Micro Final Flashcards
What are some characteristics of a virus?
They are very small and a virion, or complete virus, consists of a nucleic acid surrounded by a protein coat (capsid, the entire virus is a nucleocapsid)
They are metabolically inert and cannot reproduce on their own
They replicate within the host cell using the host cell machinery, where new copies of the genome are made and capsid is synthesized. The capsid is capable of self assembly
Infection is the process by which a virus is introduced to a host cell and reproduces
What type of virus is sars-Cov-2?
It is a single stranded RNA (ss RNA) virus
What are the steps of the bacteriophage “life” cycle?
1) Attachment to a cell (adsorption). Bacteriophages generally attach to structures such as pili, flagella, or receptors for transporters such as a maltose binding protein
2) Penetration where viral DNA enters and protein coat remains outside. Phages generally attach to a cell using a tail fiber that interacts with LPS. When tail pins make contact with the cell wall, a lysozyme-like protein makes a hole in the cell wall allowing for the injection of viral DNA
3) Replication where in the lytic cycle phages take over the host cell machinery. This involves the synthesis of viral enzymes, nucleic acid replication, synthesis of protein coats, assembly and packaging (not the most exact process), and release and lysis of host cell
What is typically observed when mapping a viral growth curve within a cell?
1) First there is an adsorption period where a virus is added and attaches to host cells
2) Then there is a latent period where early enzymes, nucleic acids, and protein coats are formed, and this is the eclipse phase
3) Then rapid growth occurs during the maturation phase where assembly and release of viruses then occurs
What are two enzymes viruses use in the “replication” process?
Lysozyme: an enzyme that makes holes in the bacterial cell wall allowing replicated virions to escape and genetic material to enter the cell
Reverse transcriptase: an enzyme in RNA viruses that replicate inside the host as DNA intermediates. Reverse transcriptase transcribes the viral RNA into a DNA intermediate
What is the purpose behind a plaque assay and why is there so much variation in the results?
Researchers use a plaque assay to create a lawn of host cells and then look for zones of clearing (plaques) that indicate viral infection to generate a virulence factor
First phages and bacterial cells are poured onto an agar plate with nutrients and the mixture is sandwiched between a top agar and nutrient agar. Then phage plaques are usually observed within a lawn of host cells.
Each plaque theoretically represents one viral particle in the initial infection, and there is much variation since the efficiency of plating (no. of plaques per virion) and culture conditions vary along with virulence and characteristics of host cells
A viral titer is used to quantify the concentration of virulent viruses, and it refers to the concentration of virus suspension as measured by the number of plaque forming units (pfu)
How can bacteriophages set up a state of coexistence with the host and become lysogenic (temperate phages can do this) rather than lytic (damaging to the host)? Why would viruses undergo the lysogenic pathway?
Bacteriophages have a lytic pathway where viral DNA can replicate, assemble, and then lyse the cell (rightward transcription, encodes for capsid/phage assembly) or they can undergo the lysogenic pathway where viral DNA can be integrated into host DNA (leftward transcription, encodes for excision/integration). A virus may undergo the lysogenic pathway when host cell replication is more likely to occur or when conditions aren’t favorable for the survival of viruses.
How does the bacteriophage lambda integrate into the bacterial genome? How does it make the choice for lytic vs lysogenic pathway?
A site specific nuclease creates staggered ends of a phage and host allowing for the integration of lambda DNA, and gaps are then closed by DNA ligase.
What genes are used in the process of choosing between lytic vs lysogenic pathway?
cI expression leads to lysogeny and cro expression leads to the lytic cycle. A lambda regulator region or “switch box” with three operators are used in this process.
The lambda repressor generally exists as a dimer where the N terminal domain binds to phage DNA and has a highest affinity for OR1 on the lambda genome. Cro additionally exists as a dimer and can bind to all three operators but has the highest affinity for the OR3 region.
How does negative control of the lytic pathway and positive control of the lysogenic pathway using the lambda repressor occur?
Negative control occurs when the lambda repressor binds to OR2 since this prevents the transcription of PR and the cro transcript downstream. This is required for lytic growth.
