i

11/15/2013

Hyalomma marginatum

(http://www.infectionlandscapes.org/2012/10/crimeancongo-hemorrhagic-fever.html)

Project supervisor: Prof F.J. Burt | Author: Nteboheleng Bafazini (2009078425)

B.MED.SC. (HONS)

MEDICAL

MICROBIOLOGY

AND VIROLOGY

DEVELOPING A STABLE CELL LINE THAT CONSTITUTIVELY EXPRESSES A NUCLEOCAPSID PROTEIN OF CRIMEAN-CONGO

HEMORRHAGIC FEVER VIRUS

This is a Mini-Dissertation Submitted to the Faculty of Medicine

Department of Medical Microbiology and Virology To fulfill the requirements for an Honors Degree

In the School of Medicine at the University of the Free State Bloemfontein, South Africa (9301)

Submitted by: Nteboheleng Bafazini

Student number: 2009078425

Supervised by: Prof. Felicity J. Burt

Table of Contents

Acknowledgements………………………………………………………………………………………………………………………..……i

List of Figures...…..………………………………………………………………………………………………………………………………ii

List of Tables...…………………………………………………………….………………………………………………………………….…iii

List of Abbreviations……………………………………………………………………………………………………………………….…iv

Abstract ............................................................................................................................................. 1

Chapter 1 ........................................................................................................................................... 2

1. Introduction ........................................................................................................................................ 2

1.1. Literature Review ........................................................................................................................... 2

1.1.1. History behind CCHFV……………………………………………………………………………………………………..2

1.1.2. The Virus………………………………………………………………………………………………………………………...4

1.1.3. Transmission and Epidemiology………………………………………………………………………………………6

1.1.4. Clinical Sign and Symptoms…………………………………………………………………………………………….8

1.1.5. Diagnosis…………………………………………………………………………………………………………………………9

1.1.5.1. Biological Specimen………………………………………………………………………………………………...10

1.1.5.2. Sample Processing……………………………………………………………………………………………………10

1.1.5.3. Diagnostic Techniques…………………………………………………………………………………….……….10

1.1.5.4. The acute phase of illness…………………………………………………………………………………………11

1.1.5.5. The convalescent phase of illness…………………………………………………………………………….12

1.1.6. Management and Treatment……………………………………………………………………………………….13

1.2. Problem Identification……………..…………………………………………………………………………………………….14

1.3. Aim………………………………………………………………………………………………………………………………………..14

1.4. Objectives………………………………………………………………………………………………………………………………14

Chapter 2 …………………………………………………………………………………………………………………………………………15

2. Methods and Materials……………………………………………………………………………………………………………….15

2.1. Introduction………………………………………………………………………………………………………………………......15

2.2. Preparation of a 15% Glycerol Stock………………………………………………………………………………………..17

2.3. Transformation of competent OverExpress C43 (DE3) E. coli cells using a heat-shock

method……………………………………………………………………………………………………………………………………17

2.4. Plasmid purification using Pure Yield™ Plasmid Miniprep System (Promega, WI, USA)……………18

2.5. Restriction enzyme digestion reaction for confirmation of positive transformation…………..……18

2.6. Analysis of restriction enzyme digestion products with 1% agarose gel electrophoresis…….……20

2.7. Plasmid purification using with QIAGEN® Plasmid Mini Kit (25) (QIAGEN, Germany)……………….20

2.8. DNA concentration………………………………………………………………………………………………………………….21

2.9. Protein expression in laboratory maintained mammalian cells………………………………………….…….21

2.9.1. Cells and culture conditions……………………………………………………………………………………….…22

2.9.2. Determining the appropriate seeding densities for transfection……………………………………22

2.9.3. Transfection of BHK and Vero cells using different transfection reagents……………………..22

2.9.3.1. Purification of pSin-GFP plasmid with the QIAGEN® Plasmid Plus Midi Kits (QIAGEN,

Germany)………………………………………………………………………………………………………………….23

2.9.3.2. Transfection of BHK cells with FuGene®6 transfection reagent (Roche, Mannheim,

Germany)………………………………………………………………………………………………………………….24

2.9.3.3. Transfection of BHK cells with Lipofectamine™ 2000 transfection reagent (Invitrogen,

Carlsbad,USA)…………………………………………………………………………………………………….……..25

2.9.3.4. Transfection of BHK cells with TurboFect transfection reagent (ThermoScientific,

Lithuania)………………………………………………………………………………………………………….………26

2.9.3.5. Transfection of BHK cells in a 24-well plate with X-tremeGENE HP transfection reagent

(Roche, Mannheim, Germany)………………………………………………………………………………….26

2.9.4. Transfection of Vero cells with different transfection reagents…………………………………….27

2.9.4.1. Transfection of Vero cells with Lipofectamine 2000 transfection reagent…………………27

2.9.4.2. Transfection of Vero cells with TurboFect transfection reagent………………………………..28

2.10. Cell processing post-transfection………………………………………………………………………………….28

2.10.1. Immunofluorescence assay (IFA) for confirmation of positive transfection of BHK cells

with pcDNA3.1TOPO-CCHFVNP plasmid………………………………………………………………………..28

2.10.2. Protein extraction and expression via sodium dodecyl sulphate polyacrylaminde gel

electrophoresis (SDS-PAGE) gel…………………………………………………………………………………….29

2.11. Preparation and use of different mounting solutions to solve problems encountered

during visualization of fluorescent cells under fluorescent electron microscope……………………31

2.11.1. Preparation a glycerol mounting solution……………………………………………………………………..31

2.11.2. Preparation of “glucose syrup” mounting solution………………………………………………………..31

2.12. Titration of Neomycin (G418) (Life technologies, NY, USA) in a 96-well plate (Nunc,

Denmark)…………………………………………………………………………………………………………………………………31

2.13. Establishing a stable cell line constitutively expressing CCHFV NP………………………………..33

Chapter 3 ……………………………………………………………………………………………………………………………………….…34

3. Results…………………………………………………………………………………………………………………………………………34

3.1. Transformation of competent OverExpress C43 (DE3) E. coli cells using a heat-shock

method…………………………………………………………………………………………………………………………………...34

3.2. Plasmid purification and DNA concentration……………………………………………………………………………34

3.3. Restriction enzyme digestion reaction for confirmation of positive transformation………………..36

3.4. Determining the appropriate seeding densities for transfection……………………………………………..38

3.5. Transfection of BHK and Vero cells using different transfection reagents…………………………….….39

3.5.1. Transfection of BHK cells with FuGene®6 transfection reagent……………………………………..39

3.5.2. Transfection of BHK cells with Lipofectamine™ 2000, TurboFect and X-tremeGENE HP

transfection reagents…………………………………………………………………………………………………….40

3.5.3. Transfection of Vero cells with three transfection reagents………………………………………….40

3.6. Immunofluorescence assay (IFA) for confirmation of positive transfection of BHK cells with

pcDNA3.1TOPO-CCHFV plasmid………………………………………………………………………………………………42

3.7. Protein expression (CCHFV NP) by BHK cells post transfection monitored through an SDS-

PAGE……………………………………………………………………………………………………………………………………….44

3.8. Effects of different mounting solutions in visualization of fluorescent cells under fluorescent

electron microscope………………………………………………………………………………………………………………..45

3.9. Titration of Neomycin (G418) (Life technologies, NY, USA) in a 96-well plate………………………….45

3.10. Stable transfection and expression of CCHFV NP………………………………………………………….48

Chapter 4………………………………………………………………………………………………………………………………………….49

4. Discussion and Conclusion…………………………………………………………………………………………………………..49

Appendix………………………………………………………………………………………………………………………………………….53

References……………………………………………………………………………………………………………………………………….58

i

Acknowledgements

This year has not been easy, not because of the work itself but for the main part due to my

health. However, it was possible by the support of everyone around me. First I would like to

thank my family for putting up with me, my health and for loving me enough to encourage and

believe in me even when I didn’t believe in myself. Your love is what kept me going till the end.

