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Microbiology - Lecture 09 Bacteriophages continued, Viruses by Dr. Boylan

[Bacteriophage powerpoint, slide #7 Protein Components of a Phage]
[Dr. Boylan] We will continue discussion of bacteriophage. Remember those are phage or
viruses that actually infect bacteria. So you could say in a way bacteria also succumb to
infectious diseases, theyre infected by viruses, by bacteriophages. And we went through
the general structure of one of the types of bacteriophage, called T2, which is a coliphage, a
phage that infects E. coli. We talked about the different components of it, the capsid head,
and inside well see, thats where the nucleic acid is. And then the tail fibers, that help the
phage attach to the surface of the bacterial cell. Remember you always have to have
adherence first adhesion. Then the next steps can occur. But everything has to stick first
to its host cells before it can do any damage at all.


[Slide #8 Phage Replication]
Replication, here we have another closer view of that T2 phage. You can see the DNA is
wrapped up, tightly coiled inside the head, the capsid head. Protein and DNA. And the rest
of it is the tail, we went into the different components of the tail yesterday. The collar, the
hair [?], the sheath, the fibers, and the prongs. So the replication of the bacteriophage is a
five-step process. We arent going to go through every step in detail, but just get the
overview of what happens when bacteriophages, these that are virulent carry out a lytic
cycle of infection.

[Slide #9 Replication of a Lytic Bacteriophage]
And here we have a lytic cycle where the phage, this is a cell wall of a gram negative
bacterium like E. coli, adhesion occurs through the tail fibers theres a receptor there. So
all these different bacteria, like E. coli and Staph aureus, they have, they are only infected
by phage for which they have receptors. These tail fibers or other components of the virus
have to find something they can adhere to, some receptor, or else they wont infect that cell.

And here we see, probably something like the cell wall, maybe a lipopolysaccharide,
sometimes they even bind to flagella or pili, something on the cell surface as far as the point
of attachment. And then after the tail fibers adhere, you can see they kind of push down a
little bit, and the prongs also, that part of the virus also penetrate the cell wall, and heres
the key step here, where you have the injection of the nucleic acid into the bacterial cell on
the bottom here. So what happens is the virus attaches, the prongs grab hold too, and then
the sheath of the tail contracts like a muscle, a syringe, and this forces the DNA right into
the bacterial cell, from the capsid, the head, into the bacterial cell by contraction of the
sheath. And we know, one big difference between bacteriophage and viruses that infect
humans, mammalians, animals, as well as viruses that infect plants, is that with phage, only
the nucleic acid gets inside the cell. Thats a big thing to remember, whether it be DNA or
RNA phage, only nucleic acid enters the cell. With the viruses that infect us and plants and
animals, the whole virus, youll see later on, does get inside our cells in order to initiate its
replication. But in phage only the DNA is shown here, penetrates. How do we know that?
How do we know that the capsid doesnt get in as well, and the proteins and the tail fibers,
etc? How do we know that? Well in a genius experiment carried out years ago by Hershey
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and Chase. Have any of you heard of these two scientists? Maybe if youve taken virology
And they said, how can we prove that maybe the whole phage does get inside the bacteria.
How can we differentiate between these two possibilities, the whole virus or just the
nucleic acid? So what they did was they got viruses and through techniques that they used,
they labeled the proteins with one type of isotope and nucleic acid with another type of
isotope. So what isotope do you think is or what element is found in proteins that is never
found in DNA or RNA? Sulfur, exactly. So you have sulfur-35 to label the proteins, and how
about, what did they use to label the proteins? Phosphorus. P-32. You never find
phosphorus in amino acids. So they labeled these viruses as shown here, nucleic acid with
P-32, proteins, capsid, and other components with sulfur-35. And then they injected the
bacterial cells. Later on they got the cells, broke them apart to see what was inside and
they found out only P-32 was inside. No sulfur at all, so that means only nucleic acid
entered the cells. With mammalian viruses that infect us, we did find sulfur as well as P-32
inside.

