Conventional Virological Assays

A. Virus isolation (VI)

Virus isolation consists of two steps;

1. The attempted recovery of virus; using in vitro cell culture, animal inoculation or egg embryo inoculation.

2. Identification of the isolate; using immunofluorescence microscopy, electron microscopy, or molecular techniques. 

Although isolation of virus followed by identification is considered to be the definitive diagnosis, VI is often laborious, expensive, and time consuming.

Purpose:

Virus isolation should primarily be attempted under specific circumstances:

1. When other detection methods fail or when trying to isolate viruses from previously unrecognized diseases.
2. If there is no other detection method of similar or greater sensitivity.
3. If the virus is required for other purposes, such as differentiation (e.g., RFLP, sequencing), characterization (e.g., typing), and/or for production of autogenous vaccines.

Techniques:

Aseptic collection and proper handling of clinical specimens is critical for VI.  Although certain viruses (e.g., parvovirus, circovirus) can withstand harsh environmental changes, this is not true for most viruses, particularly enveloped viruses. 

Samples; Virus isolation can be done on most clinical specimens, including biopsy and necropsy tissues, blood, secretions, and excretions.  Some clinical materials, e.g., urine, feces and semen, are difficult to work with because they are toxic to cell cultures. 

The quantity of sample required for VI is usually more than that needed for rapid diagnostic assays so collect and submit the adequate amount of sample.  To avoid unnecessary expense, be specific and refer to specimen selection guidelines in the User’s Guide or call the laboratory for advice.

Tissue culture: Note: (AM is providing handouts on Tissue culture).

I. Cell culture

A. Primary cell cultures

B. Diploid cell strain

C. Continuous cell lines

II. Animal inoculation

III. Egg embryo inoculation

II. Animal inoculation:

Purpose:

1. Maintaining viral stocks:

Before cell cultures were available, it was necessary to maintain virus stocks by continuous passage of virus from animal to animal.

2. Study of viral pathogenesis:

Use of laboratory animals has always been, and will continue to be, obligatory for studying the viral pathogenesis.

3. Development of viral vaccines and antiviral drugs:

The development of vaccines against hepatitis B virus would not have been possible without experimental studies with chimpanzees.

4. Study of immune system:

Understanding how immune system works or any complex organ system reacts to a virus can not be achieved without research on living animals.

5. Development of diagnostic tests:

The development of diagnostic tests for veterinary use has also benefited from research on viral diseases in experimental animals.

III. Egg embryo inoculation:

Purpose:

1. Many viruses were propagated in embryonated chicken eggs before the cell culture were available.

2. Commonly used for influenza viruses

3. Obtain high yield of virus and use in research lab.

4. Vaccine production

Egg inoculation technique:

Sterile chicken eggs are collected (after fertilization) and kept for 9-11 days incubation. A small hole is drilled in the shell and virus (0.1ml) is injected into the appropriate site for replication. The hole is sealed and incubated at 37^oC in an egg incubator for 72 hrs. After incubation, virus is harvested from respective sites of egg and centrifuged to get pure virus suspension.

A. Chorioallantoic membrane inoculation:

Herpes simplex virus, Pox virus, Rous Sarcoma virus

B. Amniotic inoculation:

Influenza virus, Mumps virus

C. Yolk sac inoculation:

Herpes simplex virus

D. Allantoic inoculation:

Influenza virus, Mumps virus, Newcastle disease virus, Avian adenovirus

Virus purification/virus titer:

Ultracentrifuge (or sucrose-gradient ultracentrifugation)

1. Take a tissue culture flask with a monolayer of cells (e.g. vero cells).

2. Infect monolayer in a small volume at a low MOI (0.01).

(I use 0.01 for HSV-1 and 0.1 for HSV-2)

3. Incubate for 1 hour, and replace with new complete medium ~ 20 ml (e.g. RPMI-

1640).

4. Wait until >90% cytopathic cells (~48 hrs) are seen. Cells should round up but still

should lightly adhere to the bottom of the flask.

5. Using a sterile pipette, pipette up and down so that all cells are collected. Put

concentrated viral supernatant into 50 ml sterile conical tubes.

6. Spin tubes at 4000 rpm for 15 min; take off supernatant leaving cell debris in tube.

7. Transfer the supernatant in Ultra-Clear Centrifuge Tubes (Beckmann) and spin at

20,000 x g for 2 hrs in a swing bucket rotor e.g. SW28 beckmann.