I would also like to thank Natalie Viljoen for helping me in every step of the way, introducing

me to every procedure and for putting up with me at all times as well as Mrs. A. van der Spoel V

DJK and Mr. L. Mathengtheng for their support whenever I needed it.

Most importantly, I would like to thank Prof. Felicity Jane Burt; my project supervisor and head

of the Virology department in the medical microbiology and virology department at the

university of the Free State. You have had an outstanding patience with me with all my

struggles, guiding me in all aspects of my course and re-assuring me that it is possible to

complete this project.

To Professor Jonathan Jansen and the Polio Research Fund group, I would love to pass my

heart-felt gratitude and honor to you for making this year a possibility by financing my studies. I

could never thank you enough.

Above all, I would love to thank my heavenly Father for granting me the strength through it all.

THANK YOU!

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List of figures

Figure 1: Life cycle of Hyalomma spp. ticks and the vertical and horizontal transmission of

CCHFV............................................................................................................................................ 3

Figure 2: CCHFV...................................................................................................................... 4

Figure 3: Transcription and translation of CCHFV RNA segments .............................................. 6

Figure 4: The geographic distribution of CCHFV........................................................................ 8

Figure 5: Clinical and laboratory course of CCHFV infection, symptoms at different phases,

laboratory analysis of liver enzymes and platelets count (PLTs) especially in fatal cases .......... 13

Figure 6: pcDNA ™3.1D/V5-His/TOPO expression vector map ................................................ 16

Figure 7: Nucleotide base pairs recognized by BamH1 and Not1 restriction enzymes

respectively................................................................................................................................. 19

Figure 8: The matrix titration of neomycin (G418) concentration and cell number to determine

the concentration resulting with complete cell death within a number of days (4) per cell

number........................................................................................................................................ 32

Figure 9: Plasmid purification results with QIAGEN® Plasmid Mini Kit (25)............................... 36

Figure 10: Gel electrophoresis analysis of double restriction enzyme digestion of

pcDNA3.1TOPO-CCHFVNP with Not1 and Bamh1 restriction enzymes for confirmation of

OverExpressed C43 (DE3) cells positive transformation with the correct plasmid ................... 37

Figure 11: Gel electrophoresis analysis of double restriction enzyme digestion of

pcDNA3.1TOPO-CCHFVNP with Not1 and Bamh1 restriction enzymes to confirm protein

expression by E. coli cells in the glycerol stock........................................................................ 38

Figure 12: Positive transfection with pSin-GFP and IFA staining of BHK cells expressing CCHFV

nucleocapsid protein (NP)...................................................................................................... 43

Figure 13: SDS-PAGE analysis of protein expressed in total protein extract from BHK cells

transfected with pSin-GFP and pcDNA3.1TOPO-CCHFVNP plus a negative control .................. 44

Figure 14: Estimated percentage of cells that remained viable after four days since they were

propagated in growth media supplemented with decreasing concentrations of G418 ............ 48

Figure 15: Positive immunofluorescence staining of CCHFV NP-expressing BHK cells……………. 48

iii

List of Tables

Table 1: Reaction mixture for a double restriction enzyme digestion of pcDNA 3.1TOPO-

CCHFVNP with BamH1 and Not1 restriction enzymes..............................................................19

Table 2: Reaction mixtures prepared for each reaction during transfection of BHK cells with

FuGene®6 transfection reagent....................................................................................................24

Table 3: Reaction mixtures prepared for each reaction during the transfection of BHK cells with

Lipofectamine™ 2000 transfection reagent.............................................................................25

Table 4: Reaction mixtures prepared for each reaction during the transfection of BHK cells with

TurboFect transfection reagent..............................................................................................26

Table 5: Preparing a 10% resolving gel for an SDS-PAGE..........................................................30

Table 6: Preparing a 4% stacking gel for an SDS-PAGE.............................................................30

Table 7: The number of times within which the plasmid was purified and the nucleic acid

concentration at each occasion..............................................................................................34

Table 8: Transfection efficiencies (in %) of different transfection reagents at different

transfection reagent: DNA ratios with BHK and Vero cell lines.................................................41

Table 9: Table 9: Symbols used to interpret results on Table 10...............................................46

Table 10: Results from the third day of neomycin titration on a 96-well plate..........................47

iv

List of Abbreviations

°C degrees celsius

µg/ml microgram per milliliter

µl micro liters

Ab antibodies

ALT alanine transaminase

APS ammonium persulfate

ASL aspartatetransaminase

BHK baby-hamster kidney

BSL bio-safety level

CCHF Crimean-Congo hemorrhaged fever

CCHFV Crimean-Congo hemorrhage fever virus

Cfu colony forming units

cm2 centimeter squared

CPE cyto-pathic effects

dH2O distilled water

DMEM dulbecco's modified eagle medium

DNA deoxyribonucleic acid

D-PBS dulbecco’s phosphate buffered saline

DRC Democratic Republic of Congo

E. coli Escherichia Coli

EDTA ethylenediaminetetraacetic acid

EIA enzyme immunoassay

ELISA enzyme linked immunosorbent assay

EtBr ethidium bromide

FBS fetal bovine serum

FITC fluorescein isothiocyanate

v

g grams

GFP green fluorescent protein

Gp glycoproteins

HCl hydrochloric acid

IFA indirect immunofluorescence assay

Ig immunoglobulin

Kb kilobases

KCl Potassium Chloride

kDa kilodaltons

L liter

LB luria broth

LB/Amp Luria Bertani broth supplemented with ampicillin

LD lactic dehydrogenase

L-Glu L-glutamine

MgCl2 Magnesium Chloride

MgSO4 Magnesium Sulphate

min minutes

ml milliliter

mol. wit. molecular weight

NaCl sodium chloride

NEAA non-essential amino acids

NEAAs’ non essential amino acids

ng nano grams

nm nanometer

NP nucleocapsid protein

o/n overnight

ORF open reading frame

P/S penicillin-streptomycin

vi

RNA ribonucleic acid

rpm revolution per minute

RT-PCR reverse – transcriptase polymerase chain reaction

SDS-PAGE sodium dodecyl sulphate polyacrylaminde gel electrophoresis

ssp species

ssRNA single-stranded ribonucleic acid

TAE buffer tris-acetate- ethylenediaminetetraacetic acid (EDTA)

TEMED tetramethylenediamine

Tryp trypsin

V volts

β beta-

1

Abstract

Crimean-Congo hemorrhagic fever (CCHF) is a widely distributed tick-borne zoonosis caused by

Crimean-Congo hemorrhagic fever virus (CCHFV). It is an important viral infection in humans

characterized by nonspecific febrile flu-like symptoms associated with headache and myalgia

that eventually progresses to a severe hemorrhagic state with a mortality rate of 30%. CCHFV is

a single stranded, tri-segmented and negative sense RNA virus of the genus Nairoviruses, in the

family Bunyaviridae. It has been described in more than 30 countries with its prevalence

coinciding with that of ixodid ticks especially of the Hyalomma spp; the principal vector of

CCHFV. CCHFV is classified as a BSL-4 pathogen with a short incubation period of 3-7 days and

no vaccine, consequently; quick, specific and sensitive diagnostic procedures are need.

