Collection and preservation of viral specimens
Specimens from virus infected animals should be collected early in the
course of the disease when maximum amount of virus is expected to be
present before any treatment and production of antibodies in the system
.Blood, feaces, sputum, nasal discharge, skin lesion etc.Depending upon
the predilection of the virus infecting certain tissues, they can be collected
at the time of post mortem. As an example, skin lesion can be collected
from animals suffering from pox diseases; trachea, bronchi and lungs
from cases suffering respiratory infections; brain and spinal cord from
cases suffering from neurotropic viruses affecting central nervous system.
Other organs like spleen, liver, kidney, lymph glands etc.can be collected
from generalized infections affecting most of the organs. Sera for
antibody testing should be collected from recovered animals after one
week of recovery.
After collection of the specimens they shoud be preserved by freezing or
freeze drying till they are used for inoculation in animals, embryonated
eggs or cell culture. The specimens can be transported to the laboratory in
thermos flask containing dry ice. If the specimen is not used immediately,
it should be preserved at (-45) ºC or lower temperature in deep freezer.
The temperature of storage cabinet with dry ice is about (-70) ºC.The
specimens should be collected in good quality screw-cap vials so that
they can withstand repeated freezing and thawing. Specimens for long
preservation and for vaccine preparation can be freeze dried under
vacuum. The freeze dried vials (sealed) can be stored in ordinary
refrigerator at (4) ºC.
Materials
Fowls infected with Newcastle disease, fowl pox, infectious bronchitis
Instruments for post mortem examination, screw- cap vials for specimens
collection, anticoagulants like sod. Citrate or EDTA or heparin, blood
agar or brain heart infusion broth, deep freezer maintaining temperature
at (-45) ºC.
Procedure
1-Collect blood from all the chickens from heart or wing vein .Place one
portion of the blood in EDTA or sod. Citrate in proportion of 9 parts of
blood and one part of EDTA for collection of plasma .The other portion
of blood will be used for collection of serum.
2-Centifuge the blood with anticoagulant at 1000 RPM for 10 minuets
and transfer the plasma (supernatant) to a screw cap vial. Place the other
portion of blood (clotted) in a slanting position in the refrigerator and
next day separate serum in screw -cap vials.
1
3-Kill the infected birds and perform post-mortem examination, collect
following specimens in screw -cap vials: Newcastle disease -lung, spleen
and kidney; fowl pox-skin lesion; infectious bronchitis: trachea and lung.
4-Inoculate brain heart infusion broth and blood agar with each sample
for bacteriological sterility test.
5-preserve the specimens including plasma and serum in a deep freezer at
(-45) ºC or lower temperature till they are needed for inoculation .All
samples should be labeled properly with details.
Preparation of virus specimens for inoculation
Tissues for virus isolation are to be grounded in a mortar and pestle with
the help of an abrasive like sand. (Good quality hard sand should be
properly washed in distilled water, treated with hydrochloric acid, washed
thoroughly, dried and sterilized before use).The diluent should be
phosphate buffer saline or nutrient broth. Normally the concentration of
tissue in the inoculum is about (10-20) %.While grinding the tissues, the
temperature should be kept as low as possible. When the tissue is likely to
be contaminated, the inoculum should be treated with antibiotics.
Materials
Frozen specimens from preserved samples, mortar and pestle, sterile
sand, PBS, syringes and needles.
Procedure
1-Take out the frozen specimens from deep freezer and thaw them at
room temperature .Transfer the specimen to a mortar, cut it into small
pieces ,add small quantity of sterile sand and PBS and grind into affine
paste, add more PBS and grind again. The amount of diluent should be
added to make it about (10-20) %.The suspension should be then
transferred to sterile tube .The suspension then centrifuged at 2500 RPM
for 20 minutes the Supernatant fluid is removed in another tube and
repeats this process 3 times to sediment the coarse particle and add
penicillin and streptomycin at the rate of 10000 units and 10 mg per ml
respectively to the Supernatant fluid and incubate at room temperature for
20 minuets. The Supernatant fluid is used as inoculum in the suitable host
system.
2-The frozen plasma and serum are removed from deep freezer and
thawed. If they are found contaminated on sterility test, they should also
be treated with penicillin and streptomycin before use as inoculum in
suitable host.
2
Methods to study Physical properties of Viruses
Equipments used for centrifugation, electrophoresis, filtration and
electron microscopy are very useful tools to study physical properties of
viruses. Ultracentrifugation is used to determine the size, shape and
density of viruses, ultafiltration to determine the size, shape and possibly
electrical charge of viruses, electrophoresis for surface potential and
possibly size of the viruses and other biological substances and electron
microscopy is used to determine size and shape and structural
arrangement of viruses or their subunits and also for counting virus
particles.Ultra-thin sections and intracellular aspect of viral multiplication
can also be studied by electron microscopy.
Cultivation of Viruses
Viruses can be cultivated only in living cells. For their cultivation, living
cells are available in:
A-Natural hosts and laboratory animals.
B-Embryonated eggs.
C-Cell culture system.
