Midterm 3 Problem Sets (17-23)

1. True or False: all viruses have capsids

True

2. True or False: all viruses have a genome

True

3. True or False: no virus can independently replicate itself

True; Why: It doesn’t have the machinery to make proteins (e.g., ribosomes)

4. What is the difference between a prion and a virus?

• A prion is a protein that results in other proteins spontaneously misfolding. It does not have a genome

  • Responsible for Mad Cow disease 

• A virus has a genome and is able to dependently replicate with host cell machinery by translating proteins for offspring.

5. What aspect of viral infection makes it possible for some plant cell viruses to be able to infect cells without a capsid?

• The virus (often as a viroid) contains single stranded RNA and has no capsid

• The viroid then enters the plant through tissue damage

• The virus can then infect other cells through the plasmodesma (cell bridges) due to the fact that the viroid is not robust 

• They can make movement proteins that allow RNA to transport between plant cells through plasmodesmata

6. Is it possible to define a universal phylogeny for viruses? Why or why not?

• No, it is not possible because:

  • 1. There are no shared genes since they all replicate from themselves 

  • 2. There is no evidence of a single origin of viruses

  • 3. The are complex and diverse

7. Measles is an enveloped virus with a -sense single stranded RNA genome.

a. When measles viruses infect hosts cells, can the genome replicate using only host cell ‘machinery’?

• No, it requires RNA dependent RNA polymerase, which the virus needs to bring with it in order to turn the -sense RNA into mRNA

  • mRNA can then be read by host cell ribosomes and translated

b. Which components of the host cell are needed for replication?

• rRNA, tRNA 

c. If you answered yes to A, are there any components that the viral genome needs to encode?

N/A

8. Feline leukemia virus is a retrovirus with a +RNA genome.

a. The viral genome encodes reverse transcriptase. What does reverse transcriptase do, and how does this function help the virus replicate

• Reverse transcriptase is used to convert the +RNA into DNA which can go into the nucleus and directly embed itself into the host DNA 

• This allows for the host cell to repeatedly transcribe the viral DNA, translating more copies of the viral proteins

b. FeLV causes white blood cell cancer (leukemia) in cats. Based on what we have learned about viruses so far:

i. What are the host and tissue tropisms for this virus?

• Host tropism is cats

• Tissue tropism is bone-meorrrow and blood forming tissues

ii. What can FeLV do once inside a white blood cell to cause the cell to become cancerous?

Once inside a white blood cell, FeLV infects it by integrating its viral DNA into the host cell's genome. It can then release inhibitions on cell replication. The reason it wants to do this is because by increasing the population of host cells, it also increases its number of targets.

1. True/False: retroelements are the remnants of retroviruses that are found in the genome.

True

2. True/False: some retroviruses aren’t able to integrate into the host genome

False

3. Order the steps through which HIV infects T cells and replicates

a. Host RNA polymerase transcribes copies of the +RNA viral genome

b. Viral reverse transcriptase makes ds DNA from the +RNA viral genome

c. dsDNA integrates into the host genome

d. Viral particles dock with cell surface receptors

e. Viral dsDNA is transported into the nucleus through nuclear pores

f. Viral proteins and +RNA genome are produced and packaged

D, B, E, C, A, F

4. The antiviral drug AZT is used to treat HIV. AZT works by blocking reverse transcriptase. In at most 4 sentences, explain how the HIV virus interacts with a T cell in the presence of AZT.

HIV virus can still bind cell surface receptors and fuse with the membrane. The +RNA viral genome can uncoat and some proteins might be able to be made. But because reverse transcriptase can’t make dsDNA, the viral genome can’t make copies of itself. This blocks the virus from replicating and producing more infectious viral particles, halting the spread of infection to other T cells.