This study was aimed at preparing a construct that can be used in the preparation of a stable

cell line constitutively expressing the nucleocapsid protein (NP) of CCHFV. A pcDNA 3.1 TOPO-

CCHFVNP plasmid was previously constructed and was used in this study for transformation of

OverExpress C43 (DE3) cells and transfection of baby hamster kidney (BHK) and Vero cells using

different transfection reagents. Protein expression was thereafter monitored via an

immunofluorescence assay (IFA) and sodium dodecyl sulphate –polyacrylamide gel

electrophoresis (SDS-PAGE). For selection of stable transfectants, the concentration of G418

that would inhibit untransfected cells growth was determined using matrix titration in a 96-well

plate. G418 concentration of 1.2mg/ml resulted with 100% untransfected cell death within two

days post selection and consequently considered appropriate for selection of positive

transfectants. Positive transfection with 20% transfection efficiency was obtained with BHK

cells that were transfected with NP and Lipofectamine 2000 transfection reagent at a 2:1

transfection reagent: DNA (ng) ratio. Cells that were positively transfected with CCHFV NP

expressed the protein and that was confirmed with an IFA using human sera from a

convalescent patient. Transfection studies were optimized with pSin-GFP, a replicon plasmid

expressing a green fluorescent protein. It was therefore concluded that CCHFV NP could

possess antigenic properties that may enable its potential use in serologic diagnosis of CCHFV

infections outside biosafety level four containment or laboratories.

2

Chapter 1

1. Introduction

1.1. Literature Review

1.1.1. History behind CCHFV

Crimean-Congo hemorrhagic fever virus (CCHFV) is part of the family Bunyaviridae and falls in

the genus Nairovirus (Clerks et al., 1981). It is the causative agent of a severe tick-borne animal

zoonosis and hemorrhagic fever: Crimean-Congo hemorrhagic fever (CCHF). Since the first

reported outbreak in 1944, cases of CCHFV infection have been documented all over the world

from western China through to southern Asia and the Middle East; southeastern Europe and in

most of Africa. All cases have been characterized with nonspecific febrile flu-like symptoms

such as headache, myalgia, chills, that eventually progress to a severe hemorrhagic state (Bente

et al., 2013; Hoogstraal et al., 1979). The geographic distribution of the virus has been reported

to coincide with that of ticks of the Hyalomma spp; and so they were designated the principal

vectors of CCHFV (Hoogstraal et al., 1979). CCHFV is a biosafety level 4 (BSL-4) pathogen with

no vaccine or a definitive treatment (Burt et al., 2007; Whitehouse CA et al., 2004); as a

consequence, its infection poses a serious threat to humans with a high mortality rate of 30%

(Bente et al., 2013; Yu et al., 2012; Grard et al., 2011). However, CCHF virus infection remains

asymptomatic in animals (Ergonul et al., 2006; Casals et al., 1978).Tick bite is however, not the

only mode of CCHFV transmission; humans are also subject to infection via contact with a

CCHFV infected patient during the acute phase of infection (human-to-human transmission)

(Abul-Eis et al., 2012; Drosten et al., 2003), or by contact with blood or tissues from viraemic

livestock, with infection manifesting post a 3-7 days incubation period (Bente et al., 2013; Papa

et al., 2002) (Fig 1).

3

Figure 1: Life cycle of Hyalomma spp. ticks and the vertical and horizontal transmission of CCHFV. Blue

arrows indicate the course of the tick life cycle. During the tick life cycle, there are a number of

opportunities for virus transmission between ticks and mammals (solid red arrows) and directly

between ticks, through co-feeding (dashed arrows). For each form of virus transfer, the thickness of the

red arrow indicates the efficiency of transmission. Infection of humans can occur through the bite of an

infected tick or through exposure to the body fluids of a viraemic animal or CCHF patient. (Bente et al.,

2013)

The virus was first encountered in the 12th century in Tadzhikistan (Hoogstraal et al., 1979)

however; CCHFV was first described in 1944 to 1945 in the Crimean peninsula where a large

outbreak of a severe hemorrhagic fever with a fatality rate of 10% and more than 200 people

infected was recorded (Casals et al. 1978; Hoogstraal et al., 1979). Thereafter, CCHFV was

reported throughout the European and central Asian republics through virus isolation from ticks

and animal blood (Hoogstraal et al., 1979) and was designated Crimean hemorrhagic fever virus

(CHFV). Simpson and co-workers (1976) reported that in 1956, CCHFV was, for the first time,

isolated from a patient in Kisangani in the Democratic Republic of Congo (DRC) by a physician

named Ghislaine Courtois and in DRC at the time, the virus was known as Congo virus (CV)

(Hoogstraal et al., 1979). It was only in 1969 when Casals et al., (1969) discovered

indistinguishable antigenic similarities between CHFV and CV and thereafter, the virus were

designated CCHFV (Hoogstraal et al., 1979; Simpson et al., 1976). In South Africa, CCHFV was

4

first encountered in 1981 when it was isolated from the blood of a boy bitten by a tick in the

North West province (Swanepoel et al., 1987) and since then, around 5-20 cases are reported

yearly (Burt et al., 2007). Of the ticks belonging to the Hyalomma spp, the Hyalomma glabrum,

H. marginatum rufipes and H. truncatum are the three most dominant species in South Africa

(Burt et al., 2007). Recently, more cases of CCHFV are reported especially in Turkey where more

than a 1000 cases are reported and confirmed annually since the first outbreak in 2002 (Bente

et al., 2013).

1.1.2. The virus

Structurally, CCHFV has a spherical virion of

approximately 80-120 nanometres (nm)

and a lipid-bilayer envelope (5-7 nm thick)

derived from its host cell. The envelope is

integrated with virus-encoded

glycoproteins forming minor surface

projections of about 5-10 nm long as well

as filamentous and circular nucleocapsid of

200-3000 nm length (Bente et al., 2013;

Whitehouse CA et al., 2004; Casals et al.,

1978). It is a Nairovirus belonging to the

Bunyaviridae family and consists of an

antisense single-stranded RNA (-ssRNA) with three segments of differing sizes, namely small (S)

which is 0.6-0.7 x106 Da, medium (M) 1.5-1.9 x106 Da and large (L) 4.1-4.9x106 Da (Clerks at al.,

1981). Post transcription and translation of each RNA segment (figure 3), the S segment

encodes for the nucleocapsid (NP) (48 x103 – 54 x103 mol.wt) and the M segment encodes a

precursor polyprotein for the two envelope glycoproteins (Gp), (GN – 72x103 -84 x103

mol.wt. and GC- 30x103 -40 x103 mol. wt.), (Clerks et al.,1981); which in conjunction with the

nucleocapsid protein; form the three major structural proteins of the virion. The L segment

encodes for the RNA-dependent RNA polymerase (Yu et al., 2012; Bente et al., 2013). Within

Figure 2: CCHFV (Published in Antiviral Research,

Volume 64, Issue 3, December 2004 pages 145-160)

Whitehouse CA et al., 2004

5

the virion, each RNA segment is enclosed in a distinct nucleocapsid and each contains three

copies of the genome (Clerx et al., 1981). In a recent study, Deyde et al. (2006) reported that

within the virus genome, overall genome RNA segment and open reading frame (ORF) lengths

and important motifs are highly conserved. However, they also observed nucleotide variations

of 20, 31, and 22% for the S, M, and L RNA segments respectively and amino acid variation of 8,

27, and 10% for the N, GPC, and L proteins, respectively (Burt et. al., 2011; Deyde et al., 2006)

which indicated substantial genetic and protein diversity. The variation is observed to be higher

in both protein and genome in the M RNA segment which encodes the glycoproteins which in

turn play a major role in virus attachment on different hosts’ cell surfaces, an understandable

distinction. Deyde et al. (2006) further stated that the number of nucleotides in the CCHFV S

segment within different strains, ranges between 1640 and 1686 nucleotides (nt) with the

major ORF from position 56-1504 (i.e. 1449 nucleotides long and encoding for 483 amino

acids.)