Cultivation of Viruses in Natural hosts and laboratory
animals
In most cases attempts are made to cultivate the viruses in embryonated
eggs or cell cultures which are more convenient and economic than
animals. However to find out the course of the disease, the clinical
symptoms, pathogenisity and post-mortem lesions, the viruses have to be
inoculated in the natural hosts and laboratory animals. Cultivation of
viruses in laboratory animals are preferred rather than in the natural hosts.
They are some viruses which can be cultivated only in natural hosts.
When viruses are grown in animals, the animals should be healthy,
disease free and free from antibodies.
In animals some viruses have preference to grow in certain tissues like
skin, respiratory system, nervous system etc.while others can grow in all
organs and tissues. The route of inoculation has to be selected according
to the possible tissue predilection of the viruses .Different routs of
inoculation maybe:
intramuscular(I/M),subcutaneous(S/C),intravenous(I/V),intraperitoneal(I/
P), intranasal(I/N)and intra cerebral(I/C).Laboratory animals for
virological studies are mice guinea pig ,rabbits,monkey,hamsters and
chickens.
3
Cultivation of Newcastle disease virus in chickens
Materials
Lung and spleen from Newcastle disease infected chickens, 4-6weeks
old disease-free chickens2, equipments for inoculation and for postmortem.
Procedure
1-take out from deep freezer lung and spleen tissues from Newcastle
disease infected chicken and prepare inoculum as described previously.
2-Inoculate (4-6) weeks old chickens with (0.4) Ml inoculum .Half of the
dose should be given I/M and other half by I/N route. Return the chickens
to the cage.
3-Examine the chickens for clinical symptoms every day. On 3rd or 4th
day of inoculation, the birds may show respiratory distress, nervous
symptoms and other clinical symptoms.
4-When clinical symptoms are evident, kill the birds with cervical
dislocation and perform post-mortem examination .Examine lesions in
trachea, lung, and other internal organs.
5-Collect trachea, lungs, spleen and brain in a screw-cap tube and store in
the deep freezer.
4
B-Cultivation of viruses in embryonated
chicken eggs
Like animals, embryonated chicken eggs also posses highly specialized
tissues and organs and are frequently utilized to grow various viruses
particularly those infecting chickens and other birds. The usual routs of
inoculation in chicken embryos are yolk sac method, chorioallantoic
membrane method, allantoic cavity and amniotic cavity routes. Routes of
inoculation in chicken embryos depend upon the viruses to be cultivated.
Cultivation of viruses in embryonated eggs is convenient and
economical method. The eggs should be used from disease -free stock.
Eggs from vaccinated flock may carry antibodies in the yolk which may
interfere in the growth of specific viruses, therefore SPF should be used.
Some factors which affect the multiplication of viruses in chicken eggs
are: age of embryo, route of inoculation, concentration and volume of
inoculum, temperature of incubation and time of incubation after
inoculation. Preliminary incubation temperature may be 38 ºC and 37ºC
after inoculation.
Embryonated eggs should be inoculated by several routes. Selection of
the routes depends upon the virus and its affinity to grow in certain
tissues. The routes of inoculation are illustrated in the following table:
Type of route
Yolk sac inoculation
Egg of embryo
(5-7)day
examples
Blue tongue ,rabies
Chorioallantoic
membrane
(10-13)day
Fowl pox, Herpes
viruses
Allantoic cavity route
)9-11)day
Amniotic cavity route
)10-12)day
Newcastle disease,
influenza viruses
Influenza viruses
After virus inoculation, the embryonated eggs are incubated and
examined daily by candling method. If embryos die within 24 hours, the
death is considered non specific and such eggs are removed from the
incubator and discarded. Some viruses like Newcastle disease virus kill
the embryos within (2-3) days. In other cases the eggs are allowed to
incubate up to (5-6) days. The eggs are daily turned upside down. On
primary isolation, some viruses may not kill the embryos or produce
various pathological changes in first one or two inoculations. To confirm
5
whether some viruses are responsible for the infection, repeated serial
blind passages are given in the eggs before discarding them as negative.
After few passages the virus may start killing the embryos or produce
other changes.
The pathological changes on embryonated eggs are:
1-Death of embryos.
2-Curling and dwarfing of embryos.
3-Haemorrhages of subcutaneous tissues.
4-Pock lesions on Chorioallantoic membrane and thickening of
Chorioallantoic membrane.
5-Development of inclusion bodies in the cytoplasm or nucleus of
infected cells.
All eggs should remain in vertical (blunt end up) except those prepared
for CAM.
Immediately after the death of the embryos, or after termination of
incubation period, the eggs should be removed from the incubator and
chilled for several hours before collection of embryos or other materials.
6
Inoculation of embryonated chicken eggs with
normal saline by yolk sac method
Materials
Seven days incubated chicken eggs, normal saline solution, egg drill
machine, egg candler, syringes, forceps, scissors, petridish, tincture of
iodine and melted paraffin.
Method
1-Candle the eggs and locate the yolk sac with a pencil. Make a mark on
the shell at about middle of the yolk sac.
2-Drill a small hole through the egg shell at the mark without piercing the
shell membrane.
