VIRUS IDENTIFICATION AND QUANTIFICATION

 

VIRUS IDENTIFICATION AND QUANTIFICATION

 

Briefing:

·      Virus isolation in cell cultures, immunofluorescence-based assay and molecular techniques to determine nucleic acid, have all been used successfully to detect and identify the viruses.

·      On the other hand, the most commonly used methods to quantify viruses can be subdivided into three broader categories:

1.   Techniques measuring viral infectivity (viral plaque assay, TCID50 and immunofluorescence foci assay)

2.   Those that examine viral nucleic acid and protein (q-PCR, immune-blotting, immune-precipitation, ELISA and hemagglutination assay)

3.   Those that rely on direct counting of physical viral particles (viral flow cytometry and transmission electron microscopy)

Methods of Virus Identification:

Microscopy in Cell Culture:

·      Main Categories

1.   Viruses that caused cell degeneration.

2.   That caused formation of inclusion bodies and cell degeneration.

3.   That caused the formation of multinucleated cells (syncytia).

4.   Those that caused no cytopathic effect (CPE).

·      Since then, cell cultures have been successfully tested routinely for in vitro isolation of viruses.

·      Presumptive identification of virus types can be made by observing morphological changes produced in host cells (CPE) caused by cytopathogenic viruses.

·      Although most laboratories combine traditional and advanced laboratory approaches to optimize viral diagnostics, virus isolation from cell cultures still remains a primary method, particularly when viable and nonviable virus need to be differentiated if a viable virus has to be isolated, and when the infection is not characteristic of any single virus.

Materials:

·      Maintenance medium (fresh cell culture growth medium as above, but in 2% serum)

·      Host cells monolayer grown on 8 well chamber slides

·      Phosphate buffered saline

·      Bouin's fixative

·      Giemsa buffer

·      Giemsa stain

·      Acetone, acetone: xylene (2:1 and 1:2)

·      Micropipettes (10 to 100 microliter)

·      Micro centrifuge tube

·      Microbiological safety cabinet

·      CO2 incubator

·      Inverted microscope

Procedure:

Seed chamber slides with host cells

 

·      Plate host cells at an approximate density (for example, 30,000 cells/chamber in an 8-well chamber slide) in growth media.

·      Gently rock chamber slides back and forth and from side to side so that cells are distributed evenly. Once cells have been seeded, allow the cells to grow overnight

·      Next day, visualize cells under a light microscope to confirm that cells are evenly distributed and reached >80% confluency

 

Prepare serial dilutions of viruses

 

·      On the day of infection, label 6 sterile microcentrifuge tubes to prepare virus dilutions

·      Fill the first tube in series with 990 μl of growth medium and remaining 5 tubes with 0.9 ml of growth medium.

·      Make a series of dilutions as follows; make a 1:100 dilution by transferring 10 μl of original virus stock to the first tube containing 990 μl of the medium.

·      Thereafter transfer 100 μl of the diluted virus to the next tube. Repeat to make a serial 1:10 dilution of the virus stock such as 10-3 through 10-7

 

Infect monolayer cells

 

·      Remove cell culture growth medium from chambers slide and add 0.5 ml of maintenance medium to each well

·      Add 100 μl of the 10-2 to 10-7 dilution to one of the wells, leaving one uninoculated control well for each set of virus tested. Place infected cells back to CO2 incubator 37oC or 34oC for monitoring CPE for one to four week

 

Giemsa stain for cell cultures

 

·      Once CPEs are visible, gently wash chambers slides with 3X PBS (5 minutes/wash) and treat them in Bouin's fixative for 10 minutes. Rinse the slides 3X in Giemsa buffer (10 minutes/wash) followed by incubation in Giemsa stain for 1 hour

·      Rinse slides briefly in Giemsa buffer and treat them in acetone for 15 seconds, acetone-xylene (2:1) for 30 seconds, acetone-xylene (1:2) for 30 seconds and finally in xylene for 10 minutes and then mount with per mount

Immunofluorescence (IF) Assay:

Briefing:

·      The immunofluorescence (IF) technique is widely used for rapid detection of virus infections by identifying the virus antigens in clinical specimens

·      IF staining is usually considered very rapid (about 1 to 2 hr) and overall gives a sensitive and specific viral identification

·      IF has been successfully used for better management of influenza virus infection and surveillance of influenza virus activity

·      Interestingly, although IF is generally considered less sensitive then

·      ELISA and PCR but the sensitivity and specificity of the IF assay depends on viral strains under consideration

·      As such IF technique is well-accepted laboratory diagnostics test,

·      however, sometimes these assays could be quite expensive, due to the cost of antibodies used

·      Additional variability may also be introduced due to non-specific binding, or cross-reactivity of commercially available antibodies

Materials and equipment:

·      Cell culture growth medium, Penicillin, Streptomycin, Gentamicin, Fungizone can be added if required

·      Phosphate buffered saline

·      FITC-conjugated secondary antibody

·      DAPI

·      5-4% Paraformaldehyde solution (pH 7.4) or methanol

·      Micro pipettes (10 to 100 microliter)

·      Micro centrifuge tubes

·      Microbiological safety cabinet

·      CO2 incubator

·      Epi-fluorescence microscope

Protocol:

·      Seed chamber slides with host cells: Cells are plated at an approximate density (for example 30,000 cells/chamber in an 8-well chamber slide) in growth media

·      Gently rock chamber slides back and forth and from side to side so that cells are distributed evenly

·      Once cells have been seeded, allow the cells to grow overnight

·      The next day, visualize cells under a light microscope to confirm that cells are evenly distributed and reached >80% confluence

Infect monolayer cells

·      Add 0.1 ml of original virus stock to each well, leaving one inoculated control well

·      Place infected cells back to CO2 incubator at 37°C or 34°C for 48 hrs.

Staining and visualization of IF

·      Forty-eight hours post-infection, aspirate the medium and then fixed cells with ice-cold methanol for 5 minutes (alternatively fix in freshly prepared

·      4% paraformaldehyde solution, and then permeabilize in 0.2% Triton X-100 for 5 min at RT)

·      The cells need to be rinsed with PBS and then incubated with a suggested dilution of antigen-specific primary antibody for 2 hours

·      Remove unbound primary antibody by washing 5X with PBS

·      Add fluorescently conjugated (FITC-conjugated secondary antibody) secondary antibody immunoglobulin G to the monolayer of cells

·      Following incubation, wash the cells again 5X in PBS, then add DAPI and visualized under phase and epi-fluorescence microscope