The Infectious Cycle
Lecture 2
Biology 4310
Spring 2021
“You know my methods, Watson”
--SIR ARTHUR CONAN DOYLE
The
Infectious
Cycle
Virologists divide the
infectious cycle into steps to
facilitate their study, but no
such artificial boundaries
occur
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Some important definitions
• A susceptible cell has a functional receptor for a
given virus - the cell may or may not be able to
support viral replication
• A resistant cell has no receptor - it may or may
not be competent to support viral replication
• A permissive cell has the capacity to replicate
virus - it may or may not be susceptible
• A susceptible AND permissive cell is the only
cell that can take up a virus particle and
replicate it
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
• Animal viruses at first could not be routinely propagated in cultured cells
• Most viruses were grown in laboratory animals
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
www.news.sanofi.us
www.vaccinews.net
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
Studying the infectious cycle in cells
• Not possible before 1949 (animal viruses)
• Enders, Weller, Robbins propagate
poliovirus in human cell culture - primary
cultures of embryonic tissues
• Nobel prize, 1954
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
Virus cultivation
Primary human Mouse fibroblast Human epithelial
foreskin fibroblasts cell line (3T3) cell line (HeLa)
continuous cell lines
Diploid cell strains (e.g. WI-38, human embryonic lung)
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University http://www.virology.ws/2009/02/09/the-amazing-hela-cells-of-henrietta-lacks/
Amazing advances in cell culture
Organoid cultures Air-liquid interface cultures
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Go to:
b.socrative.com/login/student
room number: virus
A _______ and _______ cell is the only cell that can take up a virus particle and
replicate it (fill in the blanks)
A. Naive and resistant
B. Primary and permissive
C. Susceptible and permissive
D. Susceptible and naive
E. Continuous and immortal
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
1
Cytopathic effect (CPE)
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University https://www.biorxiv.org/content/10.1101/2020.01.22.914952v2
Formation of syncytia
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Examples of cytopathic effects
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
How many viruses in a sample?
• Infectivity
• Physical: virus particles and their
components
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
Plaque assay
1930s: used to study multiplication of bacteriophages
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
Plaque assay
1952, Renato Dulbecco developed plaque assay for animal viruses
Nobel Prize, 1975
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
Plaque assay
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University virology.ws/2013/06/15/the-wall-of-polio/
Go to:
b.socrative.com/login/student
room number: virus
When doing a plaque assay, what is the purpose of adding a semi-
solid agar overlay on the monolayer of infected cells?
A. To stabilize progeny virus particles
B. To ensure that cells remain susceptible and permissive
C. To act as a pH indicator
D. To keep cells adherent to the plate during incubation
E. To restrict viral diffusion after lysis of infected cells
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
2
How many viruses are needed to form a plaque?
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Plaque purification
A method for producing virus stocks
Usually done 3 times
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
For viruses that do not form plaques: Endpoint dilution assay
TCID50
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Not all virus particles are infectious!
# of physical particles
# of infectious particles
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Particle-to-PFU ratio
• # of physical particles ÷ # of infectious particles
• A single particle can initiate infection
• Not all viruses are successful
- Damaged particles
- Mutations
- Complexity of infectious cycle
• Complicates study
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
Go to:
b.socrative.com/login/student
room number: virus
In the ‘particle to pfu ratio’, ‘particle’ can best be described as:
A. One of the proteins which makes up the virus
B. A virus which may or may not be infectious
C. A virus which is infectious
D. A virus which is not infectious
E. Elementary or composite
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
3
One-step growth cycle:
A method to study virus reproduction in cells
• Ellis & Delbruck, 1939, studies on E. coli bacteriophages
• Adsorb
• Dilute culture
• Sample
• Assay
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Single and multi-step growth cycles
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Synchronous infection - key to one-step growth cycle
To achieve this, we need to infect all the cells - but how do we know?
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Multiplicity of infection (MOI)
• Number of infectious particles ADDED per cell
• Amount of virus (PFU) ÷ # of cells
• Not the number of infectious particles each cell receives
• Add 107 virus particles to 106 cells - MOI of 10 - each cell does NOT
receive 10 virus particles
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
MOI
• Infection depends on the random collision of virus particles and cells
• When susceptible cells are mixed with virus, some cells are
uninfected, some receive one, two, three or more particles
• The distribution of virus particles per cell is best described by the
Poisson distribution
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
P(k) = e-mmk/k!
P(k): fraction of cells infected by k virus particles
m: multiplicity of infection (moi)
uninfected cells: P(0) = e-m
cells receiving 1 particle: P(1) = me-m
cells multiply infected: P(>1) = 1-e-m(m+1)
[obtained by subtracting from 1 {the sum of all probabilities for any value of k}
the probabilities P(0) and P(1)]
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Examples: 107 pfu
If 106 cells are infected at moi of 10:
45 cells are uninfected
450 cells receive 1 particle
106 cells
the rest receive >1 particle
106 pfu
If 106 cells are infected at moi of 1:
37% of the cells are uninfected
37% of the cells receive 1 particle
26% receive >1 particle 106 cells
If 106 cells are infected at moi of .001: 103 pfu
99.9% of the cells are uninfected
00.099% of the cells receive 1 particle (990)
00.0001% receive >1 particle
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
106 cells
©Principles of Virology, ASM Press
Go to:
b.socrative.com/login/student
room number: virus
If cells are infected at an MOI=10 in a one-step growth cycle
experiment, in the growth curve you will likely see…
A. Multiple bursts of virus release
B. Multiple eclipse periods
C. A single burst of virus release
D. No burst of virus release
E. Asynchronous infection
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
4
Physical measurements of virus particles
• Hemagglutination
• Electron microscopy
• Viral enzymes
• Serology
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Hemagglutination
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Measurement of viral enzyme activity
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Enzyme-linked immunosorbent assay (ELISA):
detecting viral antigens or antibodies
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University https://doi.org/10.1038/s41586-020-2012-7
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
Green fluorescent protein
Virology
VirologyLectures
Lectures
20202020
• Prof.•Vincent
Prof. Vincent
Racaniello
Racaniello
• Columbia University
• Columbia University
Deep, high-throughput sequencing
• Metagenomics
• Identification of new viruses in a sample
• Identification of new pathogens
• Human genome: 10 yr/$3B vs 1 day/$1000
(this is not DHTS)
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
Phylogenetic trees
Measure of support:
Sequences to right of
node cluster together
better than other
Root = presumed ancestor
sequences
Degree of genetic change
Scale = # changes/length of sequence
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
Polymerase chain reaction (PCR)
• Research
• Industry
• Diagnosis
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University ©Principles of Virology, ASM Press
PCR product is not the same as infectious virus
For many RNA viruses, RNA can be detected long after disappearance of infectious virus
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University https://doi.org/10.1016/j.celrep.2017.01.056
SARS-CoV-2 RNA and infectivity
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University https://www.cebm.net/covid-19/infectious-positive-pcr-test-result-covid-19/
Cycle threshold and SARS-CoV-2
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University https://www.cebm.net/covid-19/infectious-positive-pcr-test-result-covid-19/
My experience with Ct and antibody tests
https://youtu.be/Lk64Zwcj3W8 https://youtu.be/HvXCISbrK9Q
Virology Lectures 2020 • Prof. Vincent Racaniello • Columbia University
Next time: Genomes and Genetics