Positive control occurs as the repressor at OR2 helps RNAP bind and increases the transcription of PRM and cI downstream approximately ten-fold, activating the lysogenic pathway.
How does cooperative binding induce the expression of the lysogenic pathway?
The lambda repressor has the highest affinity for OR1, and this stimulates repressor binding to OR2 via protein-protein interactions between dimers. Repressor binding to OR2 induces lysogeny by blocking the expression of cro and lytic genes and stimulating RNAP binding and expression of the lysogenic pathway.
As the lambda repressor concentration increases, repressor binding to OR3 occurs via cooperative binding to a dimer far away. This is accomodated by DNA looping and it can cause lambda repressor to temporarily block its own expression.
How does Cro induce the expression of the lytic pathway?
Cro has the highest affinity to the OR3 region and this blocks the expression of the lysogenic pathway via repression along with the expression of the lambda repressor, and this allows RNAP to bind to OR1. If the repressor binds to OR2 first, expression of Cro is blocked and RNAP can transcribe lysogenic genes.
If Cro wins the lambda enters the lytic cycle and if a repressor wins the host becomes a lysogen. If Cro concentration is too high it can shut off its own expression by binding to OR1 and preventing lytic transcription.
How can the host defend against viral lyric infection?
The presence lambda repressors in the cytoplasm of a host can render it partially immune to infection by a lambda phage by blocking the lytic cycle.
How is the phage able to induce the lytic cycle in times of stress?
In times of stress the phage needs a way to induce the lytic cycle and escape the host cell. DNA damage tends to occur from heat, UV radiation, etc. and this induces the SOS Repair system which activates a recA protein from host DNA. recA can cleave the lambda repressor preventing it from cooperatively binding as a dimer and this causes the expression of more lytic genes. This allows phages to quickly assemble and release from the cell escaping the stress inducing conditions.
How can CRISPR serve as a “bacterial immune system” and retain a memory?
CRISPR/Cas systems can target either viral DNA or RNA and can interfere with viruses, plasmids, prophages, or other chromosomally encoded sequences within bacteria
When phage DNA or foreign DNA is detected, a Cas complex detects the foreign material and places a small segment of the foreign DNA into the leader end of a CRISPR locus which is then flanked by a CRISPR array to form a repeat spacer unit.
This repeat spacer is transcribed and processed into guide RNA, and this guide RNA binds to a nuclease known as CAS9. CAS9 uses this guide RNA to target foreign DNA and cleave it upon second exposure.
The PAM serves as a recognition site that stabilizes CAS9 temporarily during cleavage.
How can CRISPR/Cas9 be used for gene editing?
1) An RNA guide molecule can be programmed to match any unique DNA sequence in the human genome
2) Cas9 can then be attached to the RNA guide and find the DNA target sequence
3) The RNA can then align with the target DNA sequence and Cas9 can then attach to and cut both strands of the DNA double helix
4) DNA cuts can be amended with an extra DNA insertion made up of added homologous DNA with a desired mutation. Mutations can also be induced since broken DNA can be repaired in an error prone process
What are some differences between prokaryotes and eukaryotes, specifically when looking at mRNA and cell walls/membranes? Why is this important when looking at how eukaryotic viral infection differs from prokaryotic?
Transcription in prokaryotes is not compartmentalized and transcription is immediately followed by translation. DNA replication can also take place in the cytoplasm and no mRNA processing is required for translation.
In eukaryotes, DNA replication and transcription is limited to the nucleus and mRNA must be processed for transport out of the nucleus and translation is associated with a ribosome. There is a poly-A-tail on the 3’ end, a methylated guanosine triphosphate “cap” on the 5’ end, and splicing
How does viral infection differ between bacterial cells and animal cells?
The bacterial cell wall tends to be rigid, and this often means that the viral genome is injected into cells during infection leaving a coat on the outside. But in animal cells no wall is present and the uptake of viruses generally takes place via endocytosis, meaning a whole viral particle usually enters the cell
During uptake, nucleocapsid of virus is uptaken into an animal cell via endocytosis and the viral envelope is lost. Eventually the capsid is uncoated and a viral nucleic acid allows for virus multiplication and other related processes
What are some consequences of viral infection? How can they cause tumors or cancers after initial infection?