[Slide #10 Stages in Lytic Cycle contd]
We are going to go through these other stages in more details in the course on infectious
diseases when Dr. Levy gives the presentations. But in essence, they attach, they penetrate,
and then they have to synthesize themselves. So heres this piece of DNA, or RNA, you have
a virus, and all it wants to do is make more of itself. Thats its goal, make more nucleic acid,
make more protein. Make more nucleic acid, be it DNA or RNA, make more proteins for the
capsid and tail fibers. Thats all. Make more of itself, replicate itself. And to do that it takes
advantage of the host metabolic machinery. I talked yesterday about, you know your
brother-in-law coming for a visit like the prophage in the temperate phage. This is like
your mother-in-law coming for a visit. Once that cell is infected by the piece of DNA or
RNA, everything changes inside of that cell. Its like the DNA hijacks that metabolic
machinery of that cell, so that instead of the bacterial cell replicating more materials that it
needs, everything is directed towards what the virus needs. So the virus enters the cell,
begins production of more enzymes for nucleic acid synthesis that is viral, more proteins
that are viral. Thats all it wants to do, make more DNA or RNA, and protein, depending on
the virus. The initial proteins that are produced inside the bacterial cell that the virus
replicates are called early proteins. Early proteins. And they are usually enzymes that help
the viral replication of the nucleic acid take place. So first order of business, lets get the
nucleic acids synthesized that we are going to need for the genome. And when that is taken
care of, some of the later proteins come into play that are virally directed. And they are
enzymes used to form the proteins in the capsid and the tail fibers, or other protein
components of the virus. So thats what biosynthesis is in general. Early proteins, late
proteins. And one of the late proteins actually is our familiar enzyme called lysozyme.
These viruses have a gene often, that codes for lysozyme. So once theyve reproduced
inside the cell, formed hundreds of progeny, they want to get out of the bacterial cell. Well
heres that rigid cell wall, preventing it from being released, from escaping. So they use
lysozyme to hydrolyze that peptidoglycan bond between the sugars, lysing the cell and
releasing all the progeny viruses that then go on and infect other bacteria that happen to be
anywhere in the area. The process goes on over and over again.
Maturation is the assembly of viral parts into complete viral particles. Nucleic acids are
synthesized, proteins, capsomere proteins, etc are synthesized. Then they just kind of
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confine to each other, come together in just the right form. Its a self assembling processes.
Heres nucleic acid, its gotta be surrounded by capsomeres to form the capsid. The
capsomeres just sort of find each other in the right formation, the right structure for that
particular virus. No energy is needed, they just kind of float around in a way and form the
capsomeres of the capsid, form components of the tail, tail fibers etc, and that is finally
maturation. That means the virus is now in essence ready to infect other cells once it is
released from the bacteria. So then they are finally released by lysis, often with aid from
one of the late proteins, lysozyme.

[Slide #11 Counting Phage]
I think Dr Saxeena probably went over serial dilutions with you. To count how do we
count phage. Do we know how to count bacteria, I believe? Serial dilutions of bacterial
populations and then plating, putting bacteria on plates and then counting the colonies that
form, did he go through that?
Well in essence the same procedure is followed with phage. Except of course they dont
grow, they wont grow on petri plates or any other non living material. So how do you
count? How would you know, for example in this test tube here, theres bacteriophage. No
bacteria at all, but there all phage [in the left most test tube]. How do you know whether
there are say, 10/mL or 10,000 or 10 billion phage per mL? The way you do it is dilute it.
You have to keep diluting it 10 fold over and over again, dilute the first tube in the second,
the second in the third So here we have a series of 10 fold dilutions of phage. You take
1mL out of here for example [left most tube] and add to 9mL here [tube to the right of the
first one], so 10 fold dilution. 10 fold dilutions. And finally in this last tube, in this
particular example here, you plate 1/10
th
of a mL, always 1/10
th
of a mL, you dont want to
plate a lot, and you get lets say two circles, two holes. Each hole, or each circle, you call
them plaques. Plaques. Those are the sites, shown here on the right, those are the sites that
the virus replicated. So heres a petri plate, and this white background here is all a
bacterium. A lawn of bacteria, from one side to the other. It covers the whole plate. And in
the middle are various holes, circles called plaques. This is where you added your viral
sample, and when the viruses replicated inside the bacteria, they replicated and they killed
one cell then another, then another cell.
So each plaque here, just like a colony when you serial dilute the bacteria, each plaque is a
result of infection by this one phage. So lets say in this example shown here, they did this,
they did these serial dilutions and they found out that when they plated 1/10
th
of an mL
that only two plaques appeared on this bacterial lawn. How many phage per mL would
there be in this first tube? One, two, three 10 fold dilutions. Then you only plate here on
the petri plate 1/10
th
of a mL, and you get just two plaques. Heres the question I want you
to work on a little bit. How many phage were there per mL, 1mL, in this first tube [on the
left] if you wind up with just two plaques when you finally put that 1/10
th
mL from that last
tube [right most] onto the plate, which is shown here, but only get two plaques? So give
that a little bit of thought, if youve had serial dilutions using bacteria its the same process
that is followed. 10 fold dilutions, making it more dilute each time, so eventually you can
count them. But you see here, if you plated this sample [the 1/10 phage tube], there would
be too many to count [indicating the cartoon of the petri plate shown below the tube]. If
you plated this one, maybe, its a lot here [indicating the 1/100 phage dilution and petri
plate cartoon] you could count those as well but it looks like there are too many, they are
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each growing inside each other and next to each other so its hard to count the individual
number of plaques. Here, it is much easier [indicating the 1/1000 tube and petri plate
cartoon]. So you end up with two. How many phage were there in the first [undiluted]
tube per mL, a puzzle I might ask you, a problem that might come up in the exam,
probably.

