Exam 1 Flashcards
What type of enzymes do viruses possess and what are their functions ?
1) lysins- produced by bacteriophage to cleave host walls 2) retroviral- (HIV), injects genetic material into infected cell 3) reverse transcriptase- create cDNA 4) nucleic acid polymerase- viral genome replication 5) neuraminidases: cleave glycosidic bonds to release the virus
What allows for replication of defective viruses?
Mixed infections and a helper virus
What is a defective integrating particle (DIP)?
Defective virus integrates its own DNA into an active virus to deactivate it.
What type of virus is used for a vaccine ?
Pseudovirus-there is nucleic acid in the capsid instead of the virus
What do Hog Cholera and Classical swine fever have in common ?
Same disease, different name Both caused by pestivirus
What do Hand-foot mouth disease and Foot and Mouth disease have in common ?
Different disease, different name Hand,foot, mouth-enterovirus Foot and mouth- aphthorvirus
Class I
without reverse transcriptase-DNA, double stranded
Class II
without reverse transcriptase, DNA, single stranded
Class III
without reverse transcriptase, RNA double stranded
Class IV
without reverse transcriptase, RNA, single stranded: + sense
Class V
without reverse transcriptase, RNA, single stranded: - sense
Class VI
with reverse transcriptase, DNA to single stranded + sense RNA
Class VII
with reverse transcriptase, RNA to double stranded DNA
What is the only group allowed to classify viruses?
ICTV
Bornaviridae
single strand linear-RNA negative sense Borna disease-horses: ataxia, cats: posterior paresis
Astroviridae
single strand linear-RNA positive sense Astrovirus enteritis Avian nephritis
Prions
Scrapie-sheep and goats BSE
What are the 5 types of proteins found in viruses?
1) enzymes 2) viral non structural proteins 3) structural proteins 4) inhibitors 5) regulatory
What do viruses need to live?
A living host cell
What are three ways to cultivate a virus?
Lab animals, tissue culture, embryonated egg inoculation
What is a suspension cell culture?
Cells that do no need to attach to anything to grow
What is a monolayer cell culture ?
Continuous layer of cells that grow on the culture plate
What is a primary cell culture?
Cells directly from the parent line with the same number of chromosomes, and same chromosomes as the parent
What are advantages and disadvantages of a primary cell culture?
Advantage: used for viral vaccines, best culture used to grow viruses, close to parent line, heterogenous Disadvantage: easy to contaminate, difficult to grow, short life span, may not be the same as parent line
What is a subculture, and why do we do it?
Also called passage, but used to transfer cells to a new vessel. It is used to allow for continuous growth/provide fresh nutrients.
What does the primary cell culture become after the first subculture?
Cell line: Continuous or finite
Finite/definitive cell line characteristics are…
Possess contact inhibition, maintain original morphology/chromosomes, homogenous population, can be cultured 100x before death, slow growth rate (24-96hrs), less hassle to use, derived from embryos or subculture
Continuous cell line characteristics are…
Infinite amount of replications, growth rate is fast (12-24 hrs), not approved for vaccines, derived from cancerous cells or induced, hassle free, genetically weird
What are the 3 morphologies a cell line can be?
Fibroblastic, epithelial-like, lymphoblast-like Fibroblastic and Epithelial-like grow on a substrate Lymphoblast-like grow in suspension
Examples of culture media…
Eagle’s Basal Medium, Leibovitz L-15 Medium
Characteristics of serum in media are…
Growth: 5-10% Maintenance: 0-2% Fetal bovine serum is most commonly used Provides- growth factors, nutrients, attachment and spreading factors, adhesion factors, hormones Regulate cell membrane permeability Carrier proteins
What does phenol red pH indicator test for ?
Contamination (will be red)
How much CO2 should be used in cell culture experiments ?
4-10%
T/F-antimicrobial agents are used in cell culture
T- it prevents contamination
What does culture media provide for cells ?
Vital nutrients necessary for the cells to grow.
Ex. Leibovitz L-15 Medium
What is the serum in culture media for, and what are the two that are used ?
Adhesion, attachment, hormone, growth and spreading factors.
Molecular weight nutrients
Carrier proteins for lipid substances and trace elements
Regulate cell membrane permeability
Ex. Growth Medium: 5-10%, Maintenance Medium: 0-2%
What tasty animal to we obtain the serum from ?
Fetal bovine serum
What is the significance of a color change when using phenol red pH indicator ?
Contamination in your culture or you’re lazy and left the cell culture in the same plate for too long!
