Term
| Which virus can be barely seen with a light microscope? |
|
Definition
|
|
Term
| When was the electron microscope invented? |
|
Definition
|
|
Term
| What are the two types of electron microscops? |
|
Definition
-transmission electron microscropy (TEM)
-scanning electron microscopy (SEM) |
|
|
Term
| What focuses the electrons emitted by a electron microscope? |
|
Definition
| -electromagnetic lenses and metallic apertures |
|
|
Term
| How are magnified images visualized with an electron microscopes? |
|
Definition
| -visualized on a fluorescent screen or recorded on photographiic plates |
|
|
Term
| Compare the magnification power of EM as compared to the light microscope and the human eye. |
|
Definition
| -EM is 1000X that of the light microscrope which is 1000X that of the human eye |
|
|
Term
| What is the resolving power of the human eye? What is the resolving power of the light microscope? TEM? SEM? |
|
Definition
-human eye = 0.25 mm
-light microscope = 0.25 um
-TEM = 0.25 nm
-SEM = 10 nm |
|
|
Term
| The high resolution of EM is made possible by using a ______. |
|
Definition
|
|
Term
| Viruses can be viewed via what two methods in transmission electron microscopy (TEM)? |
|
Definition
-negative staining
-thin sectioning |
|
|
Term
| What is Negative Staining with TEM? |
|
Definition
| -staining the virus preparation with a non-penetrating stain, the stain will stain the background and not the virus |
|
|
Term
| What is the most frequently used negative stain? |
|
Definition
|
|
Term
| Are viruses light or dark in negative staining TEM? |
|
Definition
| -the virus particles are white objects surrounded by dark veavy metal atoms |
|
|
Term
| What is the simplest and most rapid method of detecting and recgonzing virus particles? |
|
Definition
|
|
Term
| What is the major disadvantage of negative staining TEM? How do we get around this? |
|
Definition
-the amount of virus in a specimen must be high
-use fecal samples or concentrate the virus |
|
|
Term
| What are the major advatage to positive staining TEM? |
|
Definition
| -simplest, fastest, allows family classification based on size, shape, and structure |
|
|
Term
| What is immunoelectron microscopy (IEM)? |
|
Definition
| -a rapid serological diagnostic test using EM |
|
|
Term
| How do we perform immunoelectron microscopy (IEM)? |
|
Definition
| -virus prep is mixed with virus specific monoclonal Ab that is labeled with small iron or gold particles, this Ab causes the viruses to clumb together and the iron/bold labelded Ab will be visible |
|
|
Term
| What are the advantages of the thin sectioning method of TEM? |
|
Definition
-permits the visualization of the site of virus replication and maturation in host cells
-preps include fixation, dehydration, and embedding in palctic |
|
|
Term
| What are the most commonly used fixatives in the thin sectioning method of TEM? |
|
Definition
| -glutaraldehyde, cacodylate, and osmium tetroxide |
|
|
Term
| In the thin sectioning TEM method, the epxy resin blocks are sectioned by a/an what? |
|
Definition
|
|
Term
| What stains are used in the this sectioning method of TEM? |
|
Definition
| -lead citrate and uranyl acetate |
|
|
Term
| What are the disadvantages of the thin sectioning method of TEM? |
|
Definition
| -takes time and is not used routinely for diagnostic purposes (positive stianing) |
|
|
Term
| What is the one requirement for scanning electron microscopy (SEM) to work? |
|
Definition
| -the specimen must be coated with metal, usually gold |
|
|
Term
|
Definition
| -the specimen is coated with gold and a beam of electrons scans the surfase of the specimen, the electrons bounce off and the energy imparted ot the metal coated surface also releases secondary electrons from the specimens and they are picked up with a detector, amplified, and directed to a screan to form an image |
|
|
Term
| What is the major difference between TEM and SEM? |
|
Definition
| -SEM gives a 3D image while TEM gives a 2D image |
|
|
Term
| What is the major disadvantage of SEM? |
|
Definition
