ID Lab Quiz 1

The dose or dilution that infects or kills 50% of subjects in the test system (aka ID50, LD50, TCID50 - tissue culture infective dose fifty).

Define the end point of a quantal assay.

Antigen or Antibody.

IFA allows for identification of either viral _____ or _____.

Obtain sample as early in the course of infection as possible.

In live animals, what step results in the greatest success in virus isolation?

An enzyme molecule.

In the immunohistochemical procedure, the monoclonal Ab is conjugated with __________.

Plastic ware. Nutrient media.

What are the basic necessities (big picture is required for growing viruses)?

Animals. Chicken embryos (eggs). Cell cultures (most important).

What are the host systems used to grow viruses?

Results: days Require: Susceptible TC, no contamination, antiserum. Variable sensitivity/specificity. (ID new emerging viruses).

What are the pros & cons of attempting to isolate a virus from a sample?

Rapid Highly sensitive Precise

What are the pros of an FA test?

ELISA. Western Blot. Agar Gel Immunodiffusion (AGID) test. Virus Neutralization Test (VNT). FA (sometimes). HI (not all viruses). Latex Agglutination test.

Name serological tests used to detect viruses.

Results: hours to days Require: suitable antigen, acute & convalescent Low to moderate sensitivity Low to high specificity. (IgM detection for clinical purposes)

Name some pros & cons of serological assays.

Glutaraldehyde Cacodylate Osmium tetroxide

Name the most common fixatives for TEM.

Primary cell cultures. Cell line cultures.

Name the two classifications of cell cultures:

Negative staining Thin sectioning

Name the two ways viruses can be viewed with TEM.

Virus isolation. Fluorescent antibody (very important - direct/indirect). Immunoperoxidase (IPX). Hemadsorption (HA - not often used). ELISA (commercially available). EM (Negative staining, Immuno-electron microscopy, used in special situations).

Name virus detection tests.

Family classification based on size, shape & structure.

Negative stain EM permits the designation of detected virus particles into its respective ________.

Results: hours Require: suitable sample & serum High sensitivity High specificity (Applicable to in-clinic use).

What are the pros/cons of using immunological assays to detect viruses?

Organ or Explant Cultures. Cell cultures.

What are the two main categories of tissue cultures?

Transmission Electron microscopy (TEM) Scanning Electron Microscopy (SEM)

What are the two types of electron microscopes?

RBC sticking to virus-specific glycoproteins called hemagglutins on the plasma membrane of virus-infected cells. These are inserted into the plasma membrane of infected cells prior to budding.

What causes hemoadsorption (HAd) in a TC culture?

Cell destruction. Inclusion bodies. Hemadsorption (Had - attachment of RBC to infected cell). Syncytia (cells come together to form one giant cell).

What changes can be observed on cell cultures?

Cell cultures.

What culture is the best type suited for & most widely used for propagating viruses?

The presence or absence of infection.

What does a quantal assay provide?

Virus in infected cells that make the tissue.

What does the FA test detect?

Electromagnetic lens Metallic apertures

What focuses the emitted electrons from the microscope onto the specimen?

Fluorescent antibody labeled added that will detect & bind to viral antigen in tissue.

What is a direct FA test?

Performed by EM using a viral suspension containing a known number of reference particles (i.e. latex).

What is a direct particle count?

A localized region of cell lysis resulting from the cell-to-cell spread of the virus replicating in a monolayer.

What is a plaque?

Dilutions (10-fold) of the stock virus inoculated into a test system (culture system) & observed for evidence of viral replication.

What is a viral infectivity assay?

Antibiotics (streptomycin/penicillin)

What is added to a cell line culture to prevent bacterial/fungal contamination?

Specific antibody added that will bind to the viral antigen, followed by fluorescent labeled antibody that will recognize & bind to the first antibody.

What is an indirect FA test?

Fixation Dehydration Embedding in plastic

What is included in the specimen preparation for TEM?

Fluorescent antibody test (FA).

What is probably the most important test to detect presence of virus in tissues & in impression smears?