3-Apply tincture of iodine to the hole and allow it to dry.
4-Inoculate 0.5ml NSS with 1 ml syringe .the needle should be inserted
full length through the hole before depositing the inoculum withdrawing.
5-Seal the hole with sterile melted paraffin and reincubate the eggs in egg
incubator and examine daily by candling for (3-4) days.
6-The yolk is harvested with the help of a (5-10) ml syringe after apply
disinfection to the shell over air sac .Break the shell over air sac with
forceps and remove the shell to a distance of about 8-10mm from the top
of the air sac, remove shell membrane and CAM from the base of air sac
of the eggs, the allantoic fluid is collected ,then the embryo is plucked by
using forceps suspended with yolk sac,the yolk sac is opened by using
scissors in sterilized petridish,the method is done in aseptic conditions.
Record observations regarding the death of the embryos and other
pathological lesions, if any.
Inoculation of embryonated chicken eggs with
normal saline by allantoic route
Materials
Embryonated chicken eggs incubated for 10 days, egg incubator, drill
machine, egg candler, NSS, syringes and needles, forceps, scissors,
petridish, tincture of iodine and melted paraffin.
7
Method
1-While candling the eggs mark an area of air sac and make another mark
on the upper end of the air sac of the eggs.
2-Drill a hole at the mark on the upper end of the air sac through the
shell. Disinfect the shell on the drilled hole with sterile precautions.
3-Inoculate 0, 2 ml NSS through the hole using 1 ml syringe.
4-Seal the hole with melted paraffin and incubate the eggs for (4-5) days.
5-For collection of allantoic fluid ,apply disinfection to the shell over air
sac .Break the shell over air sac with forceps and remove the shell to a
distance of about 8-10mm from the top of the air sac ,remove shell
membrane and CAM from the base of air sac of the eggs.
6-With the help of a 10 ml syringe, collect about 5 ml allantoic fluids
from the cavity through air sac opening and expel the fluid in a container.
Record observations regarding the death of the embryos and other
pathological lesions, if any.
Inoculation of embryonated chicken eggs with
normal saline by amniotic route
Materials
Embryonated chicken eggs incubated for (10-12) days ,egg incubator,drill
machine,egg candler,NSS,syringes and needles,forceps,scissors, petridish,
tincture of iodine and melted paraffin.
Method
1-While candling the eggs marks an area of air sac and make another
mark on the upper end of the air sac of the eggs.
2-Drill a hole at the mark on the upper end of the air sac through the shell
in the side of egg which contain embryo. Disinfect the shell on the drilled
hole with sterile precautions.
3-Inoculate 0, 2 ml NSS through the hole using 1 ml syringe with the help
of candler and turn the syringe right and left to observe movement of
embryo with movement of syringe.
4- Seal the hole with melted paraffin and incubate the eggs.
8
5-For harvesting amniotic fluid, apply disinfection to the shell over air
sac .Break the shell over air sac with forceps and remove the shell to a
distance of about 8-10mm from the top of the air sac, remove shell
membrane and CAM from the base of air sac of the eggs.
6-With the help of a 10 ml syringe, collect about 5 ml allantoic fluids from
the cavity through air sac opening and expel the fluid in a container, the
embryo is plucked ,the amniotic fluid is collected from the delicate
membrane surrounding the embryo by using syringe and expel in container.
Record observations regarding the death of the embryos and other
pathological lesions, if any.
Inoculation of embryonated chicken eggs with
normal saline by chorioallantoic membrane
route (CAM)
Materials
10-13days old embryonated chicken eggs, egg incubator, drill machine,
egg candler,NSS,syringes and needles,forceps,scissors, Petridis, tincture
of iodine and melted paraffin.
Method
1-Candle the eggs and mark the position of embryos.
2-Keep the long axis of the egg in horizontal position with embryo
uppermost ,mark equilateral triangle on one side or with each side about 1
cm.
3-Cut the egg shell at the marks of the triangle without piercing through
the shell membrane .Also make pointed cut through the shell over the air
sac.
4-Apply disinfectant on the cut areas of the shell and allow to dry.
5-Remove the shell over the triangle with the help of a needle or forceps
to expose intact shell membrane.
6-With a needle, pierce the shell membrane over the air sac and on the
side in the triangle without piercing the chorioallantoic membrane.
9
7-Creat a slight vacuum with a small rubber bulb at the hole over the air
sac by sucking the air through the bulb. Air will pass through the opening
in the shell membrane on the side of the egg allowing the CAM to drop
from the shell membrane underneath the triangle. The air sac area will
occupied by the embryo, membranes and fluids created on the side of the
egg.
8-Use 1 ml syringe and deposit 0, 2 ml inoculum through the shell
membrane over artificial air cells on CAM. Withdraw the needle.
9-Close the triangular opening in the shell with a suitable sized adhesive
tape. Also seal the hole over air sac space with melted paraffin or
adhesive tape.
10-Incubate the eggs in egg incubator and examine and turn them daily
for 3-4 days.
11-To collect the CAM; apply disinfectant on the shell over artificial air
cell. Remove the shell and shell membrane over artificial air cell with
forceps to expose CAM. Cut artificial air cell portion of CAM using
scissors, clean the membrane with NSS in a petridish and place the
membrane in a container.