T/F- you have to use 4-10% CO2 when using media buffered with a CO2-bicarbonate based buffer..
TRUE
Since we’re going to be virologists..tell me about the cell line and optimal temperatures they need…
Human/Mammals:36-37C
Insect: 27C
Avian: 38.5C
Cold-blooded animals: 15-26C
What proteases would you use for cell cultures ? Do you even care ?
Trypsin, Collagenase….. yas, because it’s on the exam. Also, one day I might say screw veterinary medicine and become a virologist.
T/F-It doesn’t matter how long you incubate cell cultures in high trypsin concentrations. What do researchers use to maintain cell integrity ?
FALSE-cells will be killed or damaged in high concentrations for too long of incubations periods.
Use enzyme-free dissociation buffers to maintain integrity
How do you dispense cells ?
Polystyrene flasks, polstyrene dishes, microwell plates, roller bottles, leighton tube
Cell cultures can be examined using…
Inverted tissue culture microscope
Cytopathic effect is…
Damage to cells during a virus invasion
Ex. Slide 34 of Cultivation of Viruses
What is the ideal way to isolate multiple viruses?
Co-cultivation: single monolayer consisting of multiple cell types, detects viral antigens using fluorescein-labeled monoclonal Ab.
Ex. Slide 41 of Cultivation of viruses
T/F- eggs are only good for food…
False! Can use to cultivate viruses
Materials: egg cander, specific pathogen free eggs, drill bits, betadine, sterile swabs
What does a blood ring indicate in an egg ?
Early embryonic death
What are the 4 routes of egg inoculation ?
Chorioallantoic, amniotic, allantoic, yolk sac
How do you do a yolk sac inoculation ?
22 gauge, 1.5 inch length needle…drill hole…place inoculum below embryo and in the yolk sac…seal hole with scotch tap or wax.
How do you do an allantoic cavity inoculation ?
26 gauge needle, drill hole above oer below air sac, inoculate allantoic cavity
How do you do an amniotic cavity inoculation ?
26 gauge needle, drill hole over the air sac, inoculate amniotic cavity
Chorioallantoic membrane inoculation (CAM) is done how?
Drill two holes in the eggshell: side and above the air sac
move air sac to side of the egg by gentle suction with rubber bulb, inoculate CAM
How do you know there is virus growth ?
Death of embryo, paralysis, stunted growth, urate deposits, hemorrhage and congestion, heamgglutins in embryonic fluids, extracellular membrane lesions
How do you inoculate mice ?
Intracerebral or intra peritoneal
T/F particles move at different rates depending on their mass
True
Isopycnic centrifugation: Buoyant density and isopycnic point. Tell me about them.
Buoyant: object has exact same density as fluid.
Isopycnic: buoyant density of particle equals that of surrounding density gradient medium.
Density Gradient Medium is…
Sucrose and Cesium Chloride
What will never happen during Isopycnic Centrifugation?
Particles will not sediment to bottom of tube
What is used to purify viruses and virus like particles ?
Chromatographic membranes
What are the two types of viral quantification tests ?
Biological: Depend on virus particle biological activity…. plaque, pock assays…various endpoint titration methods
Physical: Do NOT depend on virus particle…. electron microscope, hemagglutination, ELISA, PCR, flow cytometry
What is the most direct method to determine the concentration of viruses, but not routinely used due to
Transmission Electron Microscopy
Virus Counter 2100 is…
A memory dump question, but…..
it’s a specialized version of flow cytometry developed for nanometer scale particles. Measures intact virions.
How do you assess Antigen concetrations ?
Hemaglglutination assay
ELISA
High performance liquid chromatography (HPLC): UV analysis
Single Radial Immunodiffusion (SRID): agarose gel seeded with polyclonal antisera against a viral Ag
What is qPCR used for?
Quantifying viruses based on gene expression
What is the most accurate quantitative biological assay used ? What is the minimum PFU needed to form plaques ?.
Plaque. 1000 PFU
T/F-Plaque assays are used to obtain actual numbers of viruses
False. It is just a functional measurement
What is the Principle of Plaque assay ?
Each represents cell lysis initiated by one viral particle
What do plaques look like vs. viable cells ?
Plaques: clear circles
Viable Cells: blue
Determination of Titer calulation:
GO DO THE PRACTICE PROBLEMS
Average plaque count x reciprocal of the dilution selected
PFU/ml
What is the time for the eclipse period vs. latent period for the one-step virus growth curve ?
Eclipse: 0-12 hrs intracellular, none extracellular
Latent: 0-16 hours intracellular, extracellular
Why do we even care about Pock assay ?