| -only the surfaces of the images can be observed and the resolution is limited to about 10nm |
|
|
Term
| True or False: Viruses grow in living cells only. |
|
Definition
|
|
Term
| In the lab, what three host systems are used to grow viruses? Which system is the most important? |
|
Definition
-animals, chicken embryos (eggs), and cell cultures
-cell culture is the most important |
|
|
Term
Describe the animal inoculation route used in the following situations:
a) neurotropic virus
b) respiratory virus
c) enteric virus |
|
Definition
a)intracerebral inoculation
b) intranasal route
c) per os route |
|
|
Term
| When are eggs inoculated with virus? |
|
Definition
| -5--15 days after fertilization |
|
|
Term
| What are the different routes of inoculation of eggs with viruses? |
|
Definition
| -allantoic cavity, yolk sac, chorio-allantoic membrane, and amniotic cavity |
|
|
Term
| What are a few types of evidence of viral infection in eggs? |
|
Definition
| -death of the embryo, gross lesions, presence of hemagglutination by the fluids (allantoic and/or amniotic) |
|
|
Term
| What is a tissue culture? |
|
Definition
| -it is loosely applied to a variety of bery different procedures whose main objective is to maintain tissues alive outside of the animal body |
|
|
Term
| What are the two types of cell cultures? |
|
Definition
-organ or explant cultures
-cell culture |
|
|
Term
| What is the purpose of organ or explant cultures? |
|
Definition
| -tissue is maintained under conditions of slow growth in the hope of maintaining fairly normal histological organization that is glued to plastic and covered with nutrient media |
|
|
Term
| What is the prupose of covering an organ culture with nutrient media? |
|
Definition
| -to keep the organ alive for a variable time period |
|
|
Term
|
Definition
| -propagation of rapidly growing cells without regard to preserving cellular organization that form a monolary |
|
|
Term
| What is a primary cell culture? How is it prepared? |
|
Definition
--the first generation cell culture obtained from animal organs
-bits of tissues are incubated in trypsin that breaks up the tissue into a suspension of single cells, they grow to form a monolayer |
|
|
Term
| How long does a primary cell culutre last? |
|
Definition
|
|
Term
| Gice some examples of primary cultures used in vet med. |
|
Definition
| -chicken fibroblasts, mammalian kidney cells, testicular cell cultures, lung cell cultures |
|
|
Term
| Newly isolated ivruses (From infected animals) will grow better in what kind of culture? |
|
Definition
|
|
Term
| What is a cell line cutlure? Wha is the major difference between it and a primary cell culture? |
|
Definition
-cells that are transfermed cancer cells
-can be propagated and sub-cultured indefinitely |
|
|
Term
| How do we transform primary cells into cell lines? |
|
Definition
|
|
Term
| Give some common examples of cell lines, |
|
Definition
| -JeLA cells, Vero cells (an Africak green monkey kidney line), Crandell feline kidney (CFK), and Madin Darby Bovine Kindey (MDBK) cells |
|
|
Term
| True or False: Monolayer cell line cultures set in flasks and plates are the most popular way to isolate and grow viruses in the laboratory. |
|
Definition
|
|
Term
|
Definition
| -the cell monolayer in a tissue culture flack must be broken up into individual cells via trypsin-versene and then transferred to a new tissue culture flask in order to keep propagating the cell cultures |
|
|
Term
| What is the material most commonly used to grow tissue cultrue cells? Any special treatment? |
|
Definition
-plastic
-treated with a chemical that increases the adhesion of the cells to the plastic |
|
|
Term
| What is in Minimal Essential Medium (MEM)? |
|
Definition
| -contains all nutrients found in normal body fluids: mix of salts, certain AAs, vitamins, purines, pyrimidines, hormones, and is at an optimum osmotic pressure, bicarbonate buffer, fetal bovine serum (FBS), antibiotics |
|
|
Term
| What is the optimum pH for MEM? Does it require buffering or is it naturally at this pH? |
|
Definition
-around pH 7.4
-must be buffered |
