Theme No. J13 Clinical virology II (isolation of viruses in a chicken embryo, HIT, KFR)

To study: Repeat your notes from J03, J08 and J09.

Task No. 1: Determination of the hemagglutination unit of influenza virus in amnion fluid (Hirst´s
test)

When a virus is isolated in amniotic fluid, the result is not visible. So it is not clear, whether
the isolation was positive or not. One of ways, how to check the presence of virus in amniotic
fluid is connected with the ability of many viruses to agglutinate red blood cells in vitro. This
property of viruses is also used in HIT (see J109). Before HIT, it is necessary to titrate the
virues to measure its „power“. We measure it in so named haemagglutination units.  A
haemagglutination unit is the smallest quantity of virus which can agglutinate a given quantity of
erythrocytes.

Pipette 0.2 ml of saline into each of 8 wells in one row of a perspex panel. Add 0.2 ml of the
virus to the second well, mix, and transfer 0.2 ml to the third well. Continue in this fashion
until reaching dilution of 1:128 (well 8), pipette the excess fluid into 2% chloramine. Add 0.2 ml
of chicken erythrocytes suspension to each well. The first well serves as the control for the red
blood cells. Incubate the place for 90 minutes at room temperature.

Draw the results, and write, what dilution corresponds to 1 haemagglutination unit (1 HU).

Task No. 2 Isolation of viruses in chicken embryo

In picture a), add labels to individual structures of the egg to the first collumn. To the second
collumn write NOT USED for structures not used for viral isolation, USED for those used and USED –
POCKS  for the structure, where pocks might be visible in case of some poxviruses and
herpesviruses.

Now concentrate to your fertilized egg.

Put an egg into the ovoscope (laying) and outline the edge of the air bubble in pencil. Then wet
the shell at the site of the air bubble with tincture of iodine, and cut the shell in place of the
air bubble, keeping cca 2 mm difference between line of your cut and the line signifying the edge
of the bubble. Using sterile forceps, remove the shell top, without breaking or disturbing  the
uderlying membrane.

Place a few drops of alcohol on the „paper“ membrane to make it transparent, and examine the inside
of the egg over the ovoscope (now standing). Draw the observed structures to Picture b).

Now try to inoculate the amnionic cavity with a „virus“ (for didactic puprose, ink is used
instead). Using the ovoscope for illumination, insert a needle as if you woud have inentnion to
inoculate inside the eye of the embryo, and inject 0.1 – 0.2 ml of ink.

Now the egg woud be re-closed, the cut would be filled by parafin and the egg would be incubated.
Instead of that we will observe our results.

Using sterile forceps , break off the shell as far as the edge of the air bubble, nad then tear
both the outer and the chorioallantoic membranes. Empty the contens of the egg into the dish. Try
to identify the structures and draw your result to Picture c).


Structure         Used for is.?

Task No. 3 Assesing tissue cultures

Using a microscope at low magnification (10x objective) examine Roux-bottles or test tubes
containing tissue cultures. Draw the about 10 cells from each type of culture. In which vessels do
you see a cytopathic effect?







CPE yes – not

             CPE yes – not

                          CPE yes – not

Task No. 4 Demonstration of the shell vial assay (rapid detection of viruses)

Observe the vial and the „shell“. Read the princip of the procedure and answer following questions.

The shell vial technique is a variation on standard tissue culture in that it takes advantage of
using a living cell system and enhances viral recovery by centrifuging the clinical sample onto the
monolayer. In this technique a small bottle (vial) with a removable round glass cover slip is used
to grow the cells as a monolayer on the cover slip.

Nowadays mixed cell types can be put in a single monolayer providing a variety of cell types for
the virus to infect in a single vial. Once these monolayers are ready to be inoculated, the growth
medium is removed from the vial and the clinical sample placed directly on the monolayer. The vial
is then centrifuged, the clinical sample is removed and fresh growth medium is then added to the
vial. Although the vials can be kept until CPE occurs the CPE can't be seen unless the cover slip
is removed from the vial. Usually it is possible, using this technique, to identify the presence of
a virus before CPE occurs. A description of how a clinical sample might be handled using the shell
vial method could go something like this. Let's say that the sample is from someone suspected of
having a respiratory virus infection. Three shell vials would be set up. After 48 hours of
incubation, two of the vials would be used. The supernatants would be pipetted off and saved. The
glass cover slips would be washed gently and then fixed. One of them would be stained with a single
reagent containing influenza A and B.. Differentiation between A and B would be done by tagging the
antibodies with different fluorescent dyes. The other cover slip would be stained for respiratory
syncytial virus. The third vial is saved for staining at 72 hours for parainfluenza viruses. If the
cover slip stains positive for say influenza A the report is sent out as influenza Avirus isolated.
The supernatant from the original vial (which should contain live virus, can then be re-inoculated
into a fresh vial which is then sent off for typing of the influenza A virus.

(www.lhsc.on.ca/lab/MICRO/virology/vir_cult.htm)

What procedure is used to make the viruses from the clinical sample colonize the cells of the
tissue culture?


How can you differenciate between influenza A, influenza B and respiration syncitial virus?


Task No. 5 Determination of complement-fixation antibodies against the most common causative agents
of respiratory infections

The plate contains pairs of the sera from patients with pneumonia. In firs collumn there are
controls for serum anticomplementarity. The dilution in the secon well is 1:4. Draw the results of
reaction, write date of  colletions and evaluate results. In case of titre increase/decrease write
also how many times the titre changed.










Task No. 6 Determing the titre of antibodies against the virus of the tick-borne encephalitis using
the hemagglutination inhibition test (HIT).

The plate contains pairs of the sera from several patients with suspected tick-borne  encephalitis.
The las row contains the controls for antigen and for the red blood cells. Draw the results,
recordthe dates of sampling in the table, and evaluate the finding. The highest dilution which
prevents hemagglutination is considered the titer. In the first well the is 1:4.

Check-up questions:

1. How electronoptic and optic microscopy can be used in virology?




2. What do mean terms „tissue culture“ and „cytopatic effect“?





3. What pathogenic agents (including non-viral) can be grown in chicken embryos, and what is the
site of inoculation?





4. What viruses can be demonstrated in animals? What animals can be used?





5. What specimens and what methods are used in the virological diagnosis of influenza?





6. What methods for detection of viruses require two specimens and what is the ideal tie between
taking the first and the second?