It’s on the exam!!!!
It measure the necrotic area on chorioallantoic membrane of embryonated egg-titraion of herpesvirus and poxvirus.
pock forming units/ml
Transformation assay is….
Quantitative determination of titers of oncogenic viruses-lose contact inhibtion
focus forming units/ml
Quantal Assay is…
the measurement of presence or absence of infection.
used for certain viruses that don’t form plaques/determining the virulence of a virus in animals or eggs
What assay also measures the endpoints: ID50, LD50, Embryo LD50, Paralytic Dose50 ?
Quantal Assays
TCID50 is ?
Tissue culture infectious dose that infects 50% of the cells
Multiplicity of infection (MOI) is…
average number of virus particles infecting each cell
What type of idiot cell allows for a virus to replicate ?
Permissive Cell
What type of smart cell does not allow for a virus to replicate?
Non-permissive cell
One-step virus growth curve Eclipse period is also called what ?
Viral uncoating and replication
What is Burst size?
Number of virions released. Height curve= burst size.
What is the order and descriptions of One-Step virus growth curve ?
Adsorption: virus attaches and enters cells
Eclipse period: time interval between uncoating and appearance, intracellularly, of first infectious progeny virions. No infectious virus can be detected during this time. 2-12 hrs.
Latent Period: time before new infectious virus appears in the medium. No extracellular virions detected.
Burst size: number of infectious virions released per average cell.
What type of receptors do our cells possess for viruses to attach to?
Proteins, carbohydrates, glycoproteins, lipids, lipoproteins
T/F-sometimes an additional cell surface moleucle/co-receptor is required for entry
True
What evil virus that is rampent in St.Kitts uses more than one host cell receptor ?
HIV
Enveloped viruses are mostly…
receptor-mediated endcytosed
Clathrin-mediated endocytosis of virus by host…
1) Virion attach to host receptor
2) Adapter proteins bind to clathrin
3) Formation of Clathrin-Coated Pit (CCP)
4) Dynamim pinch off CCP that release Clathrin-Coated Vesicle (CCV)
5) Viral content delivered to endosomes
6) pH changes to acidic, viral genome released to host cell
What happens to most non-enveloped viruses ?
LYSIS
What occurs in local permeabilization ?
Allow virus capsid penetration into the cytoplasm
What other types of receptor-mediated endocytosis are there ?
*JUST REMEMBER THE NAMES*
Caveolin-mediated endocytosis
Clathrin and caveolin independent endocytosis
What do enveloped viruses only have ?
Membrane fusion/surface fusion
What is retained on the cell surface that makes it a traget to the immune system ?
Viral glycoproteins
What are the steps of antibody-dependent cell mediated cytotoxicity ?
1) antibody binds antigen on surface of target cell
2) Fc receptors on NK cells recognize bound Ab
3) Cross-linking of Fc receptors signals the NK cell to kill target
4) APOPTOSIS
What viruses are pH independent ?
HIV and measles
What are pH dependent viruses ?
HA in influenza viruses
How else can non-enveloped viruses get their viral genome into a host cell ?
Pore-mediated penetration!
How does FIP enter the host ?!
Antibody mediated attachment!
Attaches to the hosts’ macrophage via spike proteins to the CD13 receptor
Antibodies against spike proteins assist with entry into host cells through IgG-Fcgamma receptors.
Let’s talk about the uncoating of viruses…
Some animal viruses begin to uncoat after binding to external receptor, completly uncoat in cell-Poliovirus
Some uncoating is complex series with host and viral gene products-Poxvirus
Retro/reoviruses occur inside capsid
What is the process of pre-mRNA ?
Viral mRNAs are translated by cellular protein synthetic apparatus
Viral mRNA must conform to requirements of host cell translation system
Processing of primary RNA transcript/pre-RNA
mRNAs translated in cytoplasm
Viral mRNAs produced in nucleus exported to cytoplasm
Where are viruses capped ? What is the purpose of capping ?
5’ end with 7-methelyguanosine
stability, binding of mRNA to ribosomes, mark mRNA as ‘self’
How are caps synthesized ?
host cell enzymes (retroviruses, adenoviruses)
viral enzymes (poxvirus, reoviruses)
cap snatching! virus steals cap from host mRNA (influenza)
What is added t 3’ end that is important for translation ?
PolyA tail and cleavage occurs at 10-25 nucleotides downstream
What is spliced out during transcription ?
Introns bc it DOES NOT CODE FOR AMINO ACIDS
Exons do code for amino acids so they are saved!