[Slide #12 Temperate Phage, lambda]
Temperate phage. So weve looked at lytic phage in more detail then temperate so far. The
other type of phage, the temperate are the ones that inject themselves, they are always
DNA, they inject the DNA, becomes a part of the bacterial chromosome, the DNA phage in
the chromosome of the bacterium is called a prophage, infects the cell, becomes a part of it.
Replicates when the chromosome replicates. And now we have a state of, what is called on
the right here, lysogeny. So heres the virus, heres when it is lytic, we just went through
that. Here is the lysogenic cycle where the virus becomes a part of the chromosome of the
bacterial cell. And every time that bacterial cell replicates, the prophage will also replicate
along with it. Generation after generation after generation, until finally we can either
induce its excision by UV light, in other words, cause it to break out, break free of the
chromosome. Or sometimes just spontaneously. Sooner or later its going to break free
and then go through a lytic cycle and kill that bacteria or its progeny. So the prophage
eventually will go through from the lysogenic cycle to the lytic cycle once the prophage is
excised from the chromosome, be it spontaneously, after perhaps many many generations,
or UV induction or some other ways, tricks we use in the lab to get that process to go from
lysogenic to lytic. So its a lysogenic bacterium, the phage arent lysogenic but the bacteria
are lysogenic, its like a ticking time bomb once again, inside, the lysogenic stage. Eventually
this time bomb that is inside the cell will be excised and cause the death of the cell.

[Slide #13 Steps in lambda Integration and Excision]
Looking at it more closely, lambda phage, we just saw the structure before. Heres a
bacterial cell, chromosome, heres lambda phage, which is a DNA phage. And on the
chromosome of the bacterial cell we are going to focus on one particular part. One gene or
one locus of the bacterial chromosome, called the gal locus. Gal for galactose. Thats the
gene these bacteria use, have, to essentially utilize galactose for energy. Thats the gene
they turn on for that, use galactose that they are fed, break it down and use it for energy.
And here is the injection of the lambda phage DNA, it circularizes, these things always
circularize before it goes on to the next step. But note here it is going to become a
prophage in the chromosome, but note that it becomes a prophage always adjacent to the
gal locus. Always at that same site. So in this particular phage, lambda, integration of the
DNA always occurs next to what is called the gal locus. Very specific, always. Well see with
some other phage thats not the case, they might integrate here or there, no particular site.
But lambda, no. Then when you have now the prophage next to, adjacent to the gal locus,
you irradiate the bacteria, sometimes theyll just begin the lytic phase, but other times look
what happens. Instead of breaking free, when this cell here will be induced by UV light, or
once again spontaneously sometimes, instead of just a clean break of the prophage DNA,
sometimes by mistake it takes some of the adjacent bacterial DNA with it. This is
something important to remember. That sometimes it takes the adjacent gal locus with
it. It will sometimes take with it a bacterial gene. Its not supposed to, its supposed to be a
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nice, clean break, have only prophage. But in this case, the bacterial genes are also picked
up as the prophage is excised from the bacterial chromosome. And here we see an example
of it here. This particular phage that is fromed here, its also got the gal locus from the
bacteria. The point being that sometimes bacteriophage carry adjacent genes with them,
and then when they go on to infect other bacterial cells, they carry these bacterial genes
with them as well as their own. Transfer of genes among bacteria by viruses, a process
called transduction. We will discuss that more next week when we talk a little bit more
about bacterial genetics.

[Slide #14 Elements of Lysogeny]
So lysogeny, phage in the genome, prophage is formed. Bacterium carrying the prophage is
a lysogen. These are immune to further infection by similar phage because the phage
functions are repressed. The phage functions are repressed. Induction of the lysogen leads
to the excision of the prophage, replication of the phage DNA, and finally lysis of the host
bacterium. But once again, also when this excision occurs, sometimes additional bacterial
genes are taken along, a little piece of luggage taken along by the prophage DNA as well,
and they are introduced into the next bacterial cells that these phage infect.