Record observations regarding the death of the embryos and other
pathological lesions, if any.
10
C-Cultivation of viruses in cell cultures
Cultures prepared with dispersed cells are designated as cell cultures.
Tissue cultures on the other hand are those cell systems which are
established with tissue fragments or masses of cells originating from the
fragments. Several techniques such as slide cultures in which tissue
fragments are embedded in plasma clots on glass surfaces, flask cultures
in which cells are grown in suspension and cell cultures as monolayers on
glass surfaces, are employed for cultivating cell in an artificial media.
The advantages of cultivating viruses in cell culture systems:
1-They are free from antibodies, hormones and similar other host factors.
2-Different viruses produce specific metabolic and cytopathic changes.
3-Cell lines from different species and organs are available .primary cell
cultures can be prepared from different species and organs as per the
needs of specific viruses.
Most commonly used tissues for cell culture preparations are kidneys and
tests from young animals like lambs, kids, calves and piglets. Chicken
embryo kidneys and fibroblast cell cultures are also commonly used.
The preparation of tissues for cell culture involves collection of fresh
tissues in balanced salt solution (BSS),cutting the tissue in small pieces
with scissors ,and then washing with BSS .the minced tissue cells are
treated with trypsin in special flask (trypsinizing flask) with magnetic
stirrer to prepare culture with auniform layer of cells. The cells are
washed 2-3 times to free them from trypsin.Most of the cell culture work
is done with fluid medium in stationary tubes, flasks or petridishes.Flat
sided prescription and milk dilution bottles are also frequently used.
The tubes and flasks with cells and growth medium are tightly closed
before incubation. The humidity in the incubator should be about 85%
and CO2 (5-8) % .The cells will grow and form a sheet on the walls of
the tube or flask. When cell sheet is formed, it is inoculated with virus
and growth medium replaced with maintenance medium which has
minimum serum. The virus will multiply in the cells and may show
cytopathic and other changes. Cell culture monolayers are widely used
for isolation of viruses from clinical samples. Tissues from embryos grow
more rapidly than the tissues from young animals. Tissues from adult
animals
are
difficult
to
grow.
11
Three different kinds of cell cultures are:
1-primary cell cultures: They are initiated usually from tissues of
animals or chicken embryos treated with trypsin (0, 25%). They are very
satisfactory for virus cultivation and consist of both epithelial and
fibroblastic cells. Retain normal set of chromosomes and morphology for
examples:
Chicken embryo fibroblast
Chicken embryo liver cells
Chicken embryo kidney cells
2-Diploid cell cultures: They are serially propagated primary cell
cultures taken from fetal organs or tissues treated with trypsin (0, 25%),
retain normal set of chromosomes and morphology. This diploid cell
culture can be serially transferred (20-50) passage therefore can be used
for preparation of vaccines. For examples:
Fetal bovine kidney
Fetal sheep kidney
3-permanent cell lines: These cells with abnormal chromosomes
number and morphology .They are capable of maintaining indefinitely in
vitro by serial transfer. This type of cell originated from mutated cells or
from cancer, consist of epithelial cells, cannot be used this type of cells
for preparation of vaccines.
Examples of permanent cell lines:
Hella, Vero (African green monkey kidney), BHK (baby hamster
kidney) etc.
Media used for propagation of cell culture:
1-(MEM) Minimum Essential Medium.
2-Hanks solution.
3-199 media.
Components of media: media should contain the following materials:
1-Inorganic salts like Na, Mg, Ca, K essential for maintaining the growth
of cells also maintaining osmotic pressure of cell.
2- Lacto albumin hydrolysate or yeast extract.
12
3-Glucose as a source of energy.
4-Vitamins like B, B12, B6, C, Folic acid, thiamin, and choline.
5-Essential and non essential amino acids.
6-Fetal calf serum (5-10) % for growth media and (1-2) % for
maintinous media.
7-Phenol red (0, 1) %.
8-Sodium bicarbonate (4, 5-7, 5) %.
9-Antibiatics (mixture of cryst.penicillin and streptomycin).
Types of cytopathogenic effects (CPE) caused by
viruses:
There are many types of CPE which are differing according to type of
inoculated virus for example:
1-Cell rounding, aggregation and dehydration.
2-Giant cells formation (syncytia type) as seen in paramyxoviruses.
3-Formation of Inclusion bodies inside nucleus or in cytoplasm
depending on site of virus multiplication.
4- Cell transformation as in oncogenic viruses (Retro virus, papova
virus).We can see multilayer of transform cells.
13
Plaque formation
Cytopathic viruses (those which cause cell destruction) form plaques, foci
or local lesions in or on various indicator systems. No single method can
be used to plaque assay all animal viruses and method for each is beyond
the scope of this text. The virus, cell strain will vary from one facility to
the next so adaptations will have to be made with the methods discussed
below .lesion or pocks may be observed on chorioallantoic membrane of
the embryonated egg, pustules may be produced on skin or cornea of
various animals, foci or proliferation of tumors may be observed on cell
monolayer.
