Lecture 2 Flashcards
What information is encoded in a viral genome?
Replication of viral genome
Assembly and packaging of the genome
Regulation and timing of the replication cycle
Modulation of host défenses
Spread to other cells
All viral genomes must make ____ that can be read by host ribosomes.
mRNA
Is mRNA + or - polarity
+
What is the + strand?
DNA or RNA that is the coding strand (starts with AUG)
What is the - strand?
DNA or RNA that is the template strand. Does not contain open reading frame
True or false: All +RNAs are mRNAs
False. Ex: Genomic RNA vs sub-genomic mRNAs
What are the seven Baltimore classes?
- ds DNA
- ssDNA
- ds RNA
- ssRNA
- ssRNA
- ss RNA reverse transcriptase
- ds DNA reverse transcriptase
Describe Baltimore Class I viruses
Transcribe dsDNA to +mRNA
What are two Baltimore class I viruses whose genomes are copied by host DNA polymerases?
Polyomaviridae, Papillomaviridae
What are two Baltimore Class I viruses that encode their own DNA polymerase?
Adenoviridae
Poxviridae (replicates in cytoplasm, therefore needs to create its own DNA pol)
Describe Baltimore Class II viruses
Convert - or + ssDNA to dsDNA and then transcribes to mRNA
True or false: Baltimore class II viruses can be circular or linear
True
Which Baltimore class viruses use an RdRp?
dsRNA, + RNA and - RNA
What is RdRp?
RNA-dependent RNA polymerase
To make mRNAs and RNA genomes from RNA templates
Which Baltimore class viruses use reverse transcriptase
ssRNA-RT and dsDNA-RT
Describe Baltimore Class III viruses
dsRNA converted to mRNA by RdRp
Describe Baltimore Class IV viruses
mRNA can be directly translated
Describe Baltimore Class V viruses
-RNA gets converted to +RNA through RdRP
Can be segmented or non-segmented
Describe Baltimore Class VI viruses
+RNA gets converted - ssDNA by reverse transcriptase and then to dsDNA
Describe Baltimore Class VII viruses
Gapped dsDNA converted to full dsDNA and then transcribed to mRNA
Uses reverse transcriptase when turning +RNA to dsDNA (replicating genome)
What is reassortment?
Multiple viruses co-infect the same cell
Leads to rapid evolution
How were viral stocks maintained in the 1900s?
Continual passage from animal to animal
Before cell culture, how were viruses propagated?
In embryonated chicken eggs
5-14 days after fertilization, hole drilled in shell, the location of viral injection depends on site appropriate for its replication
What are the two types of cell cultures?
Primary cell cultures
- prepared from animal tissues
- limited life span
Continuous cell lines
- single cell type that can be propagated indefinitely in culture
- HeLa cells: cancer cells
- Tumor tissue or immortalized primary cells
- May not resemble the cell of origin
What is evidence of viral growth in culture cells?
Rounding up of cells, detachment, cell lysis, syncytial formation (cell fusion), nuclear shrinking/swelling, accumulation of virions/viral proteins, membrane alteration
What is the virus titer?
Concentration of a virus in a sample
What is the plaque assay?
Quantitative measure of infectious virus
Count # of infectious viral particles in a suspension
How do you calculate the titer in a plaque assay?
PFU/volume plated x dilution factor
What is a focus-forming unit assay?
Modification of the plaque assay for viruses that do not lyse cells
After viral infection, cells are permeabilized and stained with an antibody against a viral protein
What is the particle-to-PFU ratio?
of physical particles divided by the # of infectious particles
Why are viral particles not always successful at infection?
Damaged particles, mutations, empty particles, complexity of viral life cycles, antiviral defenses
What is MOI
Multiplicity of infection
Average number of cells added per cell (not the same as the # of infectious particles each cell receives)
Depends on random collision of virions and cells
How do you calculate the fraction of cells infected by one or more viral particles?
P(k) = (e^-m) (m^k)/k!
P(k) = fraction of cells infected by k virus particles
m=MOI
e = Euler’s number (approx 2.72)
k! = factorial of k
Fraction of uninflected cells = P(0) = e^-m
Fraction of cells infected with one particle = P(1) = (e^-m)m
Fraction of cells multiply infected = P(>1) = 1-(e^-m)(1+m)
How is MOI used in research?
Low MOI: experiment requires multiple cycles of infection
High MIO: when every cell in culture needs to be infected
Optimal MOI: optimize number of cells that will be infected with a single viral particle
