Guide:Dr.

Muthukumaran Sivanandham
External Mentor:Dr. K .Kumanan
Internal Mentor: S.Kirubanandan

T.Ramakrishnan

Newcastle disease is a contagious bird disease

affecting many domestic and wild avian
species

NDV is a contagious and fatal viral disease

affecting most species of birds

NDV is so virulent that many birds die without

showing any clinical signs. A death rate of
almost 100 percent can occur in unvaccinated
poultry flocks.
NDV is spread primarily through direct contact
between healthy birds and the bodily
discharges of infected birds.

ZOONOTIC RISK :

Exposure of humans to infected birds

(for example in poultry processing
plants) can cause mild conjunctivitis
and influenza-like symptoms, but
otherwise poses no hazard to human
health

The Vero lineage was isolated from kidney

epithelial cells extracted from an African
green monkey (Cercopithecus aethiops)

The Vero cell lineage is continuous and

aneuploid. A continuous cell lineage can be
replicated through many cycles of division
and not become senescent.

Vero cells are interferon-deficient; unlike

normal mammalian cells, they do not
secrete type 1 interferons when infected by
viruses. Hence they are ideal for virus
culture

NDV is purposely adapted on Vero cell line

to alter growth and virulence characteristics
so that Vero cells become a suitable
laboratory host for cultivation, mass
propagation
attenuation
and
genetic
modification of NDV.

Interest in the use of NDV as an anticancer

agent has arisen from the ability of NDV to
selectively kill human tumour cells with
limited toxicity to normal cells

Infect the Newcastle Disease Virus on Vero

cell lines
To maintain the cell lines sub culture them
and harvest the infected cells
Study the cytopatic effect of the infected
cells by Serum neutralization test and
mean death time (MDT) analysis
Quantify the virus titer for every 5
passage
Check the virus presence by RT PCR
To characterize of 354 bp fusion protein
sequence by automated sequencer

MIMIMUM ESSENTIAL MEDIUM

Eagle's minimum essential medium with
supplements were used for culturing Vero
cells and maintaining NDV on Vero cells.
Culture media was prepared by adding
5% heat-inactivated foetal calf serum
(FCS), 2% L-Glutamine, 2% sodium
bicarbonate and 1% antibiotic solution.
The growth medium was sterilized by
filtering through 0.22 m membrane filter
and stored at 4 Degree Celsius

Preparation of Trypsin Versene Glucose

solution (TPVG)
Trypsin enzyme has the capacity to detach
anchorage dependent cell lines from their
support. Its solution is often prepared in
buffer.
Preparation of TPVG
Trypsin - 0.2 g, D-glucose - 0.05 g are to
be dissolved in 100 ml of Phosphate
buffered saline (Solution A) using
magnetic stirrer. The pH is to be
adjusted to 7.5 with one normal
sodium hydroxide solution. The
solution must be sterilized
immediately by filtering through a
sterile 0.22 membrane filter,
distributed into small aliquots and
then must be stored at - 20o C until
use.

7 samples of cryo-preserved NDV 4

virus samples were obtained from
Tamilnadu veterinary university.
In these samples hemagglutination
(HA) test were conducted to check for
the presence of virus
The test results show samples 2 & 7
contain the virus at 2*10-6 dilution.

(i) 25 L of PBS was added in each

well of a plastic U-bottomed
microtitre plate.
(ii) 25 L of sample was placed in the
second well and mixed well keeping
the first row as blank.
(iii) From first well 25 L of mixture
was serially diluted .

(iv) 25 L of 0.5% (v/v) chicken RBC

was added into each well.
(v) The plate was tapped gently and
was allowed to keep at room
temperature for about 15 min.
(vi) HA was determined by observing
the hemaggluntination (button
formation) of the RBC.

A uniform layer of hemaggluntination

covering the bottom of well of the
plate was considered as positive HA
and a sharp buttoning of RBC at the
bottom of well of the plate was
considered as negative.
Its seen as only row 3 & 7 show
positive results with no button
formation.

The cryo-preseved Vero cells were

obtained from the national centre for
cell sciences , Pune.
The cells are revived by instant
thawing by quenching the vial in 40
Degree water bath
The cells were added in the growth
medium and incubated for 3 days for
formation of a monolayer of cells

The spent medium was discarded.

1 ml of TVG solution I added and kept
for 1 minute.
It is incubated for 5 minutes until the
entire cell monolayer is removed.
12ml of medium + 2ml serum is
added.
It is separated into two flask.

The spent medium was discarded.

0.5 ml of the virus sample is added.
It is then incubated for 1 hour at 37
Degree Celsius
The virus is discarded and the growth
medium is added.
It is then incubated for 3 days where
then the entire monolayer is removed
by infection

The Virus gets fully infected by 3 days of

incubation.
The virus growing inside the cells is
extracted out by freeze thawing
The cell culture is completely frozen and
the kept in room temperature until it
melts fully
This is repeated 3 times. Causing the
virus to be floating outside the cell in
the medium.

First the RNA is extracted from the

infected cell culture.
cDNA is synthesized from RNA using PCR
The cDNA is amplified again by PCR
The PCR products are run in the
Agarose gel
The PCR products are purified by elution
and then sequenced using a auto
sequencer.

Infected the NDV virus on Vero cell

lines
Have done 3 virus passage
Done HA test for the first passage.
Have extracted the RNA from the
virus sample and converted it to
cDNA and stored it.