Week 4 Module 2

Question 1

What is enumeration?

Answer 1

Determining quantity

Question 2

What is a Titer?

Answer 2

-Number of infectious virions/unit volume. For phage, titer typically given as: plaque forming units/ mL of bacterial culture or pfu/mL

Question 3

What is a Plaque?

Answer 3

-Zone of lysis that appears as clear area on lawn of host cells

Question 4

Multiplicity of infection (MOI)

Answer 4

-Ratio of infectious particles (viruses) to infection targets (host cells)

Moi = concentration of viruses or infectious particles/concentration of host cells

If 1 x 10^6 virions used to infect 1 x 10^6 host cells, moi is _1___
if 1 x 10^5 virions used to infect 1 x 10^6 host cells, moi is _0.1

Question 5

Bacteria uses_________ and Phages use__________

Answer 5

-Bacteria uses CFU/mL, and phages use PFU/mL

Question 6

What is a confluent?

Answer 6

-a uniform plate filled with bacteria, phage look like little clearings in the background of bacteria on plate.

-there are so many bacteria in bacterial culture used that a
lawn (confluent layer) of bacteria will grow that is visible to naked eye; In other words bacteria is in excess!!!

-think of ‘lawn’ of bacteria used when looking for ‘zone of
inhibition’ with antiseptics and ampicillin in Microbiology labs

Question 7

What is plating efficiency?

Answer 7

– efficiency with which virions infect
host cells in lab setting. It is typically < 100% meaning
if counted microscopically, one would see more virions
than that indicated by pfu/ml value.
-for phage, can often get plating efficiency of >50% (though <100%)
-for animal viruses, plating efficiency might be 0.1% - 1%

Question 8

What are the reasons for plating efficiency of less than 100%?

Answer 8

Some virions will fail to infect due to:
-Failure to assemble properly during maturation
-Carrying defective genomes
-Mutations that cause defects in attachment or penetration or any aspect of viral replication
-Damage during handling or storage (human intervention)

-also, if virions do infect they may not grow well due to
suboptimal (less than ideal) growth conditions.

Question 9

If you have 10 viruses will you have 10 infections?

Answer 9

-Just because you have 10 viruses doesn’t mean you’ll get 10 infections, plating efficiency is less than 100% ex. If you get 8 plaques from 10 viruses plating efficiency is less than 100%

Question 10

What is the first step of plating phage in the plaque assay?

Answer 10

-first pour bottom agar into petri dish and allow it to solidify
-bottom agar is just nutrient broth + agar (similar to what is used to plate bacteria alone)
-bottom agar may also contain antibiotic if host bacterial cells
carry antibiotic resistance marker (ex. Ampicillin)
-by having antibiotic only in bottom agar, more distinct plaques
tend to form in top agar
-bacteria and phage added to top ager

Question 11

What is the second step of plating phage in the plaque assay?

Answer 11

-in a separate tube mix host cell culture (e.g. E. coli) with suspension of virus you wish to infect it with (e.g. phage T4)
-Note: You will choose volumes of host cell culture and viral
suspension based on densities of both cultures and moi you want

Question 12

What is the third step of plating phage in the plaque assay?

Answer 12

-next, add bacterial cell/viral culture mix to another tube containing top agar
-top agar nutrient broth + agar but with lower % agar than bottom agar so it’s softer

Question 13

What are the steps of plating phage simplified?

Answer 13

1. Pouring
2. Solidification
3. Incubation for bacterial growth and Phage replication

Question 14

Size of plaques depends on?

Answer 14

adsorption efficiency, length of latent period, and burst size

Question 15

Number of plaques depends on?

Answer 15

depends on the number of infections and plating efficiency

Question 16

How long are the incubation time of plaque and bacteria?

Answer 16

24-48 hours

Question 17

What is the latent period?

Answer 17

-Latent period is when preparing everything DNA, RNA being copied, other essential stuff before virions break free
-The longer the latent period, the smaller the number of plaques

Question 18

The bigger the burst size the bigger the__________

Answer 18

plaques

Question 19

Which plates are typically counted to determine titer?

Answer 19

Plates with 10-100 plaques

Question 20

For a 10^-1 dilution with a volume of 0.1 mL what volume of media should be added?

Answer 20

For dilution take 0.1 mL virus stock into 0.9 mL of media 0.1 mL/ (0.1 mL + 0.9 mL) = 10^-1 10 x fold

Question 21

How to enumerate viruses that infect animal cells with Plaque assay?