5. A property of HIV is that the viral genome rapidly mutates, creating a population of mutant variants within chronically infected individuals. Explain why this makes HIV difficult to treat in the following two contexts:

a. Within an individual who is chronically infected

• They don’t have a specific druggable cell target, because there is such a high environmental diversity of HIV strains within the body 

5. A property of HIV is that the viral genome rapidly mutates, creating a population of mutant variants within chronically infected individuals. Explain why this makes HIV difficult to treat in the following two contexts:

b. In populations of people where HIV is being transmitted

•  You are transmitting a small subset of the diverse viral landscape — you don’t know which type of HIV is being transmitted, or which one to target/drug. Also even limited diversity can quickly become very diverse. 

6. List 2 attributes of lentiviruses that made them a good scaffold for the development of CART-T therapy:

1. Integrate genes directly into the T-cell genome (all future replicates of this T cell will also contain these genes) → long term expression

2. They can also target non-dividing T cells

7. What functions encoded by the lentiviral genome had to be removed from the DNA used by CART-T to contain the gene therapy to single cells.

• She gag on my pol till I replicate

  • Gag-Pol Peptide because this inhibits viral replication within the cell

8. What aspect of the biology of influenza A makes it necessary to design a new vaccine every year?

Influenza A has many different strains due to reassortment (NOT mutation). A new vaccine is designed each year for the predicted strains of influenza. This is not always accurate but is necessary due to the 

1. Clinically, the neutrophil to leukocyte ratio (NLR) is a widely used indicator of whether a patient is under physiological stress (Farkas, 2020). In particular, an elevated NLR is a useful early warning sign of sepsis, or a bacterial infection of the blood.

Based on this information, what we discussed in class, and your textbook reading answer the following questions:

A. Which population of blood cells is increased if NLR>3?

• Neutrophils because they are the innate first responders to an acute bacterial infection

B. Physiologically, why is this population of cells elevated in early sepsis?

• Neutrophils increase in proportion to leukocytes (meaning a higher NLR) because the body initially releases large numbers of neutrophils to fight bacterial infection, while leukocytes decrease as sepsis triggers accelerated lymphocyte apoptosis (programmed cell death)

C. What are two things this elevated population of cells does in the context of a blood infection?

• It acts as a first line of defense by: 

  • 1) Phagocytosing invaders

  • 2) Trapping bacteria in neutrophil extracellular traps (NETs)

2. Dental cleanings are associated with elevated risk of bacterial endocarditis for some patients (for example people with gum disease). Endocarditis is an infection of the heart valve due to the formation of an extracellular bacterial biofilm, called a vegetative plaque.

From the following list of immune response components, circle the innate immune components most likely to respond to the formation of a vegetative plaque of bacteria on the heart valve:

a. B cells

b. Tissue-resident macrophages

c. Neutrophils

d. Eosinophils

e. T cells

f. Complement

b. Tissue-resident macrophages

c. Neutrophils

f. Complement

3. List three ways in which NK cells are different from Neutrophils?

a. NK cells originate from lymphoid precursors, while neutrophils come from myeloid precursors 

b. NK cells are lymphocytes, while neutrophils are phagocytes 

c. NK cells specialize in recognizing and killing host cells (infected by viruses or tumor cells) through MHC I, while neutrophils circulate in the blood and destroy invading pathogens (more non-specific?)

4. If you take antihistamines for allergies, what immune cell type are you targeting, and is this type of immune cell part of the innate or adaptive immune response?

• You are inhibiting mast cells and basophils, which are part of the innate immune response. 

5. Name an immune cell type that hangs out in lymphoid tissues like MALT or lymph nodes and explain what it’s doing there.

• Dendritic cells and macrophages reside in lymphoid tissues

• Capture antigens from the mucosa 

• Triggers adaptive immunity (by presenting to B or T cells)

6. NK cells induce the cells that they target to undergo apoptosis. Why is this function important for controlling the immune response at the level of the individual?