[Slide #15 No title]
So here we have, once again both types of pathways. Lytic, weve gone through where once
again the phage DNA begins immediate replication of the phage DNA and proteins and form
new, mature lytic phages which then go on are released and do the same thing over and
over again. So we see here that ordinarily these [lytic] viruses, they do not have and
bacterial DNA with them, over here is chromosomal DNA [indicates on image of lytic cycle].
But here, with the temperate phage, the prophage integrates and it can sometimes take
along with it this blue here, can take along with it adjacent bacterial genes such as galactose
with the lambda phage.

[Slide #16 Lysogeny]
So lysogeny, ok I guess thats a repeat. So here we have a prophage. Lets just think that it is
carrying adjacent genes with it.
Heres a prophage, from the bacterial chromosome, just a prophage alone, no gal, no other
bacterial genes. Lets say it has 20 genes, but maybe only about half of them, 10 of the 20,
are involved in viral replication. The other 10 are something else. So when this DNA is
integrated into the chromosome as a prophage, it doesnt replicate, it doesnt excise. All
these genes, say 10 of the 20 genes are repressed, they are not being used to synthesize
more of the viruses because they are repressed, and as long as they are repressed the virus
will never excise. But sometimes these other genes, the other 10 not involved with phage
reproduction, are still active. These genes are not repressed, they are genes that are still
active, meaning they can lead the production of new proteins or new enzymes that do
something else not involved with phage reproduction at all. One of the most important
functions, from a medical point of view, is the synthesis of several exotoxins in bacteria.
Exotoxins, weve already seen what endotoxins are, they are toxins from the gram negative
bacterial cell wall. But, gram negative and gram positive bacteria can both produce
exotoxins, which are released from the bacteria to be toxic to our cells. Diphtheria,
botulinum, cholera, and erythrogenic toxins of some steptococci are encoded by genes on
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the prophage. Thats the key Im trying to make here. These bacteria we are going to hear a
lot about later on. The diphtheria bacteria, the one that causes botulinum, cholera, and skin
infections, scarlet fever, things like that, those bacteria would be harmless, would not cause
any infection at all in humans, unless they had the prophage, a particular prophage, that
has the genes for, for example, diphtheria toxin, botulinum toxin, or cholera toxin. And we
will see, in not just these, but in many other infections caused by bacteria are the result of
the genes that they have in the chromosome as a result of being infected and enorporating
this prophage. Of course many bacteria are infectious without prophage, their own genes
code for toxins, chromosomal genes. The key here, many infections are the result of genes
in prophage, code for toxins that kill or damage our tissue and lead to very serious
infections. If you get rid of that prophage from those bacteria, they are harmless. They
dont produce the toxins now. They still have theyre whole chromosome but they dont
have those genes from the prophage.
Well these bacteria such as shown here go from say a non-pathogenic state, harmless, to a
pathogenic state, they pick up new properties. They are avirulent, or not virulent at all, to
virulent. And when they pick up new properties as a result of this prophage integration I
just talked about, picking up genes for toxin production, they have a new property. This is
called lysogenic conversion, a term applied to the new properties that a bacterium acquires
as a result of expression of the integrated prophage genes. So once again, maybe 20 genes,
about 10 of them involved with phage reproduction, they are repressed, the other 10 genes
can do various things. Sometimes these genes like in the bacteria shown here code for
toxins that damage our tissue, because these bacteria have changed from one type to the
other, non-pathogenic to pathogenic, theyve picked up this new property. They are now
pathogens. This is called lysogenic conversion by definition. Not just toxins, but maybe
they have other new properties, might have differences in their cell wall, differences in
their flagella. But once again as a result of prophage integration, new properties acquired.
That is called lysogenic conversion and it is very important for a lot of different
infections youll hear about later on in the next course.

[Slide #17 Lysogenic Conversion]
Shown here once again [left image]. Diptheria. Heres the chromosome, phage, its called
Beta phage, infects the cell. Now, integrates [moves to right image]. Beta phage genes are
here, and heres the gene from the phage that codes for the toxin for diphtheria. Now this
bacteria which was formerly non-pathogenic is now a pathogen because it produces the
toxin that destroys our tissues and the mucous membranes in the throat, leading to
diphtheria in this particular case.

