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.