1-chorioallantoic membrane pock assay
A useful host for production of localized lesions is the chorioallantoic
membrane of the embryonated egg .the virus is serially diluted and
deposited on the membrane of replicate eggs. The membrane of uniform
cell is moist and the virus released can spread by cell contact. The
characteristic lesions produced by various viruses are of diagnostic value.
Method:
1-Three to seven days after CAM inoculation, harvest the membranes.
Disinfect the area over the original air space (blunt end).With sterile
forceps remove the shell over this space .Cut a circular area in the shell
membrane and in the chorioallantoic membrane and fold this back. Tilt
the egg and allow the contents to flow slowly out into a dish. Use sterile
forceps to control the CAM which should remain adherent to inner side of
the shell. Cut the embryo and attached membranes from the CAM. With
sterile forceps, remove the CAM and place it in a Petri dish containing
PBS.
2-Place the dish against a dark surface and count the pocks. Some are
minute and may require the aid of a dissecting microscope.
2-Monolayer plaque assay
Plaques can be seen more easily if an infected monolayer of cells in
culture is overlaid with nutrient agar. After several days, plaques will be
seen as unstained areas in the cell sheet .At the viral concentration used
each plaque can be said tobe caused by a single virus particle.
There are several important points you should realize when plaque work
is attempted .The volume of the overlay can be critical, thick overlay will
reduce the number of plaques .Other viruses may require more nutrients
and their number may be reduced with a thin overlay.
14
The media used depend on the cell culture and the virus, some viruses
require more elaborate nutrients. Serum in the overlay medium may
inhibit or enhance plaque formation. The volume of the inoculum will
vary with the virus, size of flask or bottle. work should be done in a
darker room than usual .After plaques have formed ,add 1 or 2 ml of stain
of each flask or bottle and incubate at 37ºC for 1 or 2 hr.
Cells are grown in bottles, medium is removed and asutible dilution of
virus is added in small volume. After an adsorption period nutrient agar is
added and allowed to solidify. Cultures are incubated for varying lengths
of time. Plaques may be seen by indirect light against a dark background.
A-materials
Virus suspension
MEM medium without phenol red
Antibiotic mixture
Nobel agar, sterile
Distilled water, sterile
Tissue culture flasks or bottles 3 per dilution
Flat storage trays
Water bath
B-method
1-In the meantime thaw the virus rapidly and make serial dilutions in
MEM medium.
2-Inoculate tissue cultures as follow:
a-With sterile technique, pour off the supernatant maintenance medium
into a discard container.
b-Dispense 0.2 ml of virus dilution into a replicate bottles (3 per dilution).
Manually rotate the inoculum over the surface of the cell sheet and allow
the inoculum to remain in contact with the monolayer for 1 hr. at the
appropriate temperature for the virus. Do not permit cells to dry or be
exposed to bright light during this period.
3-At the end of incubation period, set up your work area with the bottles
of culture lined up on a tray. The water bath holding the melted and
cooled agar closes by to your right.
4-With sterile technique combine MEM medium with Nobel agar
medium in a suitable container and place the container in the (45-48) ºC
water bath .Working quickly and with sterile technique.
5-With sterile technique add 10 ml of agar medium to the first culture
bottle. Do this with allow the agar to flow down the side of bottle
opposite to the monolayer, Replace the stopper and gently rotate the
bottle ,allowing the agar to flow over the cell sheet. Do not be vigorous
with this procedure in order to avoid bubbles in the agar.
15
6-Place the bottle down flat on the tray, with the agar covering the
monolayer. Repeat for each bottle in turn. Allow them to remain
undistributed in a dark room until the agar is set. This will take
approximately 1 hr.
7-Invert the bottles on the tray, this will prevent condensation from
falling in the agar, causing spread of virus over the monolayer with loss
of distinct plaques. Incubate at 37ºC.
8-Examine daily for plaques. These will appear as holes in the agar. Some
will be clear .others opaque or translucent. Some will have smoothly
defined edges, others will have irregular outline. Some will be large
others small. A particular virus will produce a particular plaque type.
Plaques counting and determination of titer
Foci are usually counted with the unaided eye, although some have to be
checked microscopically .Pocks on CAM should be observed against a
dark background or oblique illumination may be used. It must be to
distinguish non specific lesions with usually occur in the area of the
original injection site. If too high a concentration of virus was used, pocks
may become confluent. This may also be occurring if you did not rotate
the eggs after inoculation. There may be edema and hemorrhage
present.Carfully compare test inoculates with control CAMs prepared and
harvested simultaneously.
The titer is calculated by the formula:
Average number of plaques
= number of plaque forming unit (PFU) per
milliliter of original suspension
Volume inoculum×dilution
16
Determination of lethal dose 50 of Newcastle disease
virus in embryonated chicken eggs (ELD50)
Another method of virus titration is by quantal dose- response. This
method is employed with end point method of titration in which groups of
animals, egg embryos or cell culture are inoculated with certain dilutions
of virus.
Materials
Newcastle disease virus (virulent), embryonated chicken eggs, test tubes,
pipettes, syringes and needles and normal saline.