[Slide #18 Phage Typing]
One last thing about phage Id like to mention briefly is that, its called phage typing. Its like,
in a way, fingerpinting different bacteria. I start this off usually by talking about all of us
here in this room. We are all members of the same genus and species, Homo sapiens, every
one of us, are we identical? Of course not. We all are different. Bacteria are the same thing.
Maybe Staphylococcus aureus, that same genus and species, in a population of them, there
are many different, individual cells that have different properties. And some of these new
properties make the bac very path, and others make them harmless or dont do anything at
all to them. But very often, with that in mind, realizing that there are some staph, for
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example Staph aureus are very pathogenic, and others that are not. when a staph infection
occurs in a hospital, an outbreak of Staph infections, what they often want to do is find out
what particular Staph, what serial type, or strain, serial type or strain with these different
properties is responsible for the epidemic or outbreak in the hospital. So what they want
to do is track down, everybody carries Staph aureus with them, patients coming in, doctors,
nurses, dentists there, everybody. And they sample all the health personnel for the Staph
aureus and take it back to the lab. And they say, Ok, the outbreak of Staph was caused by
this particular serial type. Where did it come from, who brought it into the hospital? How
can we pinpoint, fingerprint precisely, the source of the outbreak? And one of the ways
they do this is by phage typing. All these different serial types or strains of Staph aureus
have slightly different properties. So some of them will be susceptible to some phage and
resistant to others. What they did on this plate, a lawn of Staph aureus is grown here, the
background here, kind of tan. And you cant see all of them but they put about 1, 2, 3, 4,
maybe 8, 10, 12, different phage. Spotted them individually, different bacteriophage onto
this lawn of Staph aureus and see if the phage in that drop kill them or not. If the phage did
kill them, it clears on the plaque. If the bacteria are resistant to the phage, there would still
be solid, good growth of the bacteria. So this pattern you see here, you can see obviously
there are, of all the different phages they used, five of them were really, clearly sensitive.
One of them was partially sensitive but the other ones they were resistant to. You put
another serial type of Staph aureus on a plate you might get clear zones here and here, no
zone here, so this pattern, its typing. Its identifying Staph aureus, in this case by using
phage. This is the way to track down the source of an infection. Get the particular serial
type or strain of Staph aureus and once again everybody carries Staph aureus on us, our
skin and our nose, weve all got it, but its not killing us, they arent doing any harm right
now, but some of these are very, very virulent, dangerous. So lets do what we can. Its
called epidemiology, tracking down the source of infection in a hospital or anywhere else.
CDC does it, this is what they do. They track down the source of an infection in a hospital or
an area, a geographical part of the country, But they use different techniques and often one
of them is typing, identifying the particular strain of the bacterium.
So that does it for phage. Id like to now discuss something about viruses in general, which
was the topic for today.

[Viruses powerpoint, Slide #1 Virus Properties Introduction]
So once again bacteriophage or phage are bacterial viruses that infect only bacteria. There
are RNA phage and DNA phage for bacteria. They all have only one type of nucleic acid,
RNA or DNA. They all have capsomeres, so its kind of a simple system. You saw what
happens when T2 infects E. coli, lytic cycle. And the lambda phage, the temperate phage
infects the E. coli and it goes through the lysogenic cycle, the lysogenic type of replication.

[Slide #2 Viruses]
So lets take a look, if this were a separate lecture completely on viruses, we didnt know
anything about them. Lets go back and look not just now on viruses that infect bacteria but
viruses that infect all the other living organisms on the earth, humans, plants, animals, etc.
So a lecture on viruses. What is a virus? Well probably the best definition is given by this
virologist, named Lwoff, he said a virus is a virus. Which means essentially there is nothing
else quite like a virus. It is unique. And youd have to really spend a lot of years
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understanding what they are and how they work. We know a lot, since this time of course.
But still a virus is unique, its not a bacterium, its not living. We went through the general
properties of viruses when we talked about phage. They were first identified in 1890 or so
during the golden age of microbiology. They realized when they started to grow all the
bacteria that most of the bacteria we know of cause infections, still not quite all. Most of
the bacteria in the lab they could grow, identify the source of infection using Kochs
postulates. But they realized there is something even smaller than bacteria, that they could
not see with the light microscope, and they wouldnt see it for a few more decades until the
electron microscope came along. But there had to be something smaller than bacteria,
because the infections could be transmitted from plants to plants, animal to animal, human
to human by these things that are smaller than bacteria. You couldnt see them, clear, clear
broths, bacteria free, but still something in there that if you applied it to a patient, it would
cause infections. So they called viruses poisons, because virus means poison in Latin. There
are certain viruses associated with cancer in animals. They were discovered back in the
early part of the last century. Bacteriophages, of course. Then the 1940s when the electron
microscope was really finally tuned and you could magnify things, samples of bacteria and
viruses and everything, thousands if not millions of times [magnified]. Now they could
really see these poisons, these viruses, that were there but invisible until the electron
microscope came along. And they saw the shapes of the viruses here on the right.
So you cant just say viruses are tiny things, tiny little particles, they differ in size, heres the
smallest [pointed to the parvovirus], heres one that is very, very large [poxvirus], different
length and diameter [pointed to filovirus], so we will see just a few of some of the different
types of properties that viruses have as a result of being able to visualize them with the
electron microscope. The ultimate parasite, once again. They are not growing, they depend
on living cells. They take over the machinery of living cells once they infect them and
redirect everything in that cell, be it plant, animal, to the synthesis now of viruses and thats
how they replicate.

