture medium with antibiotics
4) Look for cytopathic effects as 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 and causes plaque formation
What are the stages to plaque assays?
1) Mix virus dilution with cells. Plate and overlay cells with agarose
2) Remove agarose layer, stain cells visualise 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.