Of the three RNA segments, the S segment encodes the viral NP, the major structural protein of

CCHFV. NP is the most antigenic and highly conserved CCHFV protein and therefore the target

protein for preparation of recombinant laboratory diagnostic reagents (Yu et al., 2012; Burt et

al., 2011). Apart from its packaging of viral genomic RNA, NP also has a number of other

functions essential for viral proliferation. Yu et al., (2012) constructed the structural analysis of

CCHFV NP revealing a monomer, racket-shaped with the “head” domain and “stalk” domain

(consisting mainly of alpha-helices) as the principal parts and 40 x 50 x 95 Å dimensions.

6

Figure 3: Illustrates the Transcription and translation of CCHFV RNA segments. The S segment encodes

for the nucleocapsid protein (NP), M segment for the polyprotein which is further cleaved into

glycoprotein precursors and eventually glycoproteins Gn and Gc. Finally. The L segment which leads to

the production of RNA-dependent RNA polymerase. Bente et al., 2013

1.1.3. Transmission and Epidemiology

Transmission of CCHFV is maintained both vertically, within the principal vectors life cycle and

horizontally between ticks and certain mammalian vertebrates. Even so; the H. marginatum

marginatum ticks of the genus Hyalomma, are the most important vectors for CCHFV

transmission (Hoogstraal et al., 1979). They facilitate the vertical transmission of CCHFV within

the ticks by maintaining it throughout their developmental phases (both transovarial and

transstadial) enabling its replication and ability to carried on to the next generation during

reproduction (Bente et al., 2013). Developmental stages of the ticks are highly dependent on

climate conditions especially hot and humid conditions, which is why most, if not all outbreaks

can be associated with warm seasons (Bente et al., 2013; Ergunol et al., 2006). This is the period

when virus transmission to humans as well as animals occurs (horizontal transmission).

Transmission to humans may be via direct bite from a viraemic tick, direct contact with blood or

infected tissues from viraemic animals as well as direct contact with the blood or secretions

from CCHF patients, especially in hospitals; a condition called nosocomial infection (

7

Nabeth et al., 2004; Drosten et al., 2003, Saijo et al., 2002). In 1979, Hoogstraal and colleagues

documented CCHFV identified either serologically or directly from a number of vertebrate

animals such as cattle, sheep, goats, hares, ostriches and hedgehogs which in turn develop a

transient viraemia. CCHFV has also been isolated from 31 more species of ticks in addition to

the H. marginatum marginatum, nonetheless; the H. marginatum marginatum, distribution

proved to coincide with CCHFV prevalence hence the principal vectors and true natural

reservoirs of CCHFV (Hoogstraal et al., 1979; Ergunol et al., 2006). Farmers living in endemic

areas, medical personnel, veterinarians, and abattoir workers have a high at risk at of infection

CCHFV is a widely distributed virus (Chamberlain et al., 2005) whose first outbreak was

observed in Crimean region of Russia in the 1944 and until now it is reported in various parts of

the world (Yilmaz et al., 2009; Maltezou et al., 2010). Its prevalence is high in Africa (especially

in the Sub-Saharan Africa countries), the Middle East, eastern and southern Europe and Asia.

CCHF outbreaks have also been documented in the southern regions of European Russia, in

Mouldova, Ukraine and Transcaucasus, and in Central Asian countries; China, in Tajikistan,

Turkmenistan, Uzbekistan, Kyrgyzstan and Kazakhstan regions of the former Soviet Union

(Yashina et al., 2003, Hoogstraal et al., 1979). Fig. 5 depicts CCHFV distribution documented via

virological or serological evidence, however; it should be considered that statistics are

dependent on time of research. Various factors influence CCHFV epidemiology including

weather conditions (preferably tropical), migration of birds and people between countries,

livestock trading and most importantly the distribution of their principal vector ticks of the

Hyalomma spp, hence why the virus prevalence is high in areas where ticks are abundant

(Hoogstraal et al., 1979; Kampen et al., 2007).

8

Figure 4: The geographic distribution of CCHFV within which countries shaded yellow represents

countries with CCHF virological or serological evidence as well as vector presence while in those colored

grey; only vector presence has been documented.

http://www.who.int/csr/disease/crimean_congoHF/en/ [21/09/2013]

1.1.4. Clinical Signs and Symptoms

In infections due to viral hemorrhagic fever viruses (VHFV), clinical symptoms are very similar

regardless of the causative agent at the time and this makes diagnosis not very easy; especially

in the acute phase of infection (Drosten et al., 2003). Following the incubation period, the

infection manifest with a sudden onset of high fever, severe headache, chills, myalgia,

backache, gastrointestinal symptoms, nose bleeds and vomiting. Patients can also develop non-

localized abdominal pain and diarrhoea with feelings of confusion and aggression as the disease

progresses as well as pulmonary oedema (Burt et al., 2007). During the late phase of infection,

the symptoms are more specific with the most prominent symptom being organ manifestation

and failure, bloody urine, swollen liver, kidney failure and a haemorrhagic state; the hallmark of

CCHFV infection, commonly with a fatal outcome (Drosten et al., 2003; Hoogstraal et al., 1979).

Given the severity of the virus infection, suspected patients have to be immediately isolated

while diagnoses occurs. No vaccine against CCHFV has been developed yet, however an anti-

viral drug interfering with RNA metabolism required by the virus for replication called ribavirin

9

can be administered therapeutically. The therapeutic efficiency of ribavirin against CCHFV

infection is dependent on a quick diagnosis (Tasdelen et al., 2009). Death commonly happen

from days five to fourteen of illness but patients who recover may begin to improve on day nine

or ten even though some symptoms such as conjunctivitis and slight confusion may last for a

month or more (Burt et al., 2007).

During the first days of infection clinical pathology can be used to observe the major hallmark in

CCHFV infections as well as other hemorrhagic fever virus infections. This includes changes in

liver enzymes levels and any cellular and/or chemical composition from patients’ blood samples

including leucopenia or leukocytosis, thrombocytopenia and elevated levels of liver enzymes

such as aspartate transaminase (ASL) and alanine transaminase (ALT) (Burt et al., 2007; Ergonul

et al., 2006; Drosten et al., 2003) with the former being higher than the latter as well as lactic

dehydrogenase (LD) and more.

1.1.5. Diagnosis

CCHFV is classified under BSL-4 pathogens requiring containment level four facilities for

laboratory and animal work with the live virus. Isolation of the virus from patients’ tissues or

blood samples also requires BSL-4 laboratory. The most sensitive method for CCHFV isolation is

intracerebral inoculation of suckling mice (or 24 hour old mice) however, this procedure takes

more time to obtain diagnostic results. On the other hand, infection of susceptible mammalian

cells such as VERO cells, Hela cells and baby-hamster kidney (BHK) cells with the virus is a more

efficient procedure for quick isolation of the virus (Burt et al., [manuscript]). Mammalian cells

can also be transfected with an expression vector carrying the nucleocapsid protein (NP) of the

virus. Transfected mammalian cells can then be used in sero-diagnostics to test for anti-CCHFV

antibodies in patient’s blood via an indirect immunofluorescence assay (IFA) (Burt et al., 2011;

Saijo et al., 2002). Serodiagnosis is possible as the infection progresses. From day five since

onset of illness, IgG and IgM antibodies can be detected from patients’ blood via an IFA or an

enzyme-linked immunosorbent assay (ELISA) (Vanhomwegen et al., 2012, Saijo et al., 2005,

Drosten et al., 2003). In patients that die before seroconvertion, diagnosis is post mortem via

histopathology using liver biopsies. However, survivors demonstrate detectable antibodies

10

latest in day nine post infection. IgG remain detectable in sera for a longer period as opposed to

IgM, whose titer declines and fades away within months.