Answer 21

-can use plaque assay; Same basic idea as phage except that top and bottom agar are not used because animal cells grown in culture used different media
-viral plaques seen as clear zones in cell culture layer
-virus titer can be estimated

Question 22

How to enumerate viruses that infect animal cells with focus forming assay?

Answer 22

-can use focus forming assay – plate host animal cells/virion mix
on semi-solid media
-after short incubation time (24 – 72 hrs) stain cells with fluorescently-labelled antibody against viral specific antigen
-count number of fluorescent foci with microscope

-If you can’t see plaque specifically, you can add antibody against viral specific antigen

Question 23

How to enumerate viruses that infect animal cells with End Point Dilution Assay?

Answer 23

-can use end point dilution assay
-make serial dilutions of virus and determine concentration of
virus required either to kill or cause cytopathic effect
(observable structural change in host cell, cells look abnormal or dead) in 50% of host cells
-called TCID50 (tissue culture infective dose 50)

Question 24

What is the ideal TCID50

Answer 24

Ideal spot is between 10^-5 and 10^-6 dilution, the TCID50 is also between 10^-5 and 10^-6

Question 25

What is the virosphere?

Answer 25

– All places where viruses are found and/or interact with their hosts.
-As discussed, viruses infect all three domains of life; Bacteria, Archaea, Eukarya.
Estimated to be about 1031 viruses on Earth (vs. 1030
prokaryotic cells).

-However, because of small size, viral biomass relatively small.

Question 26

What is the hypotheses concerning viruses and evolution?

Answer 26

-evolved after cells; Are remaining cell components
-evolved before cells along with the ‘RNA world’
-viruses were first ‘pseudo’ forms of life in precellular era

Question 27

How do viruses have a profound effect on evolution of other species?

Answer 27

-viruses act in transduction and therefore influence bacterial evolution, 3 modes of genetic exchange of viruses in bacteria, transformation, conjugation, transduction
-for example, viruses known to transfer photosynthetic machinery to bacterial cells
-also, have transferred genes to bacteria that lead to antibiotic resistance
-Viral infections in bacteria can cause bacteria to evolve

Question 28

Why is it hard to determine where viruses originated?

Answer 28

-Phylogeny (tree of life) of viruses harder to determine since no universal gene marker (molecular
chronometer) exists like small rRNA.
-16srRNA is common in bacteria, archaea, and eukarya, but no universal gene marker for viruses because virus is separate from domain of life

Question 29

What is the Human Virome?

Answer 29

-Human virome is all viruses in and on the human body these include;

-animal viruses that directly infect human cells
-phage -> that infect bacteria -> that infect humans
-plant viruses ingested
-latter two categories won’t infect human cells but while they’re in the body, they’re part of the virome

-Phages are in mucosal linings of respiratory & gastrointestinal tracts

Question 30

Viruses Harmful to Humans can cause respiratory issues?

Answer 30

-can cause respiratory issues e.g. coronavirus, rhinovirus

-some viruses can be benign (see below) OR in harmful category e.g.
adenoviruses (can cause respiratory issues), polyomaviruses (can
cause UTIs), papillomaviruses (e.g. HPV can cause cervical cancer)
-pepper mild mottle virus (plant) presence can cause inflammation; associated with sensitivity to spicy foods
-phages that cause host bacteria to be antibiotic resistant
-phages that enhance pathogenicity of bacteria (makes it worse) e.g. Vibrio cholera;
phage gene encodes cholera toxin

Question 31

Viruses Harmful to Humans can cause latent infections?

Answer 31

-those that can cause latent infections e.g. herpesviruses, cytomegalovirus (CMV) which can cause mononucleosis or hepatitis

Question 32

Viruses Harmful to Humans can be benign (not harmful) or harmful some examples include?

Answer 32

-some viruses can be benign (see below) OR in harmful category e.g.
adenoviruses (can cause respiratory issues), polyomaviruses (can
cause UTIs), papillomaviruses (e.g. HPV can cause cervical cancer)

Question 33

What is a virus that can cause inflammation associated with sensitivity to spicy foods?

Answer 33

-Pepper mild mottle virus

Question 34

An example of a phage that enhances pathogenicity of bacteria is?

Answer 34

-Vibrio cholera, phage gene encodes cholera toxin

Question 35

Viruses with benign effects?

Answer 35

-Circovirus: common in poultry and pigs and therefore, GI tract of humans who consume those meats
-Adenoviruses- found in healthy people too
-Papillomaviruses infections that are asymptomatic