Procedure
1-Arrange 11 test tubes in a rack and number them 1 to 10.
2-Prepare 10-fold serial dilutions of the virus in normal saline starting
from (10-1) to (10-10).
3-Starting from the highest serial dilution of the virus, inoculate a batch
of 5 chicken embryos with each virus dilution via allantoic cavity route.
4-Incubate the embryos in egg incubator for a period of 3 days. Examine
the eggs every day for any death .Preserve dead eggs in refrigerator.
5-At the e3nd of incubation period, examine the dead and survived
embryos inoculated with each virus dilution and calculate LD50 by Reed
and Muench formula.
For the purpose of calculation of LD50 dose of the virus use the
imaginary figures shown in the table below.
tubes
Dilution
Of virus
1
10-1
Positive
response 5*/5**
2
3
4
5
6
7
8
9
10-2
10-3
10-4
10-5
10-6
10-7
10-8
10-9
10-10
5/5
5/5
5/5
5/5
5/5
5/5
3/5
0/5
0/5
*=NO. of eggs died or survived.
**= NO. of inoculated eggs with virus eggs .
17
10
Calculations:
It is clear from above table that 3 out of 5 embryos died in 10-8
The titer of virus in 10-8 dilution is (10-8) .in this case the exact 50% end
point will lie somewhere between (10-8) and (10-9) virus dilutions for
which Reed and Muench formula will be applied.
Procedure for interpolation of 50% end point of viral activity:
10-8
10-9
5/5
3/5
0/5
5
3
0
Numbers survived
0
2
5
Accumulation total
died
8
3
0
Accumulation total
survived
0
2
7
Mortality rate
8/8
3/5
0/7
60
0
Dilution of virus
Mortality rate
10-7
Numbers died
% Mortality
100
lethal dose
50
The 50%end
point
can
be
determined
by
the
following
%mortality above 50%-50%
Proportional distance =
%mortality above 50%-%mortalitybelow 50%
18
formula:
60-50
Proportional distance =
60-0
Proportional distance = 0,167 say 0, 2
Log lower dilution (dilution in which % mortality above 50%) =-8
Proportional distance (0, 2) × log dilution factor (10) =-0, 2
Sum (50% end point) =-8, 2
LD50per 0, 1 ml=10-8, 2
There are some terminologies like:
1-EID50: Embryo infective dose 50.
2-ELD50: Embryo lethal dose 50.
3-TCID50: Tissue culture infective dose 50.
19
Heamagglutination test
This test is not antigen-antibody test. Heamagglutination is a biological
phenomenon present in some viruses which have heamagglutinin antigen
on their surface like paramyxoviruses and orthomyxoviruses, the other
antigen which present on the surface of these viruses is neuraminidase
which responsible for Elution. The titer of heamagglutination is found out
before carrying out the heamagglutination inhibition test.
Purposes of use:
1-To diagnose and characterize viruses which contain heamagglutinin in
their surface.
2-To found out titer of virus.
3-To perform another test (heamagglutination inhibition test).
Factors affecting on heamagglutination test:
1-Temperature :
2-Type of washed red blood cells:
3-PH:
Materials:
Newcastle disease virus (allantoic fluid), (0,5or1) %chicken
RBCs suspension, NSS and other equipments for conducting the
test including pipettes, tubes, and microtiter plate.
Procedure:
Preparation of virus dilutions:
1-Arrange 10 tubes in a rack and label them serially 1 to 10.
2-Prepare two fold serial dilutions of the virus in normal saline beginning
with 1:2 dilution in tube NO.1 through 1:1024 dilution in tube NO.10 as
shown in the following table:
20
tubes
0,5
NSS
Virus
1
2
3
0,5 0,5 0,5
+
+
+
0,5 0,5
0,5
4
5
0,5 0,5
+ +
0,5 0,5
6
7
8
0,5 0,5 0,5
+
+ +
0,5 0,5 0,5
9
0,5
+
0,5
10
0,5
+
0,5
Mix NSS and virus in tube NO.1 and transfer 0, 5 ml to tube NO.2.Mix the
contents of tube NO.2 and transfer 0, 5 ml to tube NO.3. Continue such
mixing and transfer of virus-saline mixture up to tube N0.10 to make twofold serial dilutions.
Final
virus
dilution
2 -1
2 -2
2 -3
2 -4
2 -5
2 -6
2 -7
2 -8
2 -9
2-10
All above figures in ml.
Heamagglutination test:
1-In a microtiter plate label 11wells in a row from 1 to 11 .well NO.11 act
as RBCs control.
2- Add virus dilution from 1/2 to 1/1024 in well NO.1 to 10.the quantity
of virus dilutions in each well will be 0, 25 ml.
3-Now add 0, 25 ml RBCs suspension (0, 5) % in all the wells including
well NO.11.
4-Shake the plate well to mix the reagents and incubate at room
temperature for heamagglutination .Examine the plate every 15 minutes
up to 1 hour for uniform agglutination covering the bottoms of wells of
plate.
