BB101-Spring 2024-2025-Lecture 07 Sreelaja Nair

Viruses

If you had DNA and RNA but could not make more copies of it
and
if your DNA and RNA codes for proteins
but
you cannot synthesize these encoded proteins

Are you “alive”?

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Viruses

• Viruses contain genetic information as DNA or RNA, which is made into proteins by the
machinery of the host cell
• If viruses require a host cell to come into existence, which came first, the virus or the host cell?
• Viruses infect ALL life forms: bacteria, plants, animals
Figure 19.3 of Campbell’s Biology: a global approach
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Viruses
https://www.youtube.com/watch?v=-w4C74cu6dk

The video above is a schematised movie of the infection and lysis process. It is depicted
in in a very broad manner to convey the general idea
This same process happens when a virus infects any host cell, regardless of whether it is
a bacterial, plant or human cell
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Viruses - no envelope
• To make more copies of itself, a virus particle
known as a virion, has to enter a cell in which
the machinery for genetic material replication
and protein synthesis are functional.
• After it “hijacks” the host machinery and
duplicates its genetic material, it uses the
host machinery to package its genetic
material.
• Then it ruptures the host cell and escapes
into the host body, to find other cells into
which it will enter and repeat the whole
process.
• Viruses infect all life forms, but each type of
virus can infect only a specific species of
animal or a cell type in that animal.
• Excessive intrusion of humans into wild-life
environments has caused an increase in the
ability of viruses to cross species barriers.

Figure 19.4 of Campbell’s Biology: a global approach

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Viruses - with envelope

Genetic information in viruses can be DNA or RNA

DNA or RNA can be double or single stranded
Figure 19.7 of Campbell’s Biology: a global approach
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Viruses - vaccination
Prior infection with a milder variant provides immunity against
subsequent exposure to more virulent strains

• Vacca = latin for cow

• Dr. Edward Jenner formally demonstrated that prior inoculation with cowpox
provided protection from smallpox
• WHO declared smallpox as eradicated in 1980
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Viruses - vaccination
• Louis Pasteur (1822–1895) initially showed
attenuated pathogens provide protection against
Cholera and Anthrax (bacteria) for farm animals

• In 1879, Pierre-Victor Galtier, a veterinary professor,

serially transmitted rabies to rabbits from dogs
• ~1885, Pasteur used suspensions made from
rabies-infected dried rabbit spinal cords to show
that dogs could be protected from rabies infection

• Pasteur`s rabies strategy was post-exposure

vaccine prophylaxis , still used today

• Pasteur found the strategy to vaccinate without

ever isolating the causative agent, which would
eventually be recognized in the twentieth century to
be nonbacterial
Because rabies was 100% fatal, Pasteur went ahead and “vaccinated” humans
bitten by rabid dogs without proper animal trials
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Viruses - vaccination
• End of 19th century, yellow fever, caused by a
flavivirus spread by infected female Aedes
aegypti mosquitoes, had spread around the
world due to slave trading and global markets

• Ships were required to fly the yellow quarantine

flag - main symptom is jaundice

• South African virologist Max Theiler at the

Harvard University School of Tropical Medicine,
USA showed that the disease was viral

• Theiler showed that repeated passage in mouse

brain cultures reduced the effect of the virus on
most organs, but potentially increased its impact
on the central nervous system, which could
cause encephalitis

• Individuals could be vaccinated, but with risk of

neurotoxicity
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Viruses - vaccination

• In 1937, Max Theiler and Hugh Smith developed live attenuated yellow fever vaccine
strain 17D: 176 serial passages in mouse embryonic tissue, then monkey serum, then
minced whole chick embryo and then in chick embryo from which the brain and spinal
cord had been removed.
• After these passages, 17D lost neurotropism, viscerotropism and mosquito
competence, but still triggered an immune response in host

• Theiler showed that the Yellow Fever 17D vaccine prepared from infected whole chick
embryos was safe and effective for human use without the addition of human serum -
made it cost-effective and easier to produce in large quantities

• The Yellow Fever 17D vaccine was approved for human use in 1938 and provides
lifelong protection with a single vaccine dose

• In 1951, Theiler was awarded the Nobel Prize in Physiology or Medicine for
“discoveries concerning yellow fever and how to combat it”, the first and only time the
prize has been awarded for a vaccine
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Viruses - vaccination