Monocistronic vs polycistronic
Monocistronic: mRNa that encodes one polypeptide
Polycistronic: mRNA encodes several polypeptides
What are 5 important viral proteins ?
Enzymes, structural proteins, viral nonstructural proteins, regulatory proteins, inhibitors
How are naked and enveloped progeny virions released ?
Naked: lysis of host cell
Enveloped: budding
What 4 viruses are released by exocytosis ?
Flavivirus, Arterivirus, Coronavirus, Bunyavirus
What is a way viruses can spread cell-to-cell ?
Intercellular spread
Cell-cell plasma membrane fusion: herpesvirus, paramyxovirus, retrovirus
Tight junction: herpesvirus
Neural synapse: rabies virus
Actin or tubulin containin structures: poxviruses
Actin-containing structures, Filopodial bridges: retroviruses
Nanotube subversion: HIV-1
Virological synapse: retroviruses
What variables does virulence depend on ?
Virus: strain, portal of entry, tropism to host organs, dose of infection, immuno evasion
Host: species, immunity, receptor, physiological factors, inhibitors, fever
T/F- The higher the ID50 and LD50 the more virulent an organism is
FALSE-if it’s low it’s virulent
What are 3 other ways of assessing degree of virulence ?
Severity of illness, incubation period, degree of severity, location, and distribution of gross, histologic, and/or ulstrstructural lesions in affected animals.
What are the 5 sequential steps in viral infection…
1) entry of viruses, primary replication
2) spread, tropism, infection of target organs
3) virus-cll interactions, secondary replication
4) tissue and organ injury
5) shedding
Can viruses penetrate intact skin ? Why/Why not?
NO
Skin is awesome!!! –> dense outer layer keratin, low pH, presence of fatty acids, bacterial flora, dryness, components of innate and adaptive immunity.
What defenses do our mucuos membranes have ?
IgA, virucidal proteins
What are the defenses of the GIT ?
Mucous membrane or oral cavity and esophagus, acidity of stomach, alkalinity of intestine, layer of mucus covering the gut, lipolytic activity of bile, proteolytic activty of pancreatic enzymes, defensins, IgA, scavenging macrophages
What are the defenses of the respiratory tract ?
Mucociliary blanket, alveolar macrophages, nasal associated lymphoid tissues (NALT), bronchus-associated lymphoid tissue (BALT), temperature gradient
Do you care to tell me about the local spread on epithelial surfaces ?
Probably not, but you should….it’s not an FYI….
Virus replicates in epithelial cells at entry site, spread locally by infecting contiguous cells, produce localized infections…shed…or…spread to adjacent subepithelial tissues.
Let’s continue the talk about subepithelial invasion and lymphatic spread…
After local invasion…. the inflammatory response to virus infection, destruction of epithelium, or transport pathways like transcytosis allows for the subepithelial invasion.
This method should allow for viruses to overcome local host defense
What is critical for subepithelial spread of viruses? Also, what is apical vs. basolateral release ?
Directional shedding
Apical: virus dispersal
Basolateral: allows access to underlying tissues for systemic spread
What has targeted migration and replication of viruses within phagocytic leukocytes, pass straight through lymph nodes to enter blood stream ?
Subepithelial invasion and lymphatic spread
Viremia and the three types are…
Virus in the blood.
Primary: initial entry of virus into blood after infection
Secondary: Virus replicates in major organs, and enters circulation again
Passive: direct innoculation of virus into blood.
Active vs. Passive Viremia are…
Active: release of virions from initial site of replication to blood stream.
Passive: no initial replication elsewhere in host before
What two ways are viruses found in the blood stream ?
Macrophages, Lymphocytes
Free in plasma (parvovirus)
How are viruses eradicated from the blood stream ?
Mononuclear phagocytes in the spleen, liver, bone marrow, antibody clearance, complement-mediated clearance
What examples of virus are spread through nerves ?
Herpes simplex virus: low neuroinvasiveness of CNS, highneurovirulence. Always enters PNS, rarely CNS.
Mumps virus: neuroinvasiveness, low neurovirulence. Most infections are CNS.
Rabies: high neuroinvasiveness and high neurovirulence. Infects the PNS and spreads to CNS with 100 % lethality.
What are the 4 ways viruses spread in the nervous system ?
Axons, perinueral lymphatics, endoneural space, Schwann cells
Retrograde vs anterograde spread…
Retrograde: virus travels opposite direction of nerve impulse flow
Anterograde: virus travels in direction of nerve impulse flow
Centripetal vs Centrifugal movement…
Centripetal: toward CNS
Centrifugal: from CNS within peripheral nerves to other parts of body.