|
|
Term
| What do we add to MEM to denote pH? |
|
Definition
-phenol red
-red = alkaline & yellow = acidic |
|
|
Term
| What is a common MEM buffer? Does this change the incubation specifications? |
|
Definition
-bicarbonate bugger: sodium bicarbonate
-must be incubated at 5% CO2 |
|
|
Term
| What is another common MEM buffer other than bicarbonate buffer? What does it do? |
|
Definition
-HEPES buffer
-very effective at capturing excess hydrogen ions produced by the growing tissue culture cells |
|
|
Term
| What one thing MUST be added to MEM? |
|
Definition
| -fetal bovine serum (FBS) |
|
|
Term
| What is the purpose of adding antibiotics to MEM? Examples? |
|
Definition
-help prevent bacterial and fungal growth
-ex: streptomycin/penicillin |
|
|
Term
| At what temperature do we freeze tissue culture cells to preserve them? How about indefinitely? |
|
Definition
-negative 70 deg C or lower
-indefinitely in liquid nitrogen: -196 deg C |
|
|
Term
| Success with freezing preservationn of tissue culture cells relies on what? |
|
Definition
| -use of proper amount of protective additive, proper cooling rate, sotrage of at least negative 70 ndeg C, and rapid thawing |
|
|
Term
| Definition: Cytopathic Effects (CPE) |
|
Definition
| -cell death and other changes caused by the replication of viruses in cell culture |
|
|
Term
| Samples submitted for virus isolation from a particular domestic animal should be inoculated onto ______ derived from that particular animal. Any exceptions? |
|
Definition
-cell lines
-herpesviruses can grow in many different cell lines from different animal species |
|
|
Term
| What kind of CPE does parainfluenza cause? |
|
Definition
|
|
Term
| What are the three different cell hcanges that can result from CPE? Definition of each? |
|
Definition
-Cell destruction: death, apoptosis, vacuolization, wounding of cells, etc
-Inclusion bodies: stained bodies in the cytoplasm or nucelus of infected cells
-Syncytia: large multinucleated cells |
|
|
Term
| What are the only ways to definitively identify a viral species? |
|
Definition
| -sequencing the genome (PCR) or by diagnostic methods that employ virus specific polyclonal or monoclonal antibodies |
|
|
Term
| some viruses may not cause detectable CPE upon initial inoculation of tissue culture cells with samples recovered from infected animals. What is usually required before CPE is observed? |
|
Definition
|
|
Term
| Some viruses grow without ever causing any observable CPE. What are these called? |
|
Definition
|
|
Term
| Viruses can be quantified by what two methods? |
|
Definition
-Direct Particle Count
-Viral Infectivity Assay |
|
|
Term
| What is a direct particle count? |
|
Definition
| -direct count of viral particles by EM can be done by using a viral suspension conatining a known number of reference particles such as latex particles |
|
|
Term
| What is a viral infectivity assay? |
|
Definition
| -dilutions (usually ten-fold) of the stock virus are inoculated into a test system (cell cultures, embryonated eggs, animals, etc) and watched for evidence of viral replication |
|
|
Term
| What is a quantitative assay? |
|
Definition
| -an assay in which the infectivity is quantified by counting th enumber of infective centers such as placuqes |
|
|
Term
| How is a plaque assay made? |
|
Definition
| -inoculated confluent tissue culture cells are overlaid with a semi-solid medium (overlay medium) to localize the spread of infection to the immediate vicinity of the originally infected cell, resulting in the formation of a plque |
|
|
Term
|
Definition
| -a localized region of cell lysis resultig from the cell-to-cell spread of virus replicating in a cell monolayer |
|
|
Term
| Do all viruses produce plaques? |
|
Definition
|
|
Term
| What is a plaque-forming units? |
|
Definition
| -one PFU is equivalent to one infectious virus particle |
|
|
Term
|
Definition
| -registers the presence or absence of infection |
|
|
Term
|
Definition
| -the dose or dilution that infects or kills 50% of subjects in the test system |
|
|
Term
|
Definition