Interpretation
A positive HA test consist of a layer of uniformly agglutinated cells
covering the bottoms of the wells of the plate .Such a patterns is
designated by plus (+) sign. A negative test consist of a compact disk of
sedimented cells in the center of the bottoms of the wells of the plate
similar to control, this is designated by minus (-) sign.
21
Results
Record the end point of HA activity of the virus in the following table
.The end point of HA is the highest dilution of the virus showing
agglutination.
Well
in
perpe
x tray
Virus
dilution
1
2
1/2
1/4
3
4
5
6
7
8
1/8
1/16
1/32
1/64
1/128
1/256
9
1/512
10
1/1024
End*
point
of HA
*Score+=positive
reaction,-=negative reaction
HA titer=reverse the highest dilution of the virus showing agglutination.
22
Serological tests
Heamagglutination inhibition test
Heamagglutination test is one of the serologic tests and based on
the inhibition of viral heamagglutinin by specific antibody. The test is
used to detect indirectly the presence of a heamaggluting virus.
Because the virus is a foreign protein, it will be elicit the formation of
antibodies in the host. If serum of the host inhibits heamagglutination
by a virus which normally does so, then this means that the virus was
in the host.
Mechanism of action:
When we use specific antiserum this will lead to inhibit heamagglutinin
activity of the virus therefore when RBCs suspension added to the
reaction settled down in the bottoms of wells as dots.
Purposes of use:
1-Diagnosis of viruses by using specific antisera (alpha method).
2-To found out titer of antibodies in sera of vaccinated or infected
animals (beta method).
Materials:
Newcastle disease virus (contain 8 HA units), antiserum from fowls
vaccinated with Newcastle disease virus, normal saline solution, 1%
Fowl RBCs suspension, microtiter plateon a stand with a mirror
underneath, pipettes and other equipments for HI test.
Procedure:
Beta method:
1-Serially number 1 to 12 wells of the microtiter plate in a row.
2-Add 0,025 ml of two fold serial dilutions of the serum from 1:2 to
1:1024 in wells number 1 to 10 .well NO. 11 act as RBCs control, well
NO. 12 act as virus control.
3-Next add 0,025 ml Newcastle disease virus (contain 8 HA units)
(undiluted) in wells NO.1 to well NO. 10.
4-Mix the virus-serum reactants thorouly and allow to act for 20 minutes
at room temperature.
23
5-Now add 0,025 ml of 1% fowl RBCs suspension in all the 12 wells of
the tray, mix the contents and allow to act for(15-30)minutes at room
temperature or at incubator(37)ºC before taking the reading.
Interpretation:
The end point of the heamagglutination inhibition activity of serum is the
lowest dilution of serum in which HA activity is completely inhibited.
Calculation of HI titer:
HI titer=reverse lowest dilution of serum in which HA activity is
completely inhibited ×8 HA units.
24
Neutralization test
Neutralization test is one of the serologic tests in which serum and virus
are brought together under certain conditions and are inoculated into
susceptible host (animals, eggs, tissue culture).If antibodies for the virus
in question are absent, disease, lesions or death may result. When
antibodies are present, no such reactions are noted.
The virus sample may consist of the various fluids from embryonated
eggs, tissue culture fluids or extracts of infected tissue (brain, liver
etc.).These can be used directly or after low speed centrifugation to
remove debris. These preparations should be used fresh when possible, or
the tissue may be frozen in ampoules at -60ºC or lower.
The selection of the indicator system depends on the infectiousness and
lethality of the virus for a particular host, the cost, the ease of handling
etc. Animals (mice, hamster, and chicks), embryonated eggs (hen, duck)
or cells in culture may be employed.
There are two methods of performing neutralization test. In alpha
procedure, equal quantities of a constant amount of serum and increasing
dilutions of virus are incubated together and then inoculated into the
indicator system .In beta procedure, virus at a concentration of 1oo
TCID50 Is Incubated with two fold dilutions of serum before inoculation.
The latter method is more generally used with tissue culture because it is
more economical in the use of sera and gives a greater delicacy of test
.Also a broader range of antibody titer can be measured where constant
amount of virus is used. The temperature and length of incubation of the
serum-virus mixture will depend on the agents used although there
appears to be little agreement as to the best temperature to use in each
particular circumstance or even the need for incubating the serum - virus
mixture.
Alpha procedure
Although cell culture are used as indicator system in the procedures
described below, it should be understood that the same procedure apply
equally to doing these test in animals or eggs. One simply substitutes the
appropriate host for the cell culture tubes described below.
A-materials
Frozen virus suspension
25
Virus-specific antiserum
Uninoculated tissue cultures: four wells per dilution for virus-serum test
and virus titer control, plus uninoculated controls.
MEM media
tissue culture Microtiter plate
B- Method
1-inactivate serum at 56ºC for 30 minute to destroy heat-labile virus
inhibitors.
2-Set up 10 small test tubes in a rack .Add 0, 5 ml of serum to each tube.
3-Set up a tissue culture in flat microtiter plate with 10 rows of tissue
culture wells,4 wells per dilution and 2 rows as controls(uninoculated).