If progeny viruses are produced, cytopathic effects can occur where a cell dies after many virions are produced
Cells can also survive in the presence of progeny viruses and shed a low number of virions leading to chronic infection
Viral genome can also persist in the host cell leading to latent infection
Some viruses that have a genome that persists in the host cell and can “transform” animal cells post infection leading to uncontrolled proliferation. They are hypothesized to do this by blocking the activity of growth factors that can normally prevent growth and division in mature cells. This can contribute to cancer.
What are challenges that eukaryotic viruses run into?
They need to get inside the cell
DNA replication and transcription is limited to the nucleus so it is hard for viral DNA to enter the genome or be expressed
mRNA must also be processed in order to associate with a ribosome and allow for protein production, and this involves a poly-A tail on a 3’ end, methylated guanosine triphosphate “cap” on the 5’ end, and splicing
What are some characteristics of retroviruses and what is its replication cycle? What are three polyproteins that a viral mRNA can translate and what are each of their roles?
They contain an outer surface envelope protein and a lipid bilayer with transmembrane envelope proteins contained within them. They integrate into the host genome using RNA and replicate via a DNA intermediate using reverse transcriptase that is stored in the virion.
When the virus enters the host cell cytoplasm the genome is reverse transcribed into single stranded DNA which is then converted to double stranded DNA and integrated into the host genome.
The transcription of viral DNA then leads to the formation of viral mRNA and progeny genomes, and the viral mRNA is then translated into three polyproteins: Gag, Pol, and Env, and these are then processed into components of the viral capsid.
Gag is processed into the attachment proteins of the viral capsid, Pol is processed into a protease (cleaves amino acid chains), integrase (catalyzes integration of viral DNA with genome), and reverse transcriptase, and Env is processed into gp41 and gp120 which mediate the binding of viruses to host cells.
They are also potential vectors for gene therapy since they can integrate into the chromosome
How does replication occur in retroviruses?
1) Retroviruses enter the host cytoplasm via endocytosis and uncoat in the cytoplasm allowing free viral RNA to be present in the cytoplasm
2) Reverse transcriptase forms ds DNA from the single strand of viral RNA which then travels to the nucleus and integrates with the host DNA
3) Host machinery can then produce viral mRNA from the integrated DNA which forms three polyproteins
4) Polyproteins are used to form a nucleocapsid around viral mRNA and progeny viral RNA
5) Encapsulated viral particle then buds and releases from the host cytoplasmic membrane via exocytosis
What does reverse transcriptase do in retroviruses?
1) It can catalyze the synthesis of ssDNA with an RNA template
2) It can catalyze the synthesis of dsDNA from the synthesized ssDNA
3) It has ribonuclease H activity and can degrade the RNA strand of an RNA:DNA hybrid after DNA is synthesized from RNA
4) tRNA is used as a primer for reverse transcription
What is the infection cycle of HIV? What proteins and receptors are involved in HIV infection?
HIV attacks Th cells and enters through tears/lesions in the mucosal layer. It replicates in monocytes and Th cells and numbers of these cells can decrease below 200 per mL (500-1000 is the normal level) after an asymptomatic period that can last up to 2 years. As Th cell numbers drop, infected individuals can exhibit an increased sensitivity to infection by other pathogens and eventually weight loss, diarrhea, sarcoma, cancer, dementia, etc.
1) First attachment involves the interaction between a gp120 protein and the CD4 and CCR5 on the surface of host cells (T helper cells and monocytes). HIV resistance can be linked to variations in CCR5
2) Then the fusion of the viral envelope with the host cell membrane is mediated by gp41
3) The nucleocapsid can then insert into the host cell cytoplasm via endocytosis and allow the genome to be reverse transcribed. Host cell machinery can be used to form viral mRNAs and synthesize the virus within the host cell
How is HIV transmitted and what are key symptoms?
It enters through tears/lesions in the mucosal layer and can be transmitted through bodily fluids. Characterized by having a CD4 T cell number of less than 200/mL or one infection such as TB, wasting syndrome, pneumonia, etc.
What are key characteristics of negative strand viruses and what does their genetic material look like? What are three examples?