| -the median infective dose |
|
|
Term
|
Definition
|
|
Term
|
Definition
| -tissue culture infective dose fifty |
|
|
Term
|
Definition
| -embryo lethal dose fifty |
|
|
Term
| Will an assay have more TCID50 or PFUs? Explain. |
|
Definition
-will have more TCID50
-TCID50 is the dose that causes CPE in only 50% of patients/samples but PFUs are all of the signs of CPE, thus PFU is a larger percentage than TCID50 |
|
|
Term
| A certain proportion of viruses in any suspension are noninfectious. Why? |
|
Definition
| -have been inactivated during growth or prep, genetically defective that they lack a complete genome or have an empty capsid, low susceptybility of the asssay systems |
|
|
Term
| Which two tests are most widely used to view viral antigens directly in infected animal tissue and/or on virus isolated in TC cells? |
|
Definition
-Fluorescent Antibody test
-immunohistochemistry/immunoperoxidase |
|
|
Term
| In the FA test, one can actually observe virus specific Ab, which are tagged to a ________ dye, bound to viral Ag. What is the type of microscope used? |
|
Definition
-fluorescent dye
-fluorescent microscope |
|
|
Term
| How can a fluorescent microscope be used for FA? |
|
Definition
| -the microscope emits UV light whis is adsorbed by the fluorescent dye on the virus specific Ab, the dye them emits light of a longer wavelength in the visible specturm |
|
|
Term
| What is the most widely used dye in FA testing? |
|
Definition
| -fluorescein isothyocyanate (FITC) |
|
|
Term
| Describe the FA test in three words. |
|
Definition
| -rapid, highly sensitive, and precise |
|
|
Term
| How does a direct FA test work? |
|
Definition
| -a tisse section is on a microscope slide, then the labeled MAb is added, allowed to react to the Ag in the tissue, and rinsed before being examined by a fluorescent microscope |
|
|
Term
| How does an indirect fluorescent antibody test work? |
|
Definition
| -tissue on a slide, first rabbit anti-virus Ab, then add FITC-abeled rgoat anti-rabbit FITC labeled Ab |
|
|
Term
| What is the major difference b/n direct and indirect FA method? |
|
Definition
-direct method, the FITC-labeled Ab binds directly to the Ag
-in indirect method, the FITC-labeled Ab does not bind directly to the Ag, but recognizes another antibody |
|
|
Term
| What is the common fixative used for FA tests? |
|
Definition
|
|
Term
| What are the three differences between immunohistochemistry/immunoperoxidase and IFA? |
|
Definition
1) FITC is replaced by peroxidase enzyme
2) enzyme precipitates a substrate for color
3) slide is viewed with an ordinary light microscope |
|
|
Term
| How does peroxidase work in IHC? |
|
Definition
| -converts a soluble substance in the substrate solution into an insoluble precipitate that accumulates in the immediate vicinity of the viral Ag |
|
|
Term
| What kind of samples are used for immunohistochemical procedures? |
|
Definition
| -either fresh frozen or formalin-fixed |
|
|
Term
| How do we perform IHC test? |
|
Definition
| -a primary MAb that recognizes the virus is added to tissue and allowed to react, then a animal anti-first Ab peroxidase-conjugated Ab is added, then a substrate solution is added, the tissue is counterstained with hematoxilin and observed under a microscope |
|
|
Term
| What is the advantage of IHC? |
|
Definition
| -the location of the virus within the infected tissue can be determined precisely |
|
|
Term
| What is the major concern with VNT samples? |
|
Definition
| -VNT can only be performed if the virus has already been isolated in tissue culture cells |
|
|
Term
|
Definition
-a vial of virus is mixed with virus specific antibodies in antinserum and added to culture and observed for CPE
-if CPE appears then it is the correct virus |
|
|
Term
|
Definition
| -RBCs bind to hemagglutinins and adsorb them |
|
|
Term
| What is hemagglutination? |
|
Definition
| -viruses bind to RBC in solution and agglutinate them |
|
|
Term
| How does a hemadsorption inhibition (HAd-I) work? |
|
Definition
| -add virus specific Ab to virus so they can bind to hemagglutinins on the cell membrane |