(I use 10,000 x g for HSV-1 and HSV-2).

8. Discard supernatant. The pellet is the virus.

9. Fingerflick the tube to break up pellet and pipette up and down with a 1 ml PBS.

Combine the virus suspension into one tube.

10. 50 µl aliquots are usually appropriate and sufficient. Store at -80oC.

11. Titer stock the next day (after freeze-thaw) in quadruplicates on 96 well plates.

B. Plaque Assay:

It is a method for measuring viral infectivity and multiplication in cultured cells. Clear lysed areas or plaques develop as the viral particles are released from the infected cells during incubation.

Purpose:

- to determine the virus titers present in any clinical samples

- to measure viral infectivity

- to detect multiplication of viruses in cultured cells

- to detect cytopathic effects

Principle:

In plaque assay, monolayers of cultured cells are incubated with a preparation of virus to allow the virus to adsorb to cells. After removal of inoculum, the cells are covered with nutrient medium containing a supplement that results in the formation of gel, most commonly agar. (I use methyl cellulose instead of agar).

Why this step using gel/methyl cellulose is important?

When the original infected cells release new progeny viruses, their spread to neighboring uninfected cells in the culture is restricted by the gel or methyl cellulose. As a result, each infectious particle produces a clear circular zone of infected cells, or plaque.

Plaque forming units

When a single infectious virus is sufficient to form a plaque, the titer of virus stock can be calculated in plaque-forming units per milliliter (PFU/ml) according to the dilution of sample and number of plaques observed.

To minimize the error in calculating virus titer, a series of dilutions is used for the plaque assay and only those plates containing between 20 and 100 plaques are counted, depending upon the area of cell culture vessel.

(I count the plates containing plaques between 5 to 10 plaques while using 96-well culture plate for HSV-1 and HSV-2).

Methods:

(With reference to protocols that I follow in my lab. for HSV-1 and HSV-2 virus titers from different tissues in the infected mice):

A. Prepare your sample:

1. Harvest perfused tissue from infected mice with HSV-1 or HSV-2 and put them in 500

µl of RPMI-1640 media in microcentrifuge tubes. Keep it in ice.

2. Homogenize the tissue, and spin the tubes at 10,000 rpm for 1-2 mins to get clear viral

supernatant.

B. Serial dilutions in 96 well U bottom plates:

1. Put 200 µl of each sample in wells of first row (A) of plate. (12 samples in a plate)

2. Put 160 µl of RPMI-1640 to wells in B to H rows.

3. Transfer 40 µl of sample from A to B row. Perform serial dilution from B to C to D

upto H for each sample. Change tips every time.

C. Culture cells with monolayers (e.g. vero cells)

(Monolayers have to be ready by the time you plan to perform plaque assay. You can plate cells in 96 well flat bottom plate one day before the test or same day morning allowing it for six hours. For overnight incubation, 3 million cells are needed to plate 96-well plate while for same day incubation, 5 million cells are needed to plate 96-well plate).

1. Take out the culture plate and dump media out of plates with cells.

2. Add 100 µl of dilutions into the culture plate to respective well.

(Go from H to A, no need to change tips).

3. Incubate 1 hrs in 37^oC. (By this time, take methyl cellulose out to warm room

temperature).

Why 1 hour incubation of virus dilution in culture plate? (Dr. Carr’s favorite question). (This incubation time is sufficient for virus to adhere to receptors).

4. After 1 hour, dump the media out, gently pipette 100 µl of methyl cellulose into the

plate.

5. Incubate @ 37^oC for 24-28 hrs (next day). Read the plaques.

6. Find out the dilution that contain 5-10 plaques for each sample.

7. Calculate the plaque forming units as;

Plaque forming units (PFU/ml) per ml = Dilution factor x 10.

(Vol. used here is 100 µl; to convert into per ml multiply by 10)

C. Immunohistology:

Immunohistochemistry is a technique used to assess the presence of a specific protein or antigen in cells (cultured cells, cell suspensions) by use of a specific antibody, which binds to it, thereby allowing visualization and examination under a microscope.

Purpose:

- valuable tool for the determination of cellular contents from individual cells

- valuable tool for detecting specific antigens in tissues

- valuable tool for diagnosis of viral diseases

The following steps are generally performed in an immunohistology staining;

A. Cell Preparation

Cells to be stained can be attached to a solid support to allow easy handling in subsequent procedures. This can be achieved by several methods: adherent cells may be grown on microscope slides, coverslips, or an optically suitable plastic support. Suspension cells can be centrifuged onto glass slides, bound to solid support using chemical linkers, or in some cases handled in suspension.