1.1.5.1. Biological specimen selection

A number of factors influence the type of specimen to be used during diagnosis such as time for

sample collection. This is dependent on whether the samples are collected before or after

death. In non-fatal cases blood is collected while in fatal cases where patients died before

seroconversion, tissue biopsies (especially liver) are preferable samples.

1.1.5.2. Sample Processing

CCHFV can cause human-to-human transmissions and laboratory acquired infections with a

high mortality. However, there is still no vaccine against the virus and as a result it is a BSL-4

pathogen requiring BSL-4 laboratories. CCHFV is an important virus and it is vital for its

diagnosis to be quick and specific but in safe and appropriate environments. BSL-4 laboratories

are scarce however; there are several techniques used by laboratories without BSL-4 facilities

to inactivate the virus before handling, making it safer to work with. These techniques include

heat, gamma irradiation, Triton-X100 as well as use of certain disinfectants such as sodium

hypochlorite, formaldehyde and other lipid solvents that CCHFV is susceptible to.

1.1.5.3. Diagnostic techniques

Diagnostic assays for CCHFV include virus culture, antigen-detection enzyme immunoassay

(EIA), antibody-detection EIA, and reverse transcription–PCR (RT-PCR). Virus detection is the

main diagnostic method in the acute stage of disease and the most sensitive method is RT-PCR

(Saijo et al., 2002). In general CCHFV diagnostic approaches involve direct and indirect

techniques that include viral isolation in cell culture, viral genome or virus nucleic acid

detection through molecular techniques as well as detection of antibodies through

serodiagnostic procedure such as IFA or ELISA. Each approach however, is dependent on the

phase or stage of infection upon patient or sample collection whether it is during the acute

phase or the convalescent phase.

11

Histopathology is a diagnostic technique applied to post-mortem samples from patients and

uses needle biopsies of liver samples. Liver samples examination demonstrate lesions of

different forms such as disseminated foci of necrosis and massive necrosis as well as

hemorrhage of variable degree (Burt et al., 2007) and it can be used in conjunction with

immunohistochemical staining to detect the antigen directly.

1.1.5.4. The acute phase of illness

Diagnosis is mainly by viral nucleic acid detection, detection of antigen and virus isolation and

at the acute phase of infection antibodies have not yet reached a demonstratable threshold.

Real time RT-PCR is the molecular technique preferable for CCHFV diagnosis during the early

stages of illness before a detectable antibody response is developed and in patients who die

before seroconversion (Ergönül et al., 2006). The method has certain advantages such as rapid,

specific and sensitive which implies it can be a reliable and accurate method with the ability to

detect even small amounts of viral nucleic acid within a specimen. RT-PCR therefore enables

virus identification and confirmation through viral nucleic acid detection in serum

(Vanhomwegen et al., 2012; Drosten et al., 2003). RT-PCR also allows rapid quantification of

viral load and a viral load more than 1x108 RNA copies/ml may be indicative of a fatal outcome

(Wölfel et al., 2007). The technique is based on the principle of the amplification of a highly

conserved region of the gene (Yu et al., 2012).

ELISA confirms CCHFV diagnosis through the demonstration of the presence of viral antigen in

patient serum (Saijo et al., 2002, Vanhomwegen et al., 2012). ELISA has demonstrated a low

sensitivity especially during an acute phase of infection. Nonetheless, it is a less complicated

and user friendly method whose results are easy to interpret (Burt et al., 2011). CCHFV can also

be isolated in cell cultures using mammalian cell lines such a Hela, CER and Vero cells, in which

it propagates better in Vero cells (Saijo et al., 2002). Mostly virus confirmation in cell culture is

through examination and comparison of cytopathic effects (CPE) but because CCHFV is poorly

cytopathic, there may not be any morphological changes observed. In this case an IFA is the

alternative test for the detection and identification of the virus (Burt et al., 2011, Saijo et al.,

2002, Casals et al., 1978) upon which antigen-antibody complex is visualized through

12

fluorescent staining and is observed under a microscope. This is a quicker method of virus

isolation as opposed to isolation in mice because results may be obtained within one to five

days as compared to five to eight days or even more from mice inoculation (Burt et al.,

[manuscript]). Nonetheless; virus isolation in mice is more sensitive and vital during the

isolation of viruses at low concentrations (Burt et al., 2011) but not ideal for quick diagnosis.

1.1.5.5. The convalescent phase of illness

The convalescent phase of illness occurs mainly in survival patients and it starts around 15-20

days since onset of illness (Whitehouse et al., 2004). Antibodies can be detected during this

time. Serodiagnosis is then possible and the tests detect CCHFV-specific IgM, or a rise in IgG

titers in paired acute and convalescent sera. IgG and IgM antibodies can be found with indirect

IFA or ELISA after at least 7-9 days of illness. Of the two important Igs, IgM is short lived

(undetectable at around 5 months) while IgG can last a life time in some cases. IFA uses

fluorescein-labeled anti-human immunoglobulin conjugates to detect IgG and IgM activity on

CCHFV infected cells fixed onto an indirect IFA antigen slide. Results are observed under a

fluorescent microscope (Saijo et al., 2002, 2005).

Of the two tests, IFA and ELISA, the former is more advantageous than the latter. It is more

rapid and sensitive in detecting an immune response to CCHFV even though they both are

capable of differentiating IgG and IgM (Burt et al., 2011). They both facilitate CCHFV diagnosis

in laboratories without BSL-4 facilities since they eliminate virus culturing. Seroconversion

within patients’ blood demonstrates whether the infection is recent or current, a four-fold or

greater IgG increase in paired samples or IgM in a single specimen (Burt et al., 2011). In the

past, there were other serologic tests such as complement fixation and hemagglutination

inhibition which were used to diagnose CCHFV, but due to their observed lack of sensitivity the

tests are not as common now. Figure 5 is a representation of CCHFV infection cycle

demonstrating periods (phases of infection post onset of illness) in which certain diagnostic

tools can be applied. Represented are symptoms experienced along with every phase thereby

summarizing CCHFV infection events with more detail on the course of liver enzymes and

platelets counts in fatal cases (Weber et al., 2008). Certain facts also need to be considered

13

during diagnosis especially the fact that all hemorrhagic fever causing viruses like CCHFV, yellow

fever virus and Rift Valley fever virus have very similar clinical manifestation signs and

symptoms (Drosten et al., 2003). It is therefore important to have diagnostic assays that are

specific enough for one antigen and can quickly differentiate the viruses. On the other hand,

knowledge of the virus geographical distribution, transmission vectors and incubation period

can also make it possible to rule out a number of other related viruses thereby speeding up the

diagnosis.