[Slide #3 Virus Cultivation 1]
How do we cultivate them? You need viable cells. Back in the time of Pasteur, he was the
one who came up with the vaccine for rabies infection. He used to cultivate the rabies virus
in rabbits, living animals. Thats still one way you can do it, has to be living cells. Another
way is using embryonated eggs. Chick eggs are still often used. Shown here. The chick
embryo, we cultivate the flu virus, we want them to grow in an embryonated chick shell.
Where there are indeed within the eggs shown here chick embryos. Not only that but all the
sacks, the fluids inside, all the membranes that support the growth of the embryo are there.
So all of these are the living cells in the egg here that will support the growth and
replication of viruses. Ordinarily we dont use that except for the influenza virus. We can
not replicate the influenza virus even today by the way Ill talk about next, tissue culture.
So we still use embryonated chick eggs for rep of the flu virus. We only have a little bit of it,
we want to get more of it. You can see here this is probably an assembly line for getting the
influenza vaccine for the following year. Growing thousands if not millions of these
embryonated chick eggs and inoculating them with a little bit of flu virus to have it
replicate to millions of flu viruses which can be used for the vaccine.
The main way that today we cultivate or grow viruses is tissue culture or cell culture. Have
any of you read this book on the right here? The immortal Life of Henrietta Lacks? Im sure
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many of you have read it or at least have seen this book. It came out about half a dozen
years ago, was a best seller. The importance of Henrietta Lacks was that part of her is still
alive today. She actually passed away in the 50s, but part of her cervix, part of her cervical
tissue is still growing not just in the United States labs that study viruses but throughout
the world. Her cervical tissue is called HeLa cells, from a human carcinoma of the cervix.
She had cancer of the cervix. Cancer means of course unlimited cell growth essentially, you
cant stop these cells from growing. These cell grew so well, so abundantly, so easy to grow,
her tissue cells, that we shipped all over the world. They are a good source of cells to
replicate many many many viruses. This is called cell or tissue culture. Examples are the
HeLa cells that are used, so Henrietta Lacks. A very interesting book about her life. And of
course, even her family did not know anything about this, the use of their mothers cells in
viral studies throughout the world until maybe 15-20 years ago. The book is the story of
the family discovery about their mother never being told, what were the obligations
ethically to tell her, to tell the family, etc. Its a very good book. We can also use many many
different types of cells are used. Monkey kidney cells, the heart of an iguana, many
different organ cells are used, living cells to grow different viruses.

[Slide #4 Virus Cultivation 2]
Now how do you do this? Lets say you want to get an iguana heart to grow in tissue culture
to grow viruses and replicate viruses. Well its no good as just the heart tissue itself, so you
have to break up all the tissues in the heart of the iguana, if you want to use that. You have
to separate the cells in the heart, so first you use something like a protease, some
proteolytic enzyme that will digest the bonds between all the cells in the heart tissue in this
case. Get them separated, break them apart into individual cells if that is possible, and then
you want to get rid of the debris stuck to them. So you centrifuge them and wash them a
few times, and then centrifuge them and use buffers and salts, and then get nice pure heart
tissue cells. And then you put them in a flask such as this. Wash off the cells with buffers,
break up the tissue with proteases usually, add cells to a medium that will support their
growth. Eagles medium is the medium used to grow these cells. And we havent talked yet
about the viruses! Just the cells that we are going to use for the viruses to grow in. The
medium is called Eagles medium, he is also a scientist that died recently. But he came up
with this very, very rich medium to grow tissues like heart of iguana, kidney of a monkey,
HeLa cells. Very, very rich, many, many nutrients and vitamins and good, healthy
ingredients for growing cells in tissue. what you do is you put those cells in Eagles
medium, you put them in the bottom of a flask as shown here and then after a while you
incubate the cells in Eagles medium, these tissue cells and after a while they grow and
they start to spread out. And then they spread out, growing on Eagles medium. They hit
the sides of the flask, shown here and then they stop growing. They form a monolayer of
tissue cells. Its magic I dont know how it happens, but anyway they stop growing, and
they form a nice monolayer of living tissue cells. And now this is the growth medium in a
way for viruses. So then you put the viruses in, you seed the viruses, you open up this flask
very carefully, aseptically take the cap off. Add your viral suspension. Maybe you put in say
2 million viruses, come back in about a week, youve got 20 billion viruses! So you replicate
them in that way, replication. And if you put in just a few, maybe 10, or 20, or 30, youd
only see about 20 or 30 plaques. But ordinarily you put in a lot because the purpose of
using this is to replicate the viruses. Get much much larger numbers of viruses than you
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started off with. And later on you can purify, get the viruses after theyve replicated in the
tissue culture. So this monolayer is formed, monolayer of cells, then you add the seed or
introduce the viruses, they replicate, later on you just take them off and separate the
viruses from everything else that is in that flask.