Following are the some of the methods for cell preparation:

1. Preparation of Coated Slides

2. Cell Film Slide Preparation for Blood and Cell Suspension

3. Swabbed Slide Preparation

4. Cell Culture Slide Preparation

6. Cytospin Slide Preparation

7. Touch Prep Slide Preparation

B. Fixation

To ensure free access of the antibody to its antigen, the cells must be fixed and permeabilized. In general, fixation strengths and times are considerably shorter for cells than on the thicker, structurally complex tissue sections.

Perfect fixation would immobilize the antigens, while retaining authentic cellular and subcellular architecture and permitting unhindered access of antibodies to all cells and subcellular compartments. Wide ranges of fixatives are commonly used, and the correct choice of method will depend on the nature of the antigen being examined and on the properties of the antibody used.

Fixation methods fall generally into two classes:

i. Organic solvents: such as alcohols and acetone remove lipids and dehydrate the cells, while precipitating the proteins on the cellular architecture

ii. Cross-linking reagents: such as paraformaldehyde form intermolecular bridges, normally through free amino groups, thus creating a network of linked antigens. Cross-linkers preserve cell structure better than organic solvents, but may reduce the antigenicity of some cell components, and require the addition of a permeabilization step, to allow access of the antibody to the specimen.

Some of the fixation methods;

Acetone Fixation, Methanol Fixation, Ethanol Fixation, Methanol-Acetone Fixation,      Methanol-Acetone Mix Fixation, Methanol-Ethanol Mix Fixation, Formalin Fixation,     Paraformaldehyde-Triton Fixation, Paraformaldehyde-Methanol Fixation

C. Antibody Staining

The third step of cell staining involves incubation of cell preparations with antibody. Unbound antibody is removed by washing, and the bound antibody is detected either directly (if the primary antibody is labeled) or, indirectly using a enzyme-labeled or fluorochrome-labeled secondary reagent.

1. Direct Labeling of a Surface Antigen on Unfixed Cells in Suspension

2. Fluorescence Labeling of Surface Antigens of Attached or Suspended Tissue-Culture Cells (detail will be dealed in Immunofluorescence microscopy)

3. Fluorescence Labeling of Intracellular Antigens of Attached or Suspended Tissue-Culture Cells

D. Observation

1. Examine the cells under the microscope.
2. Record the results and taking pictures of the labeled cells.

(Note: Perform an appropriate negative control. Negative controls establish background fluorescence and non-specific staining of the primary and secondary antibodies.)

Immunohistochemistry of viral diseases:

Ebola virus in monkey hepatocyte Monkeypox virus in the skin of a monkey

Hantaan virus in capillary endothelial

cells of a hamster kidney Venezuelan equine encephalitis virus

in the olfactory mucosa of a mouse

Cell surface antigens (Retroviral antigens):

Left; HIV p24 in cytoplasm and on cell membranes of syncytial and other cells in vitro

Middle; SIV antigens in giant cell in lamina propria of colon of monkey, polyclonal Ab

Right; SIV antigens in brain lesion composed primarily of macrophages.

Diagnosis of Herpes simplex Virus

Left; epidermal multinucleated giant cells are characteristic of herpes virus infections.

Middle; multinucleate cells with dark stained inclusion bodies (Cowdry type A inclusion bodies).

Right; CMV inclusion bodies

multi multinucleate cell with dark staining inclusions.nucleate cell with dark staining inclusions. multinucleate cell with dark staining inclusions.

Viral Inclusion bodies;

Inclusion bodies are abnormal structures which appear within the cell nucleus, the cytoplasm, or both, during the course of virus multiplication. They usually have characteristic staining properties.

The inclusion bodies are concerned with the developmental processes of the virus. In some virus infections, such as molluscum contagiosum, inclusion bodies may be simply masses of maturing virus particles. In herpes simplex infections, typical inclusion bodies do not appear until after the virus has multiplied. Such inclusions may be remnants of the process of virus multiplication.

Negri bodies of rabies: these are accumulations of viral RNA in the hippocampus and cerebellar Purkinje cells.

CMV - 'Owls eye' inclusion bodies - in kidney, liver, lung.