• Viruses are obligate intracellular pathogens - cannot multiply

on their own, need a live host cell and its machinery to
proliferate

• In the 1900s, live animals and/or animal embryos were used to

culture viruses

• Later viruses were grown using animal cell cultures - still the
most efficient method
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Viruses - vaccination
In 1930s, Ernest Goodpasture, a US pathologist and medic, grew pure viruses in
culture by infecting fertilized chicken eggs

https://www.youtube.com/watch?v=NTUHX1mRfiE
BB101-Spring 2024-2025-Lecture 08 Sreelaja Nair

Viruses - vaccination
Virus inoculation into chicken eggs - live embryo culture of viruses

https://medschool.vanderbilt.edu/md-admissions/an-egg-drill-and-its-connection-with-medical-discovery-and-advancement/
BB101-Spring 2024-2025-Lecture 08 Sreelaja Nair

Viruses - vaccination
Poliomyelitis - caused by an enterovirus that in rare cases invades the
nervous system and damages motor neurons, causing permanent
disability, paralysis or death

In 1908, Karl Landsteiner and Irwin Popper showed that polio was spread
by a virus:
• Injected monkeys with a suspension of spinal cord from a polio patient
• The suspension was bacteriologically sterile
• However, following inoculation the monkeys exhibited lesions in the
spinal cord similar to those seen in humans with poliomyelitis
• Monkeys also developed paralysis of legs

Polio virus could not be grown in Chicken eggs:

• Albert B. Sabin and Peter K. Olitsky grew poliovirus in fragments of
human embryonic brain
• Risk of using this for vaccine production was neurological damage in
recipients
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Viruses - vaccination
Thomas H. Weller, John F. Enders and Frederick C. Robbins
(1954 Nobel Prize in Physiology or Medicine)
Not for vaccine development, but "for their discovery of the ability of
poliomyelitis viruses to grow in cultures of various types of tissue.”

• grew the human Lansing strain of human poliomyelitis virus in skin,

muscle and connective tissue from human fetal arms and legs
• also produced large quantities of human polio virus in cultures of
fetal intestinal tissue
• succeeded in culturing human poliovirus in cells other than neurons

~1950s two polio vaccines developed:

• an injected vaccine containing inactivated virus, originally
developed by Jonas Salk
• an oral vaccine containing live attenuated virus developed by Albert
Sabin and colleagues
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Viruses - vaccination

Whole virion, which has “forgotten” its original virulence features is used
to trigger an initial immune response

This initial response is remembered by the host body as “immune

memory” and stored in a dormant format

The stored, dormant “immune memory” reactivates when a new

infection occurs with a virulent strain

Is the whole virion necessary to acquire immunity/

protection against viral infections?
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Viruses - vaccination

PMID: 29500037
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Viruses - vaccination
Recombinant DNA technology for vaccine production

• HBV infection results in

production of intact spherical
virions ~42 nm Dane particles,
and overproduction of 22 nm
particles of exclusively HBsAg

• HBsAg is encoded by gene S and

the large form of HBsAg protein
is the most abundant form found
on the surface of infectious viral
particles crucial for HBV binding
to hepatocytes

• Cell-free production of
immunogenic HBsAg in genome-
free virus-like particles (VLPs)
paved the way for genetic
engineering for human health

Empty shell of the virus, can trigger immune response in host, but cannot
replicate and cause infection
PMID: 29500037
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Viruses - vaccination
Recombinant DNA technology for vaccine production

• preventative vaccine made of

human Hepatitis B virus
capsid protein (HBsAg)
• possible because the gene for
HBsAg was sequenced,
cloned and protein produced
in E.coli and Yeast

Empty shell of the virus, can trigger immune response in host, but cannot
replicate and cause infection
PMID: 29500037
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Viruses infect all forms of life

Organisms have evolved strategies to prevent or

overcome infections caused by viruses

One such strategy is to trigger an immune response -

requires a complex immune system

What about organisms that do not have an “immune system”?