Figure 5: Clinical and laboratory course of CCHFV infection, symptoms at different phases, laboratory

analysis of liver enzymes and platelets count (PLTs) especially in fatal cases. (Weber et al., 2008)

http://www.sciencedirect.com/science/article/pii/S1359610108000762

1.1.6. Management and treatment

CCHFV is a BSL-4 classified virus meaning it is highly pathogenic and requires careful and fully

trained personnel to handle it to avoid infection or transmission. Consequently, early diagnosis

is critical for patient therapy and prevention of potential nosocomial infections especially when

looking at hospitals (Saijo et al., 2002). Supportive therapy is the most essential part of case

management. In this case, a replacement therapy with blood products such as thrombocytes,

fresh frozen plasma and erythrocyte preparations are administered after daily check of patient

14

blood count (Jain et al., 2011). Patient isolation to avoid secondary infection is important as

well as emphasis on strict protective measures or barrier precautions practice by health

workers, when treating patients with hemorrhages from the nose, mouth, gums, vagina, and

injection sites. Currently there is no vaccine available for CCHFV, and the most universal

treatment for CCHFV infection is the broad-range anti-viral compound Ribavirin (Maltezou et

al., 2010, Tasdelen et al., 2009). Due to the lack of treatment options, better understanding of

CCHFV molecular and cellular biology is essential as it will lead to an improvement in the

development of new strategies for treatment of CCHFV infections.

1.2. 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 very limited.

Consequently, there is a need for safe diagnostic reagents which will make CCHFV diagnosis

possible even in laboratories without BSL-4 facilities.

1.3. 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 Crimean-Congo hemorrhagic

fever virus.

1.4. Objectives

1.4.1. 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.

1.4.2. Monitor protein (NP) expression to identify positively transfected cells with an IFA

and SDS-PAGE

1.4.3. Determine appropriate G418 concentration for selection of stable transfectants

1.4.4. Using G418 selection media, create a stable cell line that constitutively expresses

CCHF NP.

15

Chapter 2

2. Methods and Materials

2.1. Introduction

During preparation of a stable cell line, the gene of interest has to be cloned into the expression

vector appropriate for protein expression in mammalian cell line and its expression confirmed.

The gene encoding the nucleocapsid protein (NP) of CCHFV (the ORF of S RNA segment) was

previously amplified and cloned into pcDNA ™3.1D/V5-His/TOPO® (expression vector). The

construct was designated pcDNA 3.1TOPO-CCHFVNP and stored as a glycerol stock. In this

study, the presence of the gene was confirmed and the construct used for transfection studies.

This chapter focuses on a number of topics that include transformation of competent

OverExpress C43 (DE3) cells (Lucigen, WI, USA), plasmid purification with two different plasmid

purification kits, double restriction enzyme digestion for confirmation of positive

transformation with results by separation of products by electrophoresis on a 1% agarose gel

and preparation of a 15% glycerol stock from bacterial transformation cultures. Furthermore,

configuration of appropriate seeding densities for mammalian cell transfection with three

different transfection reagents, an IFA and SDS-PAGE gel to determine protein expression and

titration of neomycin (G418) (Life Technologies, NY, USA) concentration in a 96-well plate to

determine an appropriate concentration that will kill untransfected mammalian cells and in

turn be used for selection of positive transfectants during a stable cell line development; will be

discussed.

Plasmids (as expression vectors) can be described as naturally occurring double-stranded

circular DNA sequences extracted from bacteria, possessing the ability to replicate

independently of the bacterial genome henceforth essential for introduction of DNA fragment/

gene of interest into cells. A number of plasmids/ vectors capable of expression in mammalian

cells are available; however the choice of the appropriate vector to use is based on the

necessary features it entails and their importance in the study. The expression vector pcDNA

™3.1D/V5-His/TOPO® (5.5 kb) entails a number of features including antibiotic resistance

encoding sequences specifically SV40 early promoter and origin enabling efficient, high-level

16

expression of the neomycin (G418) resistance gene and episomal replication in cells expressing

it (SV40); which in turn is essential for selection of stable transfectants in mammalian cells. Not

only that; it also has the ampicillin (β-lactamase) resistance gene whose function is mainly for

selection of the vector expressing E. coli cells as well as a pUC-derived origin which allows high-

copy number replication and growth of the plasmid in E. coli cells. In addition, there is the

human cytomegalovirus (CMV) immediate early promoter whose function is to allows efficient

constitutive expression of the gene of interest in a variety of mammalian cells and a number of

restriction enzyme sites such as BamH1 and Not1; the two vital in this study, as well as other

features represented on figure 6 below.

Figure 6: pcDNA ™3.1D/V5-His/TOPO expression vector map; with all important features such as

restriction sites, T7 promoter, antibiotic resistance genes and more indicated.

17

2.2. Preparation of a 15% Glycerol Stock

In the previous study, the CCHFV NP gene had been cloned into pcDNA ™3.1TOPO, named

pcDNA 3.1TOPO-CCHFVNP and stored as a glycerol stock. In this study, the plasmid was

cultured, purified and the presence of the gene and its correct orientation confirmed prior to

transfection experiments. Sequence analysis had been performed previously.

2.3. Transformation of competent OverExpress C43 (DE3) E. coli cells using a heat-

shock method

Competent cells as described by Brown (Brown, 2007); refers to bacterial cell culture that has

been treated to enhance their ability to take up DNA molecules. This process is vital in bacterial

cell transformation as DNA uptake is not a constitutive characteristic of bacteria as a result,

certain methods either physical or chemical are employed to render them transiently

permeable to the plasmid. Of the methods, incubation of E. coli cells in a solution of ice-cold

50mM calcium chloride (CaCl2) is the most preferred. The salt precipitates the plasmid/DNA

onto the outside surface of the cells or alters the cell wall to improve the DNA binding onto the

surface which is then “transported” into the cell cytoplasm and hence transformation when

heat shocked for a few seconds in a water bath at 42°C.

A 100µl aliquot of chemically competent OverExpress C43 (DE3) cells was thawed on ice and

inoculated with 1µl aliquot of pcDNA 3.1TOPO- CCHFVNP. The mixture was incubated on ice for

20 minutes (min). Cells were heat-shocked in a water bath at 42°C for 50 seconds (sec) and then

back on ice for 2 min. An aliquot of 900 µl pre-warmed super optimal broth with catabolite

repression (SOC) media (see appendix 1) was added to the cells, mixed and incubated for 90

min at 37°C, with shaking at 200 rpm. The transformation culture was then plated out onto

Luria Bertani/ampicillin (LB/amp) plates (see) and incubated overnight (o/n) at 37°C. The cells’

transformation efficiency was determined using a control plasmid (pUC19) supplied with the kit

and calculated (see appendix 6) to be 4.5x108 cfu/µg of DNA. Thereafter, single colonies were

numbered and selected for a small-scale o/n plasmid preparation. Two colonies were selected,

inoculated into a 5ml LB/amp broth and incubated o/n at 37°C with shaking at 200 rpm.

18

One of the features of pcDNA ™3.1D/V5-His/TOPO® is the bla promoter which enables the

expression of the gene encoding for amp resistance. As a result, positively transformed

OverExpress C43 (DE3) cells will express the amp resistance gene and therefore propagate in LB

media and plates supplemented with ampicillin used for selection of positive transformants.

2.4. Plasmid purification using Pure Yield™ Plasmid Miniprep System (Promega, WI,

USA)

The transformation cultures from the over-night plasmid preparation were purified using the

quick protocol Pure Yield™ Plasmid Miniprep System (Promega, WI, USA), according to

manufacturer’s instructions.

