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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 BurtFaculty of Medicine
Department of Medical Microbiology and VirologyUniversity of the Free State
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
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.
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
Ver
tica
l Tra
nsm
issi
on
Ho
rizon
tal Transm
ission
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
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
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.
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
Methods & Materials1. Small scale
plasmid
preparation and
Purification
1.2. Analysis of
products with
1% agarose gel
electrophoresis2. 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.
- +
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.
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)
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
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.
Lan
e 2
Lan
e 3
Lan
e 1
Lan
e 4
Lan
e 5
Lan
e 6
Lan
e 7
Lan
e 8
Lan
e 9
≈170
≈70
≈35
≈15
≈10
SDS-PAGE
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
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.
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
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.
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.