They are single stranded RNA viruses and reproduce via exocytosis from a host cell where particles budding from a host cell membrane become enclosed in a membrane envelope. The RNA strand is negative and therefore complementary to the mRNA strand so host replication needs to occur in order for it to be transcribed. Rabies, Ebola, and Influenza are examples.
How does replication occur in negative strand viruses?
When the virus is inside the host cell, the nucleocapsid migrates to the nucleus and the negative viral RNA molecules are transcribed to positive strand mRNAs using host primers. This is accomplished using viral endonucleases (catalyzes mRNA production and processing) and replicases (catalyzes the production of an mRNA strand from an RNA template from the virus). The replicase can also be used to catalyze the production of a second negative RNA strand using a positive strand mRNA as a template and more mRNA can be produced. Poly-A tails are then added to the viral mRNAs and are also transported out of the nucleus.
The mRNA molecules produced from the viral negative template strand are translated and this leads to the production of structural proteins and the assembly of the nucleocapsid. The virus then assembles in the host cell cytoplasm and buds from the host cell.
What are key characteristics of positive strand viruses and what does their genetic material look like? Give three/four examples?
They contain single strand RNA that can be coded into polyproteins and they tend to be small and consist of an icosahedral structure. 4 examples are poliovirus, rhinoviruses, hepatitis A, and corona viruses.
How does replication in positive strand viruses occur?
The RNA contained within positive strand viruses can be directly translated into a polyprotein which forms viral proteins using host cell machinery. They can also synthesize minus strands which can be used to synthesize new plus strands. All of this takes place in the cytoplasm.
What virus is responsible for the majority of colds? What does it mean if a virus is zoonotic and what are three zoonotic coronaviruses that have been deadly for humans?
Human coronaviruses cause approximately 30% of colds and three zoonotic viruses that spread between animals and humans in particular have been deadly: MERS-CoV (which caused Middle East Respiratory Syndrome), SARS-CoV (beta coronavirus that causes severe acute respiratory syndrome) and SARS-CoV-2
SARS-CoV in particular has a high fever, aches, respiratory symptoms, difficulty breathing, dry cough and transmission is believed to require direct contact with a 5.1% fatality rate.
What is the infection cycle of coronavirus? What proteins/receptors are involved and how are they significant? What are two routes of entry and what are the specific roles of RNA-dependent RNA polymerase and three polyproteins that are initially translated?
1) Binding to a membrane fusion protein and viral entry via endocytosis occurs. For SARS-CoV-2 ACE2 is the primary membrane fusion receptor protein. There are abundant amounts of ACE2 on type 2 pneumocytes in the alveoli since ACE2 is important for regulating blood pressure, wound healing, and inflammation
2) The major route of entry is via the TMPRSS2 pathway where the ACE2 binds to the SARS-CoV-2 spike protein and TMPRSS2 (Transmembrane Serine Protease 2) cleaves this protein to allow for efficient fusion between the viral and host cell membrane and viral entry. There is also an endosomal or cathepsin pathway where Cathepsin L cleaves the spike protein
3) The positive strand can be translated by a host and also replicated via RdRp, which replicates positive mRNA into a negative strand template which can be used to form more positive strands, and polyproteins/viral components can be synthesized
4) A polyprotein can be encoded using the positive mRNA that become many smaller proteins including a protease, an RNA dependent RNAP, a Helicase, a Spike protein, and several accessory proteins such as an envelope, membrane, etc. The new viruses are then released via exocytosis
What is the role of the Nsp15 endonuclease in SARS-CoV-2 infection, and what kinds of infections are interferons used in?
It inhibits the translation of immune signaling molecules by sitting at the entry tunnel of ribosomes and cleaving host mRNAs but ignoring viral mRNAs and allowing them to be translated due to the presence of a specific leader sequence. This has the effect of blocking the translation of immune signaling molecules such as Type I and Type III interferons.
How does SARS-CoV-2 cause COVID symptoms?What conditions are risk factors for COVID and why? How do dexamethasone and Paxlovid work to mitigate COVID symptoms?