• Apoptosis is important for removing unneeded/dangerous cells without causing inflammation 

• NK cells look for lack of MHC I, which acts as a signaling mechanism for pathogen-infected cells 

1. Natural killer cells target ________ cells that ________.

a. infected; have lost MHC class I

b. infected; have lost C-reactive protein

c. bacterial; have lost MHC class I

d. infected; secrete cytokines

e. bacterial; are coated with complement

a. infected; have lost MHC class I

2. List three ways in which the innate and adaptive immune responses differ

Innate Immunity	Adaptive Immunity
Faster and more general response Present antigens to adaptive immune cells Triggers inflammation	Antigen presentation initiates response Makes antibodies which target specific antigens Takes longer to build up, but ability to make antibodies stays around (memory) Two branches Humoral Cellular

3. Matching. For each tissue context, write the letter(s) for the immune functions that take place in that context.

Immune functions:

A. Phagocytosis

B. Production of naïve B cells

C. Antigen presentation

D. B cell activation and differentiation into final differentiated cell types

E. Recognition of infected host cells

F. Inflammation

G. T cell activation and differentiation into final differentiated cell types

H. Maintenance of immunological memory

I. Cell-mediated apoptosis

Lymph nodes in a person experiencing an infection:

Bone marrow:

Epithelial tissue on a scraped elbow infected bacteria that colonizes extracellular spaces:

Sinus mucosa infected with a virus:

Lymph nodes in a person experiencing an infection: C, D, G (Antigen presentation, B cell activation and differentiation into final differentiated cell types, T cell activation and differentiation into final differentiated cell types)

Bone marrow: H, B ( Maintenance of immunological memory, Production of naïve B cells)

Epithelial tissue on a scraped elbow infected bacteria that colonizes extracellular spaces: E, F ( Recognition of infected host cells, Inflammation)

Sinus mucosa infected with a virus: I, A (Phagocytosis, Cell-mediated apoptosis)

4. List types of immune cell are most likely present in the following tissue sites/contexts. If present, whether these are differentiated into any of the following subtypes

Cell types:

A. B cells → plasma cell, memory cell 

B. T cells → T helper cell, cytotoxic T cell 

C. Macrophages

D. Neutrophils

E. NK cells

F. Mast cells

(write next to letter if applicable)

Differentiation into:

plasma cell 

memory cell

t helper cell

cytotoxic T

naïve cell: n/a

Lymph nodes in a person experiencing an infection:

Bone marrow:

Epithelial tissue on a scraped elbow infected bacteria that colonizes extracellular spaces:

Sinus mucosa infected with a virus:

Lymph nodes in a person experiencing an infection: A, B, C

Bone marrow: A (memory and naïve)

Epithelial tissue on a scraped elbow infected bacteria that colonizes extracellular spaces: B (Tc cell), C, D, F

Sinus mucosa infected with a virus: A, B (Tc cell), C, E 

5. In three sentences or less, explain why clonal expansion of B cell populations is an important property of the adaptive immune response.

• This allows B cells to clonally expand and replicate with the specific antibody required for the immune response against a specific pathogen. Normally, B cells each contain different antibody receptors, while clonal expansion allows for a single antibody subtype to be replicated. 

1. List 3 events during human development that change the microbiome, and in no more than one sentence explain the mechanism through which the microbiome is impacted.

• There are a few examples here... for instance, the transition to solid food in infancy shifts the composition of the microbiome by enriching for microbes that degrade complex carbohydrates found in fiber. Or the colonization of the gut shifts the environment from being oxic to being anoxic, which selects for anaerobic microbes.

2. In 2-3 sentences, why are Treg cells important for immune tolerance? In your answer, make sure to identify the tissues site where this cell population is found, and explain the function of Treg cells.

• Non-pathogenic microbes trigger the differentiation of Treg cells 

• T reg cells regulate the activity of helper cells, which downregulates the immune system by inhibiting inflammation by helper cells

• These cells are found in the layer underneath the epithelia, called the lamina propria

• They are important because they prevent the immune system from attacking healthy eubiota, maintaining homeostasis

3. In 2-3 sentences, explain how Curtibacterium acnes—an anaerobic Gram-positive normal member of the skin microbiota—cause acne, especially in teenagers?

• Normally, sebum is excreted from the sebaceous glands to help dead cells leave through the hair follicles

• Sebum production typically increases during puberty due to hormonal changes, which can lead to a clog of sebum and dead cells at the hair follicle 

• This facilitates Curtibacterium acnes colonization within the growing pool of sebum, resulting in an inflammatory response at the follicle, which causes acne.