High amount of virus in specimen.

What is required for a negative staining to be successful?

SEM has capacity for 3-D imaging.

What is the advantage to using SEM vs. TEM?

HA: viruses bind RBC in solution & agglutinate them. HAd attaches to the RBC but may not always cause agglutination.

What is the difference between hemagglutination (HA) & hemadsorption (HAd)?

Organ uses a piece of tissue to adhere to the growth plate. Cell cultures use a monolayer of rapidly growing cells without a preservation of the cellular organization.

What is the difference between organ/explant cultures & cell cultures?

Only the surfaces of the images can be observed. Resolution is limited to ~ 10 nm.

What is the disadvantage of SEM to TEM?

Detect the presence of serum antibodies.

What is the function of a serologic test?

It can be used on formalin-fixed tissues (detects virus in infected cells).

What is the importance of immunoperoxidase (IPX) test?

35 to 37 C

What is the incubation temperature for cell line cultures?

Phosphotungstic acid

What is the most frequently used negative stain?

Fluorescein isothyocyanate (FITC).

What is the most widely used dye for FA tests?

Primary cell cultures are cells derived from an organ & are viable for ~ 10 to 14 days. Cell line cultures can be sub-cultivated indefinately.

What is the primary difference between primary cell cultures & cell ine cultures?

A plaque formed on TC cells.

What is this?

Syncytia formed on TC culture.

What is this?

Trypsin-versene

What is used to aid in the removal of cells from the plastic in an established culture so they can be sub-cultured?

Ultramicrotome

What is used to section the epoxy resin blocks from dehydration to produce sections as thin as 0.03 um?

Virus specific monoclonal ab's that are labeled with SMALL iron or gold particles.

What is used to visualize the virus particles in IEM?

Takes time Not used routinely for diagnostic purposes.

What is/are the downfall(s) of the thin sectioning method for TEM?

Minimum essential media (MEM) - salts & osmotic pressure equals body fluids.

What kind of nutrient media is typically used for cell line cultures & it's general make-up?

Thin sectioning method.

What method is used to prepare fixed & embedded tissues or cells for TEM?

SEM

What method was used to create this picture?

ELISA

What other serologic test also uses a direct & indirect approach?

Waterproof ink: Species & individual ID. Collection date. Nature of specimen & anatomic location. Complete medical history.

What should be included on a properly labeled virus sample?

Immuno-EM

What technique was used to visualize these viruses?

PCR for viral DNA. RT-PCR for RNA viruses.

What test detects the presence of the viral genome in almost any submitted sample?

IPX

What test has been performed on this tissue?

Fluorescent antibody

What test is performed to achieve the above test if positive?

Virus Neutralization test (VNT). Hemagglutination Inhibition (HI). Hemadsorption Inhibition (HAd-I) tests.

What tests are performed to characterize & identify viruses isolated on TC cells?

CPE. Chloroform sensitivity. EM.

What tests can be performed for non-specific identification once the virus has been isolated in the sample?

IEM FA ELISA PCR

What tests can be performed for specific identification once the virus has been isolated in the sample?

Cytology (fixed in alcohol). FA test (fixed in cold acetone).

What tests can be performed from impression smears of lesions for the detection of viruses?

Autolysis inactivates many viruses.

Why should samples on deceased animals be obtained as rapidly as possible after death/euthanasia?

Immunoelectron microscopy (IEM)

______ is a rapid serological test using EM.

Suspended cell cultures (used to grow large quantities of virus - vaccine).

_______ are cultures of cells which are not allowed to become attached to any surface.

ELISA.

_______ is the most important serological test.

1. Allows for identification of either viral Ag or viral Ab. 2. IFA is more sensitive because of the layering of unlabeled Ab around Ag in the initial reaction. 3. Employs fewer conjugated antibodies.

What are the advantages of the indirect FA (IFA) test?

"Staining" the virus preparation with a non-penetrating stain: stains the background, not the virus.

Define negative staining.

Inactivated during growth or preparation. Genetically defective. Empty capsids.