Shortness of breath is a key COVID symptom. This occurs since SARS-CoV-2 binds to an ACE2 receptor on lung epithelial cells infecting them and infected cells signal the immune system and activate CD8 T cells, macrophages, etc causing inflammation. This inhibits the transfer of oxygen in the alveoli and fluid can also build up in the lungs. These factors cause shortness of breath. Conditions that are associated with high levels of inflammation can lead to severe symptoms, such as old age, diabetes, autoimmune diseases, obesity since body is “primed” to respond more strongly to SARS-CoV-2.
Dexamethasone - corticosteroid that reduces inflammatory overstimulation that is caused by SARS-CoV-2 and prevents collateral damage caused by an infected individual’s immune system
Paxlovid - contains two drugs
1) Ritonavir which can be used to slow the metabolism of antivirals in the liver since a CYP inhibitor prevents the antivirals from being metabolized too quickly by CYP enzymes in the liver
2) Nirmatrelvir which inhibits the viral protease that can separate the polyprotein precursor into individual proteins
How does the mRNA vaccine work in detail?
A vaccine is produced using mRNA that encodes for the Spike protein that binds to an ACE2 receptor as an antigen. Upon infection, this protein can then be more easily targeted by immune cells.
What is happening with the SARS-CoV-2 variants? What is the S-fuse assay and how is it involved in infection and research?
Under an S-Fuse Assay, a virus is plated with host cells and the GFP protein is split meaning fluorescence occurs when a virus, SARS-CoV-2, is fused with and ACE2 receptor on the host cell. The level of fluorescence can be used to estimate the virulence and level of binding. Variants have been shown to bind to ACE2 more strongly in an assay indicating it is more virulent.
Define the terms endemic, epidemic, pandemic, prevalence, incidence, morbidity, and mortality
Endemic - disease that is always present at a low frequency in a population
Epidemic - a larger than normal frequency of infection, likely to to rapid and direct human-human transmission
Pandemic - epidemic that occurs over a wide geographical area
Prevalence - total number of active cases
Incidence - number of new cases
Morbidity - having an illness
Mortality - death from an illness
What are some steps epidemiologists take to stop an epidemic?
1) Track diseases through systematic surveillance (reporting) or syndromic surveillance (tracking/observing emerging diseases)
2) Identify an outbreak
3) Track down a patient zero (index case) and identify close contacts
4) Determine disease etiology for emerging diseases (cause, transmission, infection)
5) Develop interventions/treatments
What are three categories of drugs against HIV and what are their mechanisms?
1) Nucleoside analogs that inhibit reverse transcriptase-AZT (azidothymidine) are one example. Analogs can be added to a growing DNA strand synthesized by reverse transcriptase but prevent the addition of the next base and this prevents integration of viral DNA with genome
2) Nuceloside reverse transcriptase inhibitors interact directly with the reverse transcriptase enzyme that alter the catalytic site
3) Protease inhibitors bind to and block the activity of HIV protease preventing polyproteins from being processed
The most effective therapy for HIV has been using several different drugs (usually a protease inhibitor and two nucleoside analogs)
What issues are associated with HIV treatment and what are some barriers to treatment?
It is expensive (approximately $10,000 per year per patient) so a large number cannot afford treatment. There are also many side effects such as fatigue, anemia, body changes, neuropathy, etc. Patients who discontinue also have an immediate rebound in viral levels and drug resistant strains can also develop.
Some barriers include politics (leaders can deny the presence of a health crisis) and false science/pseudoscience which can become widespread
How can PrEP be used in the treatment/prevention of HIV?
It is called pre-exposure prophylaxis and it is a medication that reduces the risk of contracting HIV from sex (~99% decrease) and from IV drug use (~74% decrease). PrEP is administered by taking two oral medications and one approved weekly shot. Some side effects include a headache, nausea, apetite loss, possible kidney/liver damage. Different from PEP or post-exposure prophylaxis.
What does 90/90/90 mean?
It is a UN target where 90% of infected of individuals should be aware of their HIV status, 90% of those individuals are on HIV treatment, and 90% of those individuals are virally suppressed.
Why are vaccines difficult to develop for retroviruses, specifically in the case of HIV? What is the aim of Phase III trials in South Africa and what are the targets of these studies?