4. What is the function of the mucosal layer that lines the large intestine? Name a factor that make this environment selective in terms of which species of microbes can colonize it.

• It prevents the gut microbiome from interacting directly with the epithelial cells lining the gut.

• Infection of this membranous tissue would result in inflammation, destruction of the gut, and ‘leaky gut.’ 

• This environment can select for certain species through its use of M cells, which can selectively target dysbiotic bacteria within the gut. Any microbes that make it through the mucosal layer are funneled into the M cells and directed to macrophages, allowing for an appropriate immune response.

• Another factor is different colonies within the bacteria can select for or against other bacteria through organismal interactions.

5. You are a 3rd year medical student doing a clinical rotation in Pathology. The Surgery department sends down a biopsy from the colon of a patient with suspected ulcerative colitis. Ulcerative colitis is a disease that stems from a lack of immune tolerance. You help section and stain the tissue. Based on what we talked about in class, describe the pathology of the tissue that you’d expect to observe, and identify the abundant immune cell types you’d expect to observe.

• You would probably see an abundance of T helper cells and mucosal inflammation, resulting in a thin membranous wall/leaky gut, because immune cell precursors preferentially differentiate into T helper cells in order to control the excess of bacteria

• The body can’t detect whether normal/good bacteria or bad bacteria is colonizing the gut, which means it can’t produce as many T reg cells to regulate the T helper cell response.

6. What is the function of M cells? Why are they needed in GALT but not lymph nodes?

• Any microbes that make it through the mucosal layer are funneled into the M cells and directed to macrophages, allowing for an appropriate immune response.

• The gut has a huge, continuous influx of microbes compared to lymph nodes. If the macrophages behind the m cells went into the gut then they would be eaten alive because of the seheer aboumt of bacteria and nicrobes, which means that they need to be gated by M cells. This does not happen with lymph nodes because there are not that many bacteria in the other areas of the body.

7. Dental decay is caused by dysbiosis of the oral microbiota. Cavities form because acid produced from fermentation of dietary carbohydrates by Streptococcus mutans demineralizes tooth enamel. To combat dental decay, many dentists recommend fluoride treatments, flossing, and chewing gum with the sugar alcohol xylitol in it. Based on what you have learned about the microbiome and dysbiosis, what will each of these methods do to the oral microbiome, and how might that help prevent decay?

Flouride

• antimicrobial that kills bacteria in cavities and allows it a clean slate to remineralize/heal

7. Dental decay is caused by dysbiosis of the oral microbiota. Cavities form because acid produced from fermentation of dietary carbohydrates by Streptococcus mutans demineralizes tooth enamel. To combat dental decay, many dentists recommend fluoride treatments, flossing, and chewing gum with the sugar alcohol xylitol in it. Based on what you have learned about the microbiome and dysbiosis, what will each of these methods do to the oral microbiome, and how might that help prevent decay?

Xylitol

•  prevents bacteria by acting as a non-fermentable sugar substitute that starve cavity-causing bacteria, specifically Streptococcus mutans, which cannot ferment it for energy, leading to reduced bacterial growth and adhesion to teeth

  • Bacteria, especially S. mutans, ingest xylitol but cannot break it down, leading to a "futile energy cycle" that prevents them from producing energy and growing.

7. Dental decay is caused by dysbiosis of the oral microbiota. Cavities form because acid produced from fermentation of dietary carbohydrates by Streptococcus mutans demineralizes tooth enamel. To combat dental decay, many dentists recommend fluoride treatments, flossing, and chewing gum with the sugar alcohol xylitol in it. Based on what you have learned about the microbiome and dysbiosis, what will each of these methods do to the oral microbiome, and how might that help prevent decay?

flossing

• Flossing - Physical removal of cavity forming plaques/food particles → removes sugars so that bacteria can't ferment and turn it into acid, which decays teeth 

1. Name three functions phages play in the gut, and in 1-2 sentences each, explain how these functions impact gut health.