4-Prepare ten fold serial dilutions of virus in maintenous media, adding 0,
5 ml of each virus dilution to each corresponding tube of serum and 0,0 5
ml to each replicate tissue culture well of micro titer plate( for a virus
titration. four per dilution).incubate plate at 37ºC.
5-Shake all virus-serum tubes .incubate 1 hr.at room temperature.
6-In the mean time ,set up another tissue culture plate inoculate 0, 1 ml
of serum-virus mixture to each replicate tissue culture well (10 rows of
tissue culture wells ,four per dilution)for a virus titration using different
pipette for each serum-virus dilution and place plate at 37ºC incubator.
7-Observe for CPE daily for seven day and record results.
Neutralization index
In the alpha neutralization test (constant serum plus virus dilutions)
The neutralization effect of the serum is expressed by the neutralization
index.
This is determined by tittering the virus in the presence of diluent and in
the presence of the test seum,calculating the LD50 and then subtracting
the exponent of the latter from the former ,disregarding the negative sign
.the number obtained represents the logarithm of the neutralization index.
LD50 of control titer=10-6, 5
LD50 of neutralization titer=10-4, 5
Neutralization index=10(6, 5 -4, 5)
=102
= 100
Log of Neu.index=log100=2
26
Neutralization indices of less than 10 (log less than 1) are considered not
significant, values between 10 and 50 (log 1-1, 6) are questionable,
indices over 50 (log 1, 7 or greater) are significant
27
28
Neutralization test
Neutralization test is one of the serologic tests in which serum and virus
are brought together under certain conditions and are inoculated into
susceptible host (animals, eggs, tissue culture).If antibodies for the virus
in question are absent, disease, lesions or death may result. When
antibodies are present, no such reactions are noted.
29
The virus sample may consist of the various fluids from embryonated
eggs, tissue culture fluids or extracts of infected tissue (brain, liver
etc.).These can be used directly or after low speed centrifugation to
remove debris. These preparations should be used fresh when possible, or
the tissue may be frozen in ampuls at -60ºC or lower.
The selection of the indicator system depends on the infectiousness and
lethality of the virus for a particular host, the cost, the ease of handling
etc. Animals (mice, hamster, and chicks), embryonated eggs (hen, duck)
or cells in culture may be employed.
There are two methods of performing neutralization test. In alpha
procedure, equal quantities of a constant amount of serum and increasing
dilutions of virus are incubated together and then inoculated into the
indicator system .In beta procedure, virus at a concentration of 1oo
TCID50 Is Incubated with two fold dilutions of serum before inoculation.
The latter method is more generally used with tissue culture because it is
more economical in the use of sera and gives a greater delicacy of test
.Also a broader range of antibody titer can be measured where constant
amount of virus is used. The temperature and length of incubation of the
serum-virus mixture will depend on the agents used although there
appears to be little agreement as to the best temperature to use in each
particular circumstance or even the need for incubating the serum - virus
mixture.
Alpha procedure
Although cell culture are used as indicator system in the procedures
described below, it should be understood that the same procedure apply
equally to doing these test in animals or eggs. One simply substitutes the
appropriate host for the cell culture tubes described below.
A-materials
Frozen virus suspension
Virus-specific antiserum
Uninoculated tissue cultures: fourwells per dilution for virus-serum test
and virus titer control, plus uninoculated controls.
MEM media
tissue culture Microtiter plate
B- Method
1-inactivate serum at 56ºC for 30 minute to destroy heat-labile virus
inhibitors.
30
2-Set up 10 small test tubes in a rack .Add 0, 5 ml of serum to each tube.
3-Set up a tissue culture in flat microtiter plate with 10 rows of tissue
culture wells,4 wells per dilution and 2 rows as controls(uninoculated).
4-Prepare ten fold serial dilutions of virus in maintainous media, adding
0, 5 ml of each virus dilution to each corresponding tube of serum and 0,0
5 ml to each replicate tissue culture well of micro titer plate( for a virus
titration. four per dilution).incubate plate at 37ºC.
5-Shake all virus-serum tubes .incubate 1 hr.at room temperature.
6-In the mean time ,set up another tissue culture plate inoculate 0, 1 ml
of serum-virus mixture to each replicate tissue culture well (10 rows of
tissue culture wells ,four per dilution)for a virus titration using different
pipette for each serum-virus dilution and place plate at 37ºC incubator.
7-Observe for CPE daily for seven day and record results.
Neutralization index
In the alpha neutralization test (constant serum plus virus dilutions)
The neutralization effect of the serum is expressed by the neutralization
index.
This is determined by tittering the virus in the presence of diluent and in
the presence of the test seum,calculating the LD50 and then subtracting
the exponent of the latter from the former ,disregarding the negative sign
.the number obtained represents the logarithm of the neutralization index.
LD50 of control titer=10-6, 5
LD50 of neutralization titer=10-4, 5
Neutralization index=10(6, 5 -4, 5)
=102
= 100
Log of Neu.index=log100=2
Neutralization indices of less than 10 (log less than 1) are considered not
significant, values between 10 and 50 (log 1-1, 6) are questionable,
indices over 50 (log 1, 7 or greater) are significant.
31