1) Live attenuated viruses can integrate into a host cell’s DNA and cause disease
2) HIV has a high mutation rate and it is difficult to make a vaccine against all clades
3) mRNA vaccine technology is limited since HIV along with many other retroviruses are so diverse meaning multiple proteins will have to be targeted and a lot of mRNA will have to be used but the amount of mRNA that can be incorporated in a vaccine is limited
What did the Frontier Phase III trials in South Africa suggest about the use of vaccines for HIV?
HVTN 702 trial enrolled 5400 HIV-uninfected people at risk for infection to test the safety/efficacy of the RV144 HIV vaccines. Two vaccines were tested, the ALVAC-HIV containing a viral vector with 3 HIV genes and a protein vaccine supplied by GSK which should create an immune response to a protein GP120 which facilitate entry into host cells.
This was the most successful HIV trial to date and the efficacy was 31% against a single HIV clade.
What are the symptoms of Ebola and how is it transmitted? What are isolation and safety protocols and what was significant about the 2015 Ebola outbreak?
It is highly lethal where 50%-90% of infected individuals die usually due to organ failure/tissue death from inflammation. Symptoms are fever, bleeding, vomiting, muscle/joint pain, skin rash, diarrhea. Fruit bats are considered to be a vector and it is transmitted via bodily fluids, consumption of infected meats, contact. Incubation is a 2-21 day period
In 2015, African health care workers were unfamiliar with the disease and mistook it for Lassa Fever/Malaria. There was a rapid spread to cities, stocked/staffed health care facilities were not present, WHO and other groups did not recognize the serious nature of the outbreak, and there was a failure of some to respond. The 2015 Ebola outbreak was widespread wheras in the past it was limited by scope and geography
How can antibody therapy be used to treat Ebola? Is there an Ebola vaccine and does it work?
Ebola patients were recently treated with antibodies from survivors which was shown to be effective, and a Canadian company is developing a way to produce antibodies for Ebola in plants.
rVSV-ZEBOV is also a recombinant vaccine that consists of a vesicular stomatitis virus which is engineered to express a glycoprotein from Zaire ebolavirus to provoke an immune response to Ebola and it was approved by health care workers in 2019.
What are NTDs? How many people are affected by them and what are risk factors?
WHO defines them as disease that share a set of features that allow them to persist in conditions of poverty where they cluster and frequently overlap. Over 1 billion people suffer from one or more
What is dengue/hemorrhagic fever? What type of mosquito usually transmits it, and how many viruses cause dengue? What are the moderate and severe symptoms?
It is a mosquito-borne disease and half the world’s population lives in areas where dengue viruses can be transmitted. It tends to be transmitted by the Aedes agytpi mosquito. There are four distinct but related viruses and recovery from one provides lifelong immunity to that one but only partial protection from the other three. Vector control is the main area of prevention/control.
Infants/young children may have a fever with a rash and older children/adults may have either a mild fever or a disease with a severe headache, pain, muscle/joint pains, rash. In severe cases it can lead to convulsions, liver enlargement, and haemorrhagic phenomena (bleeding through sweat glands), and circulatory failure
What is the lifecycle of dengue (from mosquito to person and back to mosquito)?
Mosquitos usually acquire the virus after feeding on the blood of an infected person. After the virus is incubated for 8-10 days, an infected mosquito can transmit the virus to susceptible individuals for the rest of its life, and female mosquitos can also transmit the virus to offspring which can infect humans. Virus circulates the blood for 2-7 days (when they have fever) and this is when mosquitos acquire the virus. Dengue can also be transmitted from mother to child during pregnancy.
How is dengue treated and what are some of the best prevention methods? How is this impacted by the increase in Aedes aegypti?
No treatment but symptoms can be managed and with intensive therapy, mortality can be reduced to less than 1%. Maintenance of fluid volume is most critical but pain/fever can also be treated.
Vector control via environmental management and chemical methods have also been key. This includes solid waste disposal and improved water storage practices such as covering containers to prevent egg laying by female mosquitos. Led by community based programs
Vaccine was also approved for children in 2022 for those with previous infection in areas where dengue is endemic
