Guide:Dr.Muthukumaran SivanandhamExternal Mentor:Dr. K .KumananInternal 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 µm 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 μ 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 μL of PBS was added in each well of a plastic U-bottomed microtitre plate.

(ii) 25 μL of sample was placed in the second well and mixed well keeping the first row as blank.

(iii) From first well 25 μL of mixture was serially diluted .

(iv) 25 μL 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.