A 3 ml bacterial culture procedure was followed. To pellet the bacterial cells, cultures were

centrifuged at a maximum speed of 16000 x g (applied in all centrifugation steps). The pellet

was re-suspended via pipetting in 600µl of distilled water (dH2O). Into the suspension, a 100µl

aliquot of cell lyses buffer was added mixed, then a 350µl aliquot of pre-chilled (4°C)

neutralization buffer followed by a thorough mix and centrifugation for 3 min. The supernatant

was transferred into the PureYeild™ Minicolumn inserted into a collection tube, centrifuged for

15 sec and the flow-through discarded. To wash, an aliquot of 200µl endotoxin removal wash

mix was added into the column and centrifuged for 15 sec followed by 400µl of column wash

solution and centrifugation at similar conditions. Thereafter, the DNA was eluded from the

column with a 30µl aliquot of nuclease free water into a clean 1.5 ml micro centrifuge tube via

a 15 sec centrifugation. The DNA concentration was determined and then stored at -20°C. (All

reagents used were provided with the kit.)

2.5. Restriction enzyme digestion reaction for confirmation of positive transformation

To confirm positive transformation and insertion of the CCHFV-NP encoding gene in pcDNA

™3.1D/V5-His/TOPO®, a double restriction digestion reaction was carried out. Restriction

enzymes are enzymes with the capability to cleave DNA at specific restriction sites producing

short DNA fragments called restriction fragments. Each enzyme has a unique specificity for a

certain number of nitrogenous base pair sequence and their application can be used in cloning

19

and verifying DNA sequences. BamH1 and Not1 (Promega, WI, USA) were used in the study, of

which both are type II restriction enzymes, meaning they recognize the same restriction site of

6 and 8 base pairs respectively (fig 7). Buffer E (Promega, WI, USA) within which BamH1 activity

is 100% while Not1 activity is 25-50% was used (www.promega.com, Restriction Enzyme

Activity in Promega 10X Buffers, Reaction Temperature and Heat Inactivation). The cloning site

of pcDNA ™3.1D/V5-His/TOPO® is flanked with, among others, Bam1 and Not1 restriction sites

and as a result the two enzymes were selected to excise the NP gene. Table 1 depicts the

preparation of the restriction enzyme digestion reaction mixture. After a quick spin, the

reaction mixture was incubated in a water bath at 37°C for 2 hours (hrs). Two control reactions

of single restriction digestion of the plasmid with BamH1 and Not1 respectively were also set up

to linearize the plasmid and confirm its size as well as to confirm that both restriction enzymes

are functional.

Figure 7: Nucleotide base pairs recognized by BamH1 and Not1 restriction enzymes respectively

(restriction sites).

Table 1: Reaction mixture prepared for a double restriction enzyme digestion of pcDNA

3.1TOPO- CCHFVNP with BamH1 and Not1 restriction enzymes:

Reaction constituent Volume (µl)

1 x Buffer E 2

Purified pcDNA 3.1TOPO- CCHFVNP 2

BamH1 (10 U/µl) 1

Not1 (10 U/µl) 1

Nuclease free water 14

Total volume 20

20

2.6. Analysis of restriction enzyme digestion products with 1% agarose gel

electrophoresis

This is an analytical procedure used commonly to separate and analyze DNA fragments

generated by restriction enzymes as well as determining the size of DNA molecules ranging

from 500 to 30,000 base pairs. This technique analyzes or separates DNA molecules by their

speed which is influenced by the molecule size and charge (negative) as well as their ability to

migrate towards the positive pole as a current is applied through the gel.

A 1% agarose gel (see appendix 4) containing ethidium bromide for products visualization was

prepared and set up in Tris-acetate-EDTA (TAE) buffer (see appendix 3) of pH 8.5. Digestion

products of 20 µl aliquots were loaded in 2ul of a 6x blue loading dye (see appendix 3). A DNA

ladder, O’GeneRuler DNA Ladder Mix #MS1173 (Fermentas, Illinois, USA) encompassing DNA

fragments ranging from 100 to 10, 000 bp was used as a standard marker. The process was

performed using a Bio-Rad PowerPac Basic system (Bio-Rad, California, USA) at 80 Volts (V) for

60 min and the products observed using a UV transilluminator. Thereafter, glycerol stocks of

15% glycerol were prepared by aliquoting 850µl of bacterial transformation culture and 150µl

of glycerol into cryotubes (Nunc, Roskilde, Denmark) and stored at -80C.

2.7. Plasmid purification using with QIAGEN® Plasmid Mini Kit (25) (QIAGEN, Germany)

For mammalian cell transfection, plasmid purity is of great importance and the plasmid

purification kit to be used had to guarantee to yield an endotoxin free plasmid for transfection

henceforth the use of the QIAGEN® Plasmid Mini Kit (25) (QIAGEN, Germany) according to

manufacturer’s instructions.

Prior to purification, a small overnight (o/n) bacterial culture was prepared by inoculating pre-

warmed 5 ml LB/amp broth with 10 µl of the glycerol stock and then incubated at 37C with

shaking at 200 rpm. Cells were harvested from the o/n bacterial culture by centrifugation at

6,000 x g for 15 min at 4C. The supernatant was discarded and cell re-suspended in 300µl

aliquot of buffer P1 (containing RNase A and lyseBlue reagent). Subsequent to that, an aliquot

of 300µl buffer P2 was added to the mixture, mixed thoroughly by inversion and then incubated

at room temperature for 5 min. A 300µl aliquot of pre-chilled (4C) neutralization buffer P3 was

21

then added, vigorously mixed and incubated on ice for 5 min and then centrifuged at 16,000 x g

for 10 min at 4C. A Qiagen-tip 20 was equilibrated with 1 ml of buffer QC allowed to flow

through into the collection tube. After centrifugation, the supernatant was applied to the tip

and allowed to flow through. Then 4ml of buffer QC was used to wash the tip by allowing it to

flow through and the DNA was eluted into a clean 1.5ml eppendorf tube with 800µl of QF

buffer. DNA was then precipitated with 560µl of room temperature isopropanol, centrifuged at

16, 000 x g for 30 min at 4C and the supernatant carefully discarded (via pipetting). The pellet

was washed with 1ml room temperature 70% ethanol and centrifuged at 16,000 x g at room

temperature. Then the supernatant was discarded, pellet air-dried on the bench, re-dissolved in

30 µl of nuclease free water and stored at -20C.

2.8. DNA concentration

DNA concentration was determined using the NanoDrop 2000 spectrophotometer (thermo-

scientific, Illinois, USA) with its purity based on the absorbance ratio at 260nm and 280 nm.

2.9. Protein expression in laboratory maintained mammalian cells

Protein folding similarly to all physical and chemical reactions is driven by the necessity to

achieve and maintain a state of minimum thermodynamic free energy with respect to the

surrounding solvent molecules in particular within mammalian cells (Twyman, 2004). For a

protein to be functional, it first has to attain its ideal and correct native conformation and

posttranslational modifications which bacterial cells are not very ideal as some modifications

are unique to mammalian cells henceforth the use of mammalian cells to allow proper folding

which in turn results in proper protein expression. In order to accomplish this, expression

vectors viable for mammalian cells such as pcDNA3.1D/V-His-TOPO possessing the appropriate

features that include mammalian cell promoter along with a polyadenylation are used to attain

correct posttranscriptional management of the gene (mRNA) before translation.

22

2.9.1. Cells and culture conditions

Laboratory maintained Baby hamster kidney (BHK) cells stored as aliquots at passage level six in

liquid nitrogen were used.

BHK cells were propagated according to standard procedures in T75 flasks (75 cm2 cell culture

Corning® flasks) (Corning Incorporated, NY, USA) at 37°C, in pre-warmed Dulbecco's Modified

Eagle Medium (DMEM) (Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum

(FBS) (Delta Bio-products, Johannesburg, SA), 1% penicillin-streptomycin (P/S) (10,000 U/µg per

ml, Sigma, USA), 1% 200mM L-glutamine (Lonza, Verviers, Belgium) and 1% of 100x Non-

essential amino acids (NEAA’s) (Lonza, Verviers, Belgium). Cultures were passaged (1:5) every

48hrs using standard trypsination procedures (see appendix 5) (500µl of 2.5% trypsin Lonza,

Verviers, Belgium).