Transcytosis is a cellular process that selectively takes up macromolecules, including phage. This may enable phages to interact with development of the immune system and potentially defend tissue from bacteria.

Lysogeny is likely common in the gut, so prophages may be important in preventing superinfection of bacteria (my enemy’s enemy is my friend). If superinfections do occur, it’s possible that it can result in new functions via transduction

When new bacteria and phage are ingested, the microbial ecology of the gut changes. New phages may infect healthy-gut bacteria and decrease their population. The loss of gut health is called dysbiosis

Toxin release can also have negative impacts on gut health if lysogenized bacteria produce toxins, such as Shigella and E. coli.

2. Living or non-living? Identify one way in which viruses display characteristics of a living entity, and one way that viruses display a characteristic of a non-living entity.

• Viruses have genomes and can evolve

• Viruses do not have the capability to reproduce on their own, they require a host organism. 

3. List four discoveries and/or technologies that we discussed in class that emerged from research on bacteriophage

1. CRISPR-Cas gene editing

2. Phage therapy for antimicrobial resistance

3. Phage to deliver chemotherapy

4. Antibody discovery

4. Name two ways in which bacteria can defend themselves against phages, and in 1-2 sentences, explain the molecular mechanism through which this defense works.

• Bacteria and archaea have evolved defense mechanisms: 

  • Genetic resistance: altered receptor proteins → phage cannot bind 

  • Restriction endonucleases: cleave viral DNA lacking methylation → host genomes have methylation so they are spared 

  • CRISPR integration of phage DNA sequences → adaptive immunity 

• How exactly does CRISPR confer adaptive immunity?

  • A piece of phage DNA gets copied as a “spacer” into the host genome 

  • This creates a spacer-repeat-spacer-repeat sequence that acts as a “record” of past infections 

  • If the bacterium survives the infection, reinfection of the same phage causes transcription of the phages into CRISPR RNA (crRNA)

  • The crRNA joins a Cas complex to recognize and cleave the phage DNA (since its complementary), rendering immunity 

Note: not all bacteria have this ability

1. What is the difference between and endotoxin and an exotoxin?

• Endotoxins are expressed on bacterial membranes and are basically lipopolysacharide proteins that can contact with other organisms and are released upon death to induce inflammation (MAMPs). They can be mutagenic. 

• Exotoxins are toxins that are released by the bacteria to damage or lyse external cells.

2. Name a type of exotoxin, and in a sentence or two, explain its mechanism of action.

• Botulinum toxins

  • They inhibit neurotransmission by blocking acetylcholine receptors, which prevent transmission of nerve impulses to muscle

3. Are pathogens that are highly infectious always highly virulent? Why or why not.

• No, they are not always highly virulent because there can be pathogens that spread easily like a cold, but are not very severe (e.g. cause minor symptoms).

4. Give me an example of a disease vector.

• Mosquitoes, rodents, humans

5. Give me example of a fomite and explain how the vehicle that makes up this fomite might help to transmit a pathogen.

• Water (vehicle) in a contaminated well (fomite) 

• Water from a contaminated well will be consumed by multiple different people, spreading the disease amongst a population and resulting in sickness.

6. What is the difference between direct contact and vertical transmission as mechanisms of disease transmission?

• Direct contact is when two organisms directly transmit or pass on pathogens

• Vertical transmission is when a pathogen can cross the placental barrier, resulting in transmission between mother to baby (within).

7. Name a trait or capability that ‘obligate’ or primary bacterial pathogens tend to have, that opportunistic bacterial pathogens probably don’t have, and explain how this trait or capability contributes to pathogenicity

• Obligate pathogens tend to have effector proteins that go into the target cell and somehow alter them to ease pathogenic entry to the cell. This is not seen in opportunistic pathogens as much, because opportunistic pathogens are inherently only able to effect individuals who are in some way compromised. This effector trait contributes to pathogenicity because it increases the infectivity of the pathogen by increasing the number of pathogens that are able to go into the cell.