3+4: Nucleic Acids, Lipids, Carbohydrates Flashcards
Characteristics of Viral Nucleic Acids:
- Carries the genetic information, determines viral properties
- Relatively small (3–300kb)
- dsDNA, ssDNA, dsRNA, ssRNA
- linear or circular
- continuous or segmented
virions may contain alien nucleid acids like:
- Polyomaviruses, pestiviruses: host cell nucleic acid
- Arenaviruses: ribosomes
- Retroviruses: onc, src genes
Investigation of RNA infectivity can be done by?
- positive sense
- negative sense
What is positive sense?
5´to 3´ direction. Happens fast. It goes straight to mRNA, without the transcription process
- i. e: Picornavirus
What is negative sense?
3´ to 5´ direction. Slow. Need viral enzymes for transcription
What are the different types of electrophoresis?
- Agarose-gel electrophoresis
- sometimes polyacrylamide-gel electrophoresis
Restriction endonuclease analysis consist of:
- dsDNA
- Endonucleases
What are endonucleases?
- Bacterial defense enzymes
- recognize certain sequences
- sticky end - blunt end
What happens in Restriction endonuclease analysis?
- Enzyme cleavage
- Physical mapping
Characteristics of Enzyme cleavage:
- more exact size
- smaller fragments - handling
- -> restriction fragment length polymorph
What are Restriction fragment length polymorph. used for?
- Identification and taxonomy
- Epidemiological investigations
What are the function of Physical mapping?
- Localization of cleavage sites
- partial digestion with endonucleases
Molecular cloning of viral DNA consists of?
- Propagation of virus DNA fragments in bacterial plasmids
- Mass DNA production: quicker, cheaper
- Expression plasmids: protein production
- safe bacteria are needed.
What is the role of Viral Proteins?
- defense and targeting of the genome
- shape of the virion
- enzymes for multiplication
- receptors
Grouping of proteins:
- Structural proteins: surface proteins, core proteins.
- Non-structural proteins
What are/are the function of surface proteins?
Structural proteins
- capsid, envelope
- Shape, antigenicity, adsorption, HA activity, enzymes for penetration or or release of virions
What are/are the function of Core proteins?
- Na protection, Stabilization, enzymes for replication
Where can you find the Non-structural proteins?
- coded in the viral genome
- not integrated in the virion
- present only in the vegetative virus
Types of Non-structural proteins:
- Early, immediate-early proteins
- Late proteins
What are/are the function Early, immediate-early proteins?
Non-structural proteins
- regulation of the cell, inhibition of cellular defence
- enzymes for replication
What are/are the function Late proteins?
Non-structural proteins
- enzymes for structural protein maturation, virus assembly
- (the majority of the late proteins are structural)
Name the methods of protein investigation:
- SDS-Polyacrylamide-gel electrophoresis (PAGE)
- Immunobloting (Western blot)
- Immunoperoxidase staining
- Monoclonal antibody production
Characteristics of SDS-Polyacrylamide-gel electrophoresis (PAGE):
- concentrated virus suspension
- ## SDS + Mercapto-ethanol = Linearization (polypeptides)
Staining used in SDS-Polyacrylamide-gel electrophoresis (PAGE)?
Silver, Coomassie Brilliant Blue dye.
- Polypeptide map
- characteristic to the virus
- -> number and size of viral polypeptides
Function of Immunobloting (western blot)?
Transfer of viral proteins from PAGE into nitrocellulose filter
When do we use Immunoperoxidase staining?
- SDS-PAGE
- Western blot
- histology sections
Function of Immunoperoxidase staining?
- Detection of antigens
- Diagnostics, identification
- mechanism and timing of protein production
- epitope investigation
Characteristics for Monoclonal antibody production?
- MAb produced by a single clone of B cells
- specific for a particular epitope
- Cloning
- Use for diagnostics and virus analysis
What happens if you mix MAb with myeloma cells?
Mitosis, but no AB production
Testing of the clones:
Monoclonal antibody production
ELISA, IF, IPO
–> epitope specific MAbs
What are viral lipids?
- enveloped viruses
- acquired from cellular membrane strictures (budding)
What is the cellular membrane structures of the Viral lipids?
- phospholipid, cholesterol
- virus specific proteins, glycoproteins
- -> poxviruses, ASFV
What are viral carbohydrates?
- ribose, deoxyribose, in the NA
- Glycoproteins, on the surface
pox: in the core to