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Viruses - how bacteria vaccinate themselves

Clusters of Regularly InterSpaced Palindromic Repeats

PMID: 36656942
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Viruses - how bacteria vaccinate themselves

Clusters of Regularly InterSpaced Palindromic Repeats
• Bacteria acquire viral genome sequences
upon infection and insert it into their
chromosome as spacer sequences

• Spacer sequences from different

viruses are separated by
bacterial DNA repeats

• Spacers are the “memory” of a

bacteria’s encounter with specific
viruses - acquired during an
unsuccessful infection
• This “memory” enables the
recognition and neutralization of
the invading genetic material
upon subsequent infections
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Viruses - how bacteria vaccinate themselves

Clusters of Regularly InterSpaced Palindromic Repeats
• Bacteria acquire viral genome
sequences upon infection and insert
it into their chromosome as spacer
sequences

• CRISPR Associated genes (cas

genes) convert the spacer+repeat
into RNA
• Also makes cas protein, which is a
nuclease (cuts DNA/RNA)
• The spacer+repeat RNA+cas
protein complex “knows” where to
target because of base
complementarity between RNA
sequence and DNA
• This complementarity recognizes
the nucleic acid of the invading
virus when encountered again
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CRISPR - 2020 Nobel Prize in Chemistry

CRISPR discovery CRISPR mode of action CRISPR application

Francisco Mojica Emmanuelle Charpentier Jennifer Doudna Feng Zhang

2005 2011 2012 2013

Francisco Mojica originally identified the spacer DNAs from viral genomes
in archaea and bacterial genomes when he was a PhD student

Mojica coined the term CRISPR

Why is CRISPR such a big deal?

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• DNA can be “broken” in many ways

• Naturally occurring DNA mutations cause human diseases
• DNA mutations made in the lab in animals help mimic human
diseases in model organisms
• Chemicals and radiations could “break” DNA and were engineered
to be efficient
If DNA were a dart board, till 2012, we could ensure that the dart
would hit somewhere on the board
The randomness of the hit meant that one could never target a
specific site on the DNA

With CRISPR
we can hit
bulls eye
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What makes CRISPR specific to a particular site?

If you know the sequence of DNA

You can predict the sequence of the RNA

You can make that RNA in vitro (guide RNA)

Mix the RNA with cas9 protein

The RNA-cas9 protein complex “knows where to go”

This occurs because of sequence complementarity between

RNA and DNA

Allows a particular RNA sequence to target a specific DNA sequence

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Gene editing by CRISPR

• Genome editing by CRISPR-Cas
requires a single guide (sg) RNA
to direct the Cas9 endonuclease
to a specific region of the
genomic DNA
• Requires a PAM sequence
(Protospacer Adjacent Motif), next
to the site of cleavage
• Once it reaches the site on the
DNA, Cas9 cuts the DNA
resulting in a double strand break

The cuts caused by cas9

need to be repaired
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Gene editing by CRISPR - disrupt the gene

DNA breaks caused by cas9 are repaired without external information

Non homologous end joining

- NHEJ

Few bases can get added or removed at the cut site -

PMID: 36656942 alters the original information in a disruptive manner
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https://www.youtube.com/watch?v=4YKFw2KZA5o
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Gene editing by CRISPR - repair the gene

DNA breaks caused by cas9 are repaired with the aid of external information

Homology Repair
• Requires a new piece of DNA
• The sequence of this new piece of DNA is the
“correct” one
• The new DNA sequence is used as a template to
recover the information lost when cas9 cut the DNA
• If cas9 cuts at two places on the DNA, the entire
DNA segment in between the cuts can be fully
replaced

Allows bad information on the DNA to be corrected

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CRISPR is a tool which provides humans an

unprecedented power to change the most important data

What does this power enable us to do as a species?

Should we do it?
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The Immune System

1. Multiple layers of protection

2. Prevent
3. Eliminate

Innate immunity = all

animals and plants

Adaptive immunity = only

in vertebrates

Figure 43.2 of Campbell’s Biology: a global approach

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Innate Immunity - Antimicrobial peptides (AMPs)

AMPs are short protein fragments -

• made when proteins are degraded
• some are encoded by genes

• Upon infection with a pathogen, AMPs penetrate into the phospholipid membrane and
disrupt it by interacting with the lipid bilayer
• This creates holes in the membrane of the pathogen and causes it to lyse
• AMPs can also interact with cellular proteins and nucleic acids inside the pathogen
and disrupt their function
• AMPs are also produced by bacteria - to eliminate competition for a “niche”
• Bacterial AMPs in human gut are important in our immune system
PMID: 34496967
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Innate Immunity - Antimicrobial peptides (AMPs)

How specific are AMPs to one type of pathogen?