A certain proportion of viruses in any suspension are noninfectious, why?

pH 7.4. Phenol Red indicator, bicarbonate or HEPES buffer.

Culture media must be buffered. What is the optimum pH, indicator used & buffers?

Cell death & other changes caused by the replication of viruses in cell culture.

Define Cytopathic effect (CPE).

Terms used to determine the infectivity of the original viral suspension since ordinarily infection with a single infectious particle is sufficient to form a plaque (1 PFU = 1 infectious virus particle).

Define a plaque forming unit (PFU).

Sample is homogenized. Supernant is filtered. Antibiotics were added to the filtrate. Filtrate is inoculated onto TC cells (species specific) & cultured for a few days - blind passages.

Describe the first step in specimen processing for virus isolation in the laboratory.

Tissue 0.5 cm thick (10% buffered formalin).

Explain considerations when obtaining a biopsy of tissue for submission to isolate virus.

Viremia: EDTA, citrate or heparin (never freeze whole blood). Serum: Red top tube - clot for hours in the fridge. (5-10 mL LA, 0.5-2 mL SA).

Explain how a blood sample should be handled for virus detection.

Swabs: nasal, ocular, throat = TM. Small sections of trachea, lung, bronchial & mediastinal LN in TM.

Explain samples submitted from nasal & trans-tracheal samples:

Vesicular fluid & epithelium overlying vesicles. Biopsy from the margin of lesions. Skin scraping (submit in glass slide or in TM).

Explain the types of samples obtained from skin/mucosa.

The specimen must be coated with a metal. Gold (typically)

For SEM the specimen must have what done to it & what substance is typically used for this?

HeLA cells. Vero cells. Crandell feline kidney. Madin Darby bovine kidney.

Give examples of monolayer cell culture lines.

Fluorescent screen Photographic plates

How are images from an EM visualized?

Direct particle count (via EM). Viral infectivity assay (dilutions of virus inoculated & observed for evidence).

How are viruses quantified?

Antibody added that will detect & bind to viral antigens in the tissue. A secondary labeled antibody (peroxidase labeled) is added that will bind to the first antibody. Substrate is precipitated by the peroxidase.

How is IPX ran?

1. Virus specific antibodies (antiserum) are mixed with virus then added to the cell culture. 2. The monolayer is observed for CPE. 3. No observable CPE then assumed that antibodies neutralized virus & prevented replication.

How is a viral neutralization test performed?

Characteristic lesions (pyogranulomas, necrosis, perivascular cuffing, inclusion bodies). Immunoperoxidase (IPX - direct identification of the virus).

How is a virus detected histopathologically?

Good with proper technique: (-70 C or lower) store indefinitely with liquid nitrogen. Proper amount of protective additive (glycerin or DMSO). Rate 1 C/min. Rapid thawing.

How well do cell lines freeze?

A stream of electrons with short energy wave-lengths & the shorter the wavelength then different visible colors make up the image.

State briefly how an EM is able to produce an image (how is the high resolution of EM possible?)

False.

T/F. All viruses grown on a monolayer plaque assay produce plaques.

True

T/F. Negative staining is simple & rapid in detecting/recognizing virus particles.

Infective centers (i.e. plaques, pocks or foci).

The infectivity of an enumerative method (quantitative assay) is quantified by counting the number of _______.

Hemadsorption on TC cells.

This is an example of hemadsorption or hemagglutination?

Fluorescent antibody (FA) test. Enzyme Linked Immunosorbent Assay (ELISA). Immunohistochemistry (IPX). Immuno-Electron microscopy (IEM).

What tests can be performed to characterize & identify viruses that are performed directly on infected animal tissue &/or on virus isolated in TC?

Cold on ice. Frozen.

What two ways should viral samples be submitted:

Fetal bovine serum

What type of serum is added to most cell culture media?

TEM with negative staining.

What was used to visualize these viruses?

The vicinity of the infected cells (reason its good for determining location of virus).

Where does the precipitate of the virus occur in IPX?