|
|
Term
| What is the major concern with samples for hemagglutination inhibition testing?? |
|
Definition
| -can only be performed if virus has already been isolated in tissue culture cells |
|
|
Term
| How does one run a hemagglutination inhibition test? |
|
Definition
-virus pre is added to an antibody solution that is though to be specific for the virus, then RBC is added
-if a button, hemaglutinins have bound by Ab and it is the virus
-if a veil, there was no binding and it is not the virus |
|
|
Term
| Is the ELISA sensitive? What about specific? |
|
Definition
| -highly sensitive and specific |
|
|
Term
| What are the two main uses for ELISA? |
|
Definition
-can be used to ID and quantitate viral specific Ag that has been solublizied and attached to the bottom of a plastic well or to membranes
-can be used to capture and quantitate viral Ag in serum or other body fluids |
|
|
Term
| What principle is the basis of the ELISA test? |
|
Definition
| -an Ab can be coupled with an enzyme and still retain obth its immunological and enzymatic activity |
|
|
Term
| What does "ELISA" stand for? |
|
Definition
| -enzyme linked immunosorben assaty |
|
|
Term
| What enzymes are primarily used in ELISA tests? |
|
Definition
-alkaline phosphatase
-horseradissh peroxidase |
|
|
Term
| What is the substrate for horseradish peroxidase? For alkaline phosphate? |
|
Definition
-ortho-phenyldiamine (OPD)
-p-nitrophenyl phosphate |
|
|
Term
|
Definition
| -the presence of viral nucleic acid in the sample |
|
|
Term
| The importance of the PCR lies in its ability to do what two things? |
|
Definition
-amplyfy very small amounts of viral DNA or RNA from samples
-rest a large number of samples through automation |
|
|
Term
| What do you need to perform a PCR? |
|
Definition
| -primers that can recognize the specific viral genome |
|
|
Term
|
Definition
| -primers that recognize the specific viral genome are added, these bind to the genome of the virus present in the sample, and a polymerase enzyme amplifies the portion of hte genome located b/n the 2 primers millions of times so that the DNA product can be visualized |
|
|
Term
| How does real-time PCR (qRT-PCR) work? |
|
Definition
| -the reaction works the same way as PCR except the primers are fluorescently labeled so when the genome is replicated and the primers release, they glow and show in realt-ime the amount being amplified thus it is quantitiative |
|
|
Term
| Is PCR sensitive? What about specific? |
|
Definition
| -very sensitive and specific |
|
|
Term
| How can you get a false positive diagnostically with PCR? |
|
Definition
| -a latently infected cell may give a positive result for a virus that just happens to be present in the sample but the virus is not necessarily causing the disease |
|
|
Term
| What can cause a false negative with PCR? |
|
Definition
| -if the virus mutates and the primers no longer recognize the mutated genome |
|
|
Term
| How can we apply PCR to RNA viruses? |
|
Definition
| -have to use a RT-PCR, add in reverse transcriptase to transcribe the RNA genome into a DNA molecule before a PCR can be performed |
|
|
Term
| What do serologic assays usely measure? |
|
Definition
| -virus specific antibodies, usually IgG |
|
|
Term
| What is the difference between the virus neutralization test and Agar Gel Immuno-Diffusion test? |
|
Definition
| -CNT can tell you which virus serotype infected the animal but AGID can only tell you which virus it is |
|
|
Term
| Is a seropositive animal necessarily infected? |
|
Definition
| -No, circulating Ab may be the result of vaccination, past infection, ingested maternal Ab, or ongoing infection |
|
|
Term
| What is seroconversion? What does it indicate? |
|
Definition
-the acute sample is seronegative and covalescent sample seropositive
-indicates that the disease is due to an infection with particular virus being tested fro |
|
|
Term
| What is a significant increase in titers? |
|
Definition
| -courcold or greater rise in serum Ab titer b/n the 2 samples |
|
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