Experimental logic
Infect animals with a pathogen

Detect the AMP produced

Check that the AMP is able to neutralize that specific pathogen

Need the AMP to be genetically encoded

Allows one to “tag” the resultant AMP and “see” it

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Green Fluorescent Protein and its variants

GFP is a genetically encoded fluorescent protein - has a high quantum yield
Variants can be made by mutating the original gene

https://www.youtube.com/watch?v=Z4vJ8rQCNgg&t=1s
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Fluorescently tagging proteins in cells and animals

PMID: 19553219
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Innate Immunity - Antimicrobial peptides (AMPs)

How specific are AMPs to one type of pathogen?
Experimental logic
Infect animals with a pathogen

Fuse the AMP gene with GFP gene and “express” it in the animal

AMP is produced fused with GFP protein and will fluoresce when
translated by cells in the animal

Infect the animal with pathogens

Check if the cells are fluorescing

If fluorescence is detected = cells in the animal produced that AMP

against the pathogen it was exposed to
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Innate Immunity - Antimicrobial peptides (AMPs)

How specific are AMPs to one type of pathogen?
Done in Drosophila (fruit flies), using two bacteria as pathogens and two
AMP genes as “read outs”

Pathogens used
fungus Neurospora crassa
bacterium Micrococcus luteus

AMP genes GFP tagged

drosomycin
defensin

Figure 43.4 of Campbell’s Biology: a global approach

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Innate Immunity - Antimicrobial peptides (AMPs)

How specific are AMPs to one type of pathogen?
Experimental logic: must test two things - specificity and function
1. Generate mutant flies that cannot naturally produce AMPs - allows you
to create a functional assay
2. The mutants will only make the AMPs you “express” in the flies, this
will be the AMP::GFP fusion gene you have made - allows you to
control production of one AMP at a time
3. Infect the mutant flies expressing one of the AMP::GFP fusion gene
with one type of pathogen
4. AMP::GFP made in response to the pathogen can be seen by
fluorescence - allows you to check if the AMP made is unique for a
pathogen
5. Allow the infected flies to live. Survival of the flies will be the assay to
test if the AMP produced can kill the pathogen - this is the functional
assay
Logic loop complete
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Innate Immunity - Antimicrobial peptides (AMPs)

How specific are AMPs to one type of pathogen?

Flies infected with fungus Neurospora crassa

What can you interpret and conclude from the graph?

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Innate Immunity - Antimicrobial peptides (AMPs)

How specific are AMPs to one type of pathogen?
Mutant flies expressing drosomycin::GFP infected with fungus Neurospora crassa

Interpretation for drosomycin:GFP experiment

1. ~80% wildtype flies infected with N. crassa live 120 hours and beyond
2. ~90% mutant flies infected with N. crassa begin dying at ~48 hours
3. ~75% mutant flies infected with N. crassa and expressing drosomycin::GFP survive to
120 hours and beyond
4. ~75% mutant flies infected with N. crassa and expressing defensin::GFP begin dying
at ~72 hours
Figure 43.5 of Campbell’s Biology: a global approach
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Innate Immunity - Antimicrobial peptides (AMPs)

How specific are AMPs to one type of pathogen?
Mutant flies expressing drosomycin::GFP infected with fungus Neurospora crassa
Interpretations
1. ~80% wildtype flies infected with N. crassa live
120 hours and beyond
2. ~90% mutant flies infected with N. crassa begin
dying at ~48 hours
3. ~75% mutant flies infected with N. crassa and
expressing drosomycin::GFP survive to 120
hours and beyond
4. ~25% mutant flies infected with N. crassa and
expressing defensin::GFP begin dying at ~72
hours
Some relevant conclusions:
1. Drosomycin production upon infection with N. crassa allows flies to fight the infection
and survive - fact
2. Drosomycin may be produced specifically against N. crassa (need the fluorescence
data as well) - inference subject to additional experiments - logical leap based on
facts
How will you test this inference?
Figure 43.5 of Campbell’s Biology: a global approach