Why are viruses so good at getting through the blood-brain barrier ?
1) Increase permeability of endothelial cells via TNF
2) Breakdown of endothelial cell junctions through Matrix-metalloproteinase (MMP)
3) Monocytes!
Virus Shedding ! Yay… what is shedding ?
Infectious virions is crucial to maintenance of infection in populations
What is critical to virus transmission ?
Virus shed
Acute vs Persisten infection (shedding)
Acute: intensive over short period of time
Persistent: low shedding for months to years
What are the routes of viral shedding from host ?
Oropharynx & GI, Respiratory, Mucous membranes, Oral & genital fluids, blood, urine, milk, skin
Tropism is…
affinity of a virus for a particular host tissue
What determines viral tropism ?
Receptors of host cell, attachment proteins, viral enhancers, cellular protease requirement, temperature of replication, acid lability and protease digestion, transcriptional control of tropism, anatomic barriers, host organ response to infection
Types of virus injury to skin (6)…
1) Vesicle-small distinct elevation with fluid
2) Ulcer-opening in the skin caused by sloughing of necrotic tissue extending past the epidermis
3) Nodule, tumor-palpablel solid, elevated mass with distinct borders. tumors extend deep.
4) Warts- benign skin growths
5) Papules-solid elevations without fluid with sharp borders
6) Erythema- reddening of skin, from systemic viral infections
Types of injury to GI tract…
1) Destructions of enterocytes from replication, hypersecretion
2) GI disease, malabsorpstion, diarrhea
3) Dehydration, acidosis, hemoconcentration
Injury to respiratory tract (8)…
*There is tropism for different parts of the respiratory tracts*
1) loss of ciliary activity
2) loss of mucous layer lining
3) multifocal destruction of epithelium
4) inflammation
5) exudation
6) influx of inflammatory cells
7) obstruction of air passages
8) hypoxia & respiratory distress
9) secondary bacterial infections
Types of injury to the CNS (6)…
Lytic infections cause..
1) encephalitis or encephalomyelitis
2) neuronal necrosis
3) neurophagia
4) perivascular cuffing (perivascular infiltrations of inflammatory cells)
5) Progressive demyelination
6) neuronal vacuolation
Injury to endothelium (4)..
1) Petechial and ecchymotic hemorrhages
2) Disseminated intravascular coaglation [DIC]
3) Edema
4) Infaraction
Describe DIC…
Something males have….ha….but really…
1) Complication from viral infection of blood vessels
2) widespread activation of clotting cascade = formation of blood clots in small vessels
3) excess clots can necros organs
4) once clots are degraded severe bleeding can occur
What two ways can fetuses get infected ?
Amnionic via vagina
Placental via viremia
What is teratogenesis ?
Abnormal development or slowing of development in embryo or fetus –> may result in death or malformations
Susceptibility to teratogens: varies with species, and decreases with age
Cell injury to embryo..
1) cerebellar hypoplasia
2) arthrogryposis
3) porencephaly
4) congenital hydranencephaly
Explain virus induced Immunopathology…
1) Required tissue injury mediated by host response to virus infection
2) Depends on balance between protective and destructive effects of host response
3) Infected cells not immediately destroyed and becomes chronic
4) Host can survive with minimal symptoms if virus cleared quickly (opposite happens when there is a large immune response)
The role of Tcells…is this immuno? no!…it’s Viro…
T-cells can destroy virus infected cells or release cytokines
CD8+ destruction of infected cells can destroy liver
CD4+ and CD8+ can create chronic inflammation in persistent viral infections
CD4+ release more cytokines than Cd8+
What are cytokines and examples ?
Small proteins for cell signaling. Mediate and regulate immune process, and cause inflammation.
Ex/ Monokines-mononucler phagocytic cells
Lymphokines-Th cells/lymphocytes
Interleukins-leukocytes (mediators)
The role of Innate Immunity is…
TLRs-persistent viral infections allows for these cells to release PRO-inflammatory cytokines thus producing inflammation.
Free radicals, nitric oxide, and superoxide inhibit replication….but will cause cell damage if produced in XS
Toxicity from antibody response..
Inflammatory reaction from antibody binding to cell that engages IgG and Fc to release mediators that leads to complement cascade
What is vasculitis caused by ?
Antibodies failing to neutralize virus which triggers the complement cascade
What type of infection suppresses the humoral and cell-mediated immune system ?
Systemic infection