2.9.2. Determining the appropriate seeding densities for transfection

To determine appropriate seeding densities for transfection of BHK cells, flat-bottomed tubes

(Nunclon™ surface, Nunc, Denmark) or 24-well plates (Nunc, Denmark) were used. Different

seeding densities were used with each. After cells were passaged, a 1:10 dilution of cell

suspension in trypan-blue staining solution was prepared and cells were counted under a

microscope (Olympus CKX41SF) (Olympus Corporation, Tokyo, Japan), using an improved

Neubauer Chamber cell counting method. Within each tube or well, sterile cover slips were

added. Cells were seeded at 2.5x105 and 3.0 x105 cells per tube or 1x105 and 1 x104 cells per well

in 2ml and 1ml DMEM respectively. Cells seeded into tubes were incubated o/n at 37C

whereas plates were incubated in a humidified (±99%) 5% CO2 incubator at 37°C. The

confluence of attached cells (aimed at ±80-90% confluence) was then examined under a

microscope.

2.9.3. Transfection of BHK and Vero cells using different transfection reagents

Different transfection reagents have different transfection efficiencies, different toxicity levels

and their effect may differ with different cell lines. In an attempt to configure which

transfection reagent was more efficient with BHK cells, different transfection reagents were

23

tested. A transfection reagent with at least more than 50% transfection efficiency at a certain

pre-determined DNA: transfection reagent ratio was considered appropriate for BHKs. pSin-GFP

supplied by Prof Huie, was used as a transfection control to optimize transfection conditions

prior to cell transfection with pcDNA3.1TOP-CCHFVNP. During each transfection reaction, a

tube or well of non-transfected BHK cells was set up. This served as a negative control

necessary for distinction of specific from non-specific binding during an immunofluorescence

assay (IFA).

2.9.3.1. Purification of pSin-GFP plasmid with the QIAGEN® Plasmid Plus Midi Kits

(QIAGEN, Germany)

A large scale plasmid preparation was performed, for purification of both pSin-GFP and

pc3.1TOPO-CCHFVNPA using the QIAGEN® Plasmid Plus midi kit, according to manufacturer’s

instructions.

A 5ml o/n small scale plasmid (pSin-GFP) preparation was prepared by inoculating 5ml of 2x TY

broth supplemented with kanamycin (see appendix 1) with 10µl of pSin-GFP glycerol stock and

incubated o/n at 37C with shaking at 200 rpm. Post 24hrs, 50ml of pre-warmed 2xTY/Kan

broth was then inoculated with 50µl of the small scale o/n bacterial culture and incubated

under similar conditions. To pellet and harvest bacterial cells, the bacterial suspension was

centrifuged at 4C for 15 min at 6000xg. The pellet was re-suspended in 2 ml of buffer P1 and

into the solution, 2ml of buffer P2 was added, gently mixed until viscous and incubated at room

temperature for 3 min. A 2ml aliquot of buffer S3 was added to the lysate and mixed by

inversion. A QIAfilter Cartridge was set-up by placing into a 50ml falcon tube and the lysate was

then transferred into QIAfilter and let stand for 10min at room temperature. The cell lysate was

then filtered through a plunger into a falcon tube. An Aliquot of 2ml buffer BB (binding buffer)

was added to the lysate, mixed and transferred to the QIAGEN Plasmid Plus spin columns set-up

on the QIAvac 24 Plus and the liquid eluted with about 300 mbar vacuum. Thereafter, columns

were washed twice with 700µl aliquots of buffer PE with a final centrifugation at 10,000xg for

1min at room temperature. The DNA was eluted with 200µl of nuclease free water. The nucleic

acid concentration was determined using the NanoDrop 2000 spectrophotometer with its

24

purity based on the absorbance ratio at 260 nm and 280 nm. The plasmid concentration was

taken into consideration with less or more volume added accordingly, in order to maintain the

amount of DNA required per transfection reaction. Similar transfection reactions were set up

with both pSin-GFP and pcDNA3.1TOPO-CCHFVNP.

2.9.3.2. Transfection of BHK cells with FuGene®6 transfection reagent (Roche,

Mannheim, Germany)

Flat-bottomed tubes and 24-well plates were set up and cells seeded accordingly (refer to 2.9.2)

24 hrs prior to transfection.

Seeding densities: 3x105 cells/tube

1x105 cells/well

Growth media: DMEM supplemented with 10% FBS, 1% Pen/Strep (10,000 U/µg per ml), 1%

200mM L-glu and 1% of 100x NEAA’s. In each tube and well, added 2ml and 1ml of growth

media was added respectively.

Table 2: Reaction mixtures prepared for each reaction during transfection of BHK cells with

FuGene®6 transfection reagent:

FuGene®6

transfection

reagent: pSin-GFP

ratio

FuGene®6

transfection

reagent volume

(µl)

pSin-GFP plasmid

volume (µl)

*Dilution media

volume (µl)

3:1 0.6 1 18.4

3:2 0.6 2 17.4

6:1 1.2 1 17.8

*Dilution media: serum-free DMEM supplemented with 1% L-glu

The transfection reagent (FuGene®6) was diluted in dilution media and incubated at room

temperature for 5min prior to the addition of plasmid. The plasmid was added, mixed and

incubated at room temperature for 15min to allow the transfection reagent: DNA complex to

form. The cells media was discarded and replaced with transfection media (D-MEM

supplemented with 5% FBS, 1% 200mM L-glu and 1% of 100x NEAA’s, no Pen/Strep). After

25

incubation, the transfection mixture was added drop-wise to respective tubes or wells, mixed

by shaking gently and incubated o/n at 37C, accordingly.

2.9.3.3. Transfection of BHK cells with Lipofectamine™ 2000 transfection reagent

(Invitrogen, Carlsbad,USA)

Cells were seeded accordingly in 24-well plates and flat-bottomed tubes in 1ml and 2ml growth

media respectively. They were then incubated for 24 hours at 37C in a humidified (99%) CO2

incubator. After incubation, transfection was carried out according to manufacturer’s

instructions.

Seeding densities: 3x105 cells/tube

1x105 cells/well

Table 3: Reaction mixtures prepared for each reaction during the transfection of BHK cells

with Lipofectamine™ 2000 transfection reagent:

Lipofectamine™

2000

transfection

reagent: pSin-

GFP ratio

Lipofectamine

™ 2000

transfection

reagent

volume (µl)

Dilution

media

volume

(µl)

Incu

bat

ed a

t ro

om

tem

per

atu

re f

or

15

min

pSin-GFP

plasmid

volume

(µl)

*Dilutio

n media

volume

(µl)

Total

volume/

reaction

mixture

(µl)

2:1 4 50 2 50 106

3:1 6 50 2 50 108

* Dilution media: Opti-MEMM (Life Technologies, UK)

The transfection reagent was diluted in 50µl of Opti-MEM and incubated for 15min at room

temperature. The DNA was also diluted similarly. After incubation, both dilutions were mixed

and incubated at room temperature for 20min to allow the transfection reagent: DNA

complexes to form. Media in cells was changed to transfection media. Post incubation,

transfection complexes were added drop-wise to the cells in tubes and wells, mixed and then

incubated o/n at 37C.

26

2.9.3.4. Transfection of BHK cells with TