Reverse Genetics in Virology Flashcards
What is reverse genetics?
Causing a change in phenotype by changing the genes and then finding the link to the observed phenotype
what type of virus is the rift valley fever virus?
Bunyavirus
What is the non-pathogenic mutant of RVFV?
MP12
How would you determine which of 23 changes was the one causing a change in phenotype?
Study each change separately and work out which change causes the low pathogenicity phenotype using forward genetics
how would you determine which segment has the important mutations?
Switch the segments around between WT and MP12. The version which has a segment from MP12 and is least virulent is the mutation
What does NS2 do?
Blocks IFN production which allows virus growth. KOs make massive amounts of interferon
What are LATE motifs?
PPXY and PTAP involved in HIV release
what else can reverse genetics be used for other than investigating gene function?
Including the GFP unit as an independent transcriptional unit for tracking the cell tropism of Measles.
How can we mutate large DNA viruses?
Compile DNA genome on a BAC
Manipulate with recombinant techniques
cut WT genome so it cannot replicate
Allow site-specific recombination
What is engineering RNA viruses harder?
Their genomes cannot be engineered directly, DNA versions of the entire virus sequence must first be generated.
how do we engineer + sense viruses?
Generate cDNA on a plasmid and mutate
Transfect into cells that will transcribe the genome as a ss RNA (mRNA)
Viral proteins are translated and new genomes are replicated
Why can we not engineer - sense genomes like + sense?
Cannot transfer the cDNA into cells that will transcribe the genome as ssRNA. need to swap the polarity of the molecules
How do we convert - sense to + sense?
Wrap in N protein
Bind polymerase and transcribe
Translate + RNAs into protein then engineer as you would + sense
What does assembly of the ribonucleoprotein require?
PB1, PB2, PA and NP
What is GISRS?
Global Influenza Surveillance Response System
