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PRODUCTION OF NEWCASTLE

DISEASE VIRUS USING VERO CELLS


FOR DEVELOPMENT OF VACCINE
RN RIDUAN BIN RUSIMIN (1110357)

Abstract

Figure below shows growth curve of Vero cells with time of


infection (TOI) at 80 hours and the indication of Cytophatic effect
(CPE) under microscope and 96 well plates.
Growth Profile (Vero Cells)
7.00E+05

Cell Concentration(cells/ml)

Experiments were carried out in T-flask to investigate the optimum level


of serum concentration and multiplicity of infection for virus
propagation. The optimum condition was achieved by Response
Surface methodology (RSM) with Face Centered Central Composite
Design (FCCCD) with 3 centre points using Design Expert software
version 6.0.8. The response for the optimization experiment was Tissue
Culture Infectious Dose 50% (TCID50) with 11 experimental runs.
Growth profile for Vero cells was plotted to find the suitable Time of
Infection (TOI) for the NDV.

Result & Discussion

6.00E+05
5.00E+05
4.00E+05
3.00E+05
2.00E+05
1.00E+05
0.00E+00
0

12 24 36 48 60 72 84 96 108 120
Time (hours)

Problem Statement
The current method used for production of ND vaccine is by
propagating the NDV in egg embryo, but this method is only sufficient
for small scale production of vaccine. To solve this problem, vaccine
production by propagating NDV virus in mammalian cell is the most
ideal method as it allowed easier upscaling for the process involved
with spinner flask or bioreactor.

Project Objectives
1. To propagate NDV in Vero cells for development of ND vaccines.
2. To determine the optimum condition needed for the virus
propagation in T-flask.

Research Methodology
Frozen Vero Cell line
was thawed and cultured
until passage 3 (p3)
NDV was infected on semi confluent p3 of
Vero cells until p5. On each passage, virus
culture was harvested, stored at 20oC and
infected again until it reached passage 5. (p5)
16 samples was taken

The recording of CPE effect in TCID50 table on day 2 and day 5


(No CPE reading on day 1)

Design of Experiment (DOE) table with recorded response and


3D surface response graph.
Run

Block

Serum
Concentration (%)

MOI
(pfu/cells)

TCID50 (TCID50/ml)

Block 1

2.00

11.00

2.16 x 109

Block 1

2.00

11.00

1.48 x 109

Block 1

2.50

2.00

9.78 x 107

Block 1

1.50

11.00

2.58 x 109

Block 1

2.00

2.00

4.42 x 107

Block 1

2.50

20.00

3.97 x1010

Block 1

2.00

20.00

2.06 x 1010

Block 1

2.50

11.00

1.34 x109

Block 1

1.50

20.00

9.03 x 109

10

Block 1

2.00

11.00

3.07 x 109

11

Block 1

1.50

2.00

1.08 x107

Validation of parameter gained from the Design Expert software


Serum

Multiplicity

Virus

Infectivity

Concentration

of Infection

Titre (TCID50/ml)

2.48 %

20.00

2.547 x 1010

2.48 %

20.00

1.29 x 109

Selected
Solutions

Validation

Growth Profile of Vero Cells


was done to determine
suitable time for NDV
infection.
MOI (multiplicity of
infection) using TCID50
assay was done.
Optimum value was
determined using DOE, MOI
and serum concentration for
NDV was propagated in TFlask
Optimization and
Validation of
data

Conclusion
1. All of the objectives stated in this project was successfully
achieved.
2. The difference in TCID50 response in the validation
experiment was due to possible human or technical error.
3. The optimum value gained for MOI was 20.00 and serum
concentration was 2.48%.
4. These result is an important information when large scale
production of NDV is considered.

Acknowledgment:
Special thanks to DR. Raha, Bro Azmir Ariffin and DR. Munirah for
the completion of this project.
Adapted from IRIE 2010 template

DEPARTMENT OF BIOCHEMICAL & BIOTECHNOLOGY ENGINEERING


KULLIYYAH OF ENGINEERING
INTERNATIONAL
ISLAMIC
UNIVERSITY
MALAYSIA
FINAL
YEAR PROJECT
(FYP)
2 SEMESTER
1 2010/2011

ELECTRICAL & COMPUTER ENGINEERING