[Slide #5 Heterogeneity of Viruses]
Heterogeneity of viruses, they vary in size, in nanometers not micrometers. Here are some
in diameter, I think the smallest one is this parvovirus here. it has a diameter of about 20
25 nM, this one right here [parvovirus]. And you would never see this with a light
microscope. Here are the other ones, and here is the, well learn a lot more about the
herpesvirus, that causes cold sores, and other viruses that cause infections in the mouth or
oral cavity, especially herpesvirus. But we will also talk about flu and the common cold, etc.
Heres a herpesvirus which is a very large virus, about 250nM in diameter. And here is the
largest virus, the poxvirus, and smallpox is one example of it. This virus is unique from all
the other viruses here, its got its own unusual shape which is called by different names but
lets say brick shaped. There might be other names coming up. All the other viruses have
one or two types of morphologies Ill briefly discuss. So the different size that differ in
morphology, you can see the differences in shapes among these viruses, and they differ in
chemical composition, either DNA or RNA genome plus protein.

[Slide #6 Viruses and Host]
The host range virus refers to the actual host. What type of animals can these viruses
infect? Some have a host range of only humans. Other viruses like influenza we know can
infect humans, can infect birds, can infect pigs. And so that is called a host range. What are
the hosts for this particular virus? What animals can they infect. Some are only human,
some are only human, have a host range of only human. Many other viruses of course
infect humans and animals, especially the flu virus, and that is why youll see later on, that
we have flu epidemics. Because of the fact that influenza virus can infect not just humans
but birds and pigs. Youve heard of swine flu and bird flu, yeah the same virus infects them
that infects us.
Effect on the host with regard to disease. Some are generalized, cause many different types
of symptoms of infection of the blood, multiple organs. for example ones shown here are
generalized, they have a whole systemic effect, a body-wide effect. Many different organs
can be infected by viruses, some viruses like the ones shown here. They dont restrict
themselves. So, ones that are more specific. So that is called tropism. What are the viruses
likely to adhere to, what is their tropism. Is it general, can they infect many many types of
cells? Or is it very specific, for example, rabies and polio. They like to cause infection only
with the nerve system. So for example, the rabies virus would be called neurotropic.
Neurotropic. Rabies viruses have a tropism, an affinity for nerve tissue. Influenza,
respiratory system. So, specific, not general. And then Hepatitis virus, Hepatitis A, B, C, D,
and E, are hepatotropic, they only infect liver cells.


[Slide #7 Properties Common to Viruses]
So, properties common to viruses. Either DNA or RNA genome. Capsid. They multiply or go
through a replication cycle only in living cells. And here is the key. After entering host
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cells, the initial step of viruses, of mammalian viruses or plant viruses, is to separate the
genome from its capsid. Uncoating. So once again, remember that with viruses that infect
us, the whole virus gets inside the cell. Not just the nucleic acid, the whole virus. The
capsid and its genome get into our cytoplasm. And it is the role of the genome to cause the
replication of the virus. So that genome inside the capsid now inside of our cells has to be
released from the capsid or the protein shell which is enclosed. It has to be freed. And this
process is called uncoating. Uncoating, where this genome is released from the capsid and
is free in the cell to direct its replication.

[Slide #8 Some Other Points About Viruses]
Some other points about viruses. A virion is a name for an infectious virus. Viruses are
called filterable agents because they can go through filters that bacteria cannot go through
because they [the viruses] are smaller. Many viruses have envelopes, I will talk about soon.
And many viruses carry accessory enzymes to aid in their replication.
So it may not be just DNA or RNA in the capsid. HIV for example, another virus, they carry
also with them a little bit of luggage. They say ok we are going to expose our nucleic acid
inside this host cell. But in order for it to start replicating, Im going to have with me an
enzyme like a polymerase, a DNA polymerase or an RNA polymerase thats going to help
me right away initiate replication of my own genome. So some viruses also carry with them
accessory enzymes to aid in their replication, to have it begin almost instantaneously once
the genome is freed up and uncoated from the capsid.

[Slide #9 Features to Distinguish Viruses from Bacteria]
Features to distinguish viruses from bacteria. Here are bacteria, mycoplasma, these are all
bacteria, the first three. So distinguish viruses from some bacteria, some different types of
bacteria. Here are bacteria in general, and most of the bacteria we will discuss. Heres one
weve already mentioned, once again many times, mycoplasma. Mycoplasma, what is
unusual about that, what you can see, no cell wall. Dont forget about that for the exam.
This bacterium has no cell wall. Other bacteria have a cell wall. Other bacteria grow on
artificial media, they divide by binary fission, they have both DNA and RNA, they have a cell
wall. Mycoplasma are bacteria, they have the first three features but they do not have a cell
wall.
Rickettsiae and Chlamydiae, Rickettsiae is a bacterium, causes typhus and rocky mountain
fever, dont worry about that now, well get to that later. Chlamydiae is the genus of
bacteria that causes the number one sexually transmitted disease today, Chlamydia
infections, genital infections, STD, venereal disease. The unusual property of these two
bacteria is that they do not grow on artificial media. As far as we know, these are the only
two bacteria that do not grow on artificial media. Meaning, they are kind of like viruses,
These two bac genera can only grow inside of our cells. They dont attach to the surface of
our cells like other bacteria can and replicate there. They have to some way be transmitted
from one person to another, get inside of our cells, and replicate only inside of our cells.
Thats the only way they will survive. Thats a property that they have in common with
viruses. Now, they are a bacteria. But they cant be grown on artificial media, like viruses,
and they can only replicate inside of our cells. So viruses can not grow ion artificial media,
dont divide by binary fission, they divide by replication inside of cells, they have only one
type of nucleic acid and they have no cell wall.
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[Slide #10 Classification of Viruses - Genome]
And I only have about two more slides then Ill give you a break because I know some of
you are going somewhere I guess, upstairs, 6
th
floor. Lets finish with this slide,
classification of viruses according to their genome, DNA or RNA. So you dont just look at
the virus morphology and say ok its this shape or that shape, its this size or that size.
There are ways, and we put viruses into many different families. We classify viruses.
Different groups. What kind of properties do we use to identify different viruses? Well the
genome itself is one. Type of nucleic acid. Is it a DNA or RNA virus? Thats a big one, of the
genome. Thats essential to know about a virus, whether its DNA or RNA. Strandedness, is
it double stranded or single stranded? Many viruses, either RNA or DNA have only single
stranded, not many, but some, single stranded DNA or single stranded RNA. So thats one
feature we can use to characterize viruses. Molecular weight. You can tell from that slide
we saw initially that some viruses are very small, some were very large. And the larger
viruses, like hepatitis, and herpes, and especially smallpox, have very, very large molecular
weight genomes, whereas the parvovirus, the smallest virus, has a very low molecular
weight genome. Fewer genes too, lower molecular weight, smaller genome, fewer genes,
than the larger viruses like smallpox or herpes. Linear or circular. Some of the genomes of
viruses are linear, others are circular. Remember the bacterial chromosome is circular, the
plasmid is circular. Some viruses are linear, stretched out, and some are circular. So these
are ways to characterize viruses by their genome. Some genomes are continuous, that
means just one piece, others are segmented. The HIV virus is a single genome, one genome
of RNA, but it has two separate RNA segments. Two segments, but one genome made up of
two separate segments, not hybridized, two separate double stranded, [mumbled], two
separate strands of RNA, double stranded.
The influenza virus has one genome, eight different segments. So once again we will talk
about the influenza genome and its segmented property and discuss how that helps
produce the flu epidemics and pandemics that happen from time to time.
And finally, we end up with polarity, positive or negative. This is a property that can only
be applied to genomes of viruses that are single stranded RNA or DNA.
Lets just focus on polarity, as a positive or negative, for single stranded genomes. Lets talk
about first an RNA virus, an RNA virus, single stranded genome. One strand of RNA. Is it
positive polarity or is it negative polarity? What is the difference between the two? And
there are both types. By definition, a single stranded RNA virus has a positive polarity if its
RNA is recognized by the host it infects as messenger RNA. That is essentially it. So some
viruses inject their single stranded mRNA into a cell, it goes right to the ribosome of that
cell, and it is recognized as messenger RNA and it begins the production of more viral
proteins.
Other viruses have negative polarity, single stranded RNA. They inject their negative strand
into the host cell, and it is not recognized as messenger RNA. So something else has got to
happen, well talk about later. Something else has got to happen to it to make it infectious.
But by definition, positive polarity means the RNA is recognized by the cell as messenger,
and negative polarity, single stranded RNA is not recognized as messenger RNA, does not
go to the ribosome, does not direct translation, at least immediately. OK so we will wind up
there and pick up tomorrow.