1. B.Med.Sc. (Hons) Medical Microbiology & Virology
Research Project Presentation
Title: DEVELOPING A STABLE CELL LINE THAT CONSTITUTIVELY
EXPRESSES A NUCLEOCAPSID PROTEIN OF CRIMEAN-CONGO
HEMORRHAGIC FEVER VIRUS(CCHFV)
By: Nteboheleng Bafazini
Supervised by: Prof. FJ Burt
Faculty of Medicine
Department of Medical Microbiology and Virology
University of the Free State

2. The History Behind CCHFV
 First encountered in the 12th century in Tadzhikistan
 1944 to 1945: First described in the Crimean peninsula and named Crimean hemorrhagic fever virus
(CHFV)
 1956: Initially isolated from a patient in Kisangani, Congo and named Congo virus (CV)
 1969: Indistinguishable antigenic similarities between CHFV and CV were discovered. thereafter,
the virus was designated CCHFV
 1981: First encountered in South Africa- isolated from the blood of a boy bitten by a tick in the North
West province
 Since then,5-20 cases are reported yearly

3. The Virus
 The causative agent of a severe tick-borne animal zoonosis and hemorrhagic fever with a 30%
mortality rate
 Classification:-
 Family: Bunyaviridae
 Genus: Nairovirus
 Genome:-
 -ssRNA with three segments of different sizes:
 Small segment (S): encodes nucleocapsid protein (NP).
 Medium segment (M): encodes envelope glycoproteins.
 Large segment (L): encodes RNA polymerase.

4. Transmission & Epidemiology
 Epidemiology:
 Its prevalence coincides with its
principal
vector’s distribution:
H. marginatum marginatum ticks
 Documented in more than 30 countries:
 Africa, the Middle East, eastern and
southern Europe and Asia
HorizontalTransmission

5. Symptoms, Diagnosis & Treatment
 Symptoms:
 Occur post a 1-7 days incubation period.
 Include: sudden onset of high fever, headache, chills, myalgia, backache, elevated liver
enzymes levels, organ failure and eventually progress to a sever hemorrhage.
 Diagnosis:
 Serological: detection of IgG & IgM antibodies using an immunofluorescence assay (IFA)
 Molecular: viral nucleic acid detection using PCR
Treatment: No vaccine.
 Intravenous ribavirin therapy.
 Supportive therapy

6. Problem identification & Aim
 Problem Identification:
Preparation of reagents for assays for CCHFV diagnosis requires culturing the virus which in
turn requires laboratories with BSL-4 facilities. However, these types of facilities are limited.
Consequently, there is a need for safe diagnostic reagents which will make CCHFV diagnosis
possible even in laboratories without BSL-4 facilities.
 Aim:
The aim of this study is to prepare a construct that can be used in the preparation of a stable cell
line that constitutively expresses the nucleocapsid protein of CCHFV

7. Objectives
 Transfect mammalian cells with an expression vector carrying CCHFV NP using different
transfection reagents and compare transfection efficiency of different transfection reagents and
different cell lines.
 Monitor NP expression to identify positively transfected cells with an IFA and SDS-PAGE.
 Determine appropriate G418 concentration for selection of stable transfectants
 Using G418 selection media, create a stable cell line that constitutively expresses CCHFV NP.

8. Methods & Materials
 In the previous study: CCHFV NP was cloned into pcDNA ™3.1TOPO and the construct named
pcDNA 3.1TOPO-CCHFVNP
 The presence of the gene was confirmed prior to transfection experiments.
 Cell culture:
 Baby hamster kidney cells
 Vero cells
 Transfection Reagents: Selection antibiotic:
 Fugene6 TurboFect Geneticin (G418)
 Lipofectamine 2000 X-tremeGene HP

9. Methods & Materials
1. Small scale
plasmid preparation
and Purification
1.2. Analysis of
products with 1%
agarose gel
electrophoresis 2. Determining appropriate
seeding densities & transfection
of BHK cells using different
transfection reagents.
3. Cell processing post-
transfection for confirmation
of positive transfection:
 Immunofluorescence assay
(IFA)
SDS-PAGE 5. Confirmation of
stable transfection with
an IFA
1.1. Double
restriction enzyme
digestion with Not1
& BamH1
4. Titration of
Neomycin (G418)
concentration in a 96-
well plate and
Development of a stable
cell line.
- +

10. Establishing a stable cell line constitutively
expressing CCHFV NP
 BHK cells were transfected with pcDNA 3.1TOPO-CCHFVNP using Lipofectamine™ 2000
transfection reagent at a 2:1 transfection reagent: DNA ratio.
 After 24 hours of incubation, growth medium was discarded and replaced with 2% FBS growth
media supplemented with G418 (1.2mg/ml) .
 After six days, BHK cells transiently/stably expressing NP were trypsinized and transferred
into six well plates with 2ml selection media.
 Four days later, cells were trypsinzed, cultured in T25 flasks
 IFA was performed in 8-well multitest slides.
 Selection media was changed every 48hrs.
 Stably transfected BHK cells were then maintained in selection media.

11. Results
Plasmid purification and DNA
concentration Restriction enzyme digestion reaction for
confirmation of positive transformation
 DNA concentrations:
 Appropriate seeding density: 3x105
cells/ml
 Transfection efficiency (TE):
 Highest TE with NP: 20% (Lipofectamine
2000)
 Lowest TE with NP: 0% (all reagents)
 Highest TE with GFP: 80% (TurboFect)

12. Results
A: BHK cells expressing GFP.
B (I & ii): GFP expressing cells (Light
microscope)and B (ii) confocal
microscopy
C: CCHFV NP expressing cells
(confocal microscope )
C (I & ii) confocal microscope &
under a fluorescent microscope.
IFA

13. Results
Lane 1: Spectra Broad Range Pre-stained protein
Lane 2&3: Lipofectamine 2000 (2:1 ratio)
transfected BHK cells with pcDNA3.1TOPO-
CCHFVNP, pellet and supernatant respectively.
Lane 4&5: TurboFect (2:1) transfected BHK cells
with pcDNA3.1TOPO-CCHFVNP, pellet and
supernatant respectively.
Lane 6&7: TurboFect (2:1) transfected BHK cells
with pSin-GFP, pellet and supernatant respectively.
Lane 8&9: Negative control (untransfected cells),
pellet & supernatant respectively.
≈170
≈70
≈35
≈15
≈10
SDS-PAGE

14. Results
Titration of G418 concentration
 1.2mg/ml G418 concentration was
selected as appropriate for selection of
positive transfectants.
 Stably transfected BHK cells
Positive immunofluorescence staining of
CCHFV nucleocapsid protein-expressing
BHK cells in suspension

15. Discussion
 Optimal seeding density to facilitate transfection in 24hours was determined to be
3x105cells/ml
 Mammalian cell transfection: each transfection reagent showed a different efficiency at a
different transfection reagent: DNA ratio.
 The optimal ratio (2:1) was used for GFP and NP
 pSin-GFP was used for optimization of transfection experiments
 Lipofectamime 2000 had the highest transfection efficiency NP under optimized conditions.
 SDS-PAGE gel: 53kDa and 26.9kDa bands were expected for CCHFV NP and GFP
respectively.
 No bands corresponding to those were evident on the SDS-PAGE gel.
 Factors such as too little protein expression and the inability to keep BHK cells going for long to
allow more protein expression could be attributed to the problem.

16. Discussion
 With an IFA, a green fluorescence was observed, regardless of cell loss experienced.
 During G418 titration:
 high G418 concentration resulted in quick cell death regardless of the number of cells per well (Rows A & B)
 As the concentration decreases, cell number becomes significant: more cells and less G418 results in more
cell death due to overgrowth as opposed to G418 intoxification in highly diluted cells
 Two days post selection with 1.4mg/ml & 1.2mg/ml G418, all untransfected BHK cells died

17. Conclusion
 Therefore BHK cells were capable of taking up plasmid using a selected transfection reagent
(Lipofectamine 2000) and in turn expressed the protein encoded by the gene cloned into the
expression vector
 BHK positive transfectants expressed CCHFV NP, detected by human raised anti-CCHFV antibodies
using an IFA
 CCHFV NP transfected construct could be used in the development of a stable cell line using a pre-
determined neomycin (G418) concentration to select for positive transfectants
 Continuously passaging the clones would lead to a clone of cells constitutively expressing CCHFV
NP, which can in turn be used in the development of diagnostic assays or in epidemiology research.
 Further work will be performed to scale up the experiments and confirm protein expression with an
SDS-PAGE.

18. Acknowledgements
 I would like to thank everyone who helped make this year and work a success through their endless
outstanding support:
 My family
 My “sitter” , helper and advisor: Natalie Viljoen
 My remarkable supervisor for her constant inspiration and motivation: Prof. FJ Burt
 My Honor degree sponsors for making this dream a reality :
 Prof. J. Jansen
 Polio Research Fund
 The department of Medical Microbiology and Virology
 Others: Mrs L. Mathengtheng for his help at all times
 Mrs A. van der Spoel V DJK for her support
 Above all, my heavenly Father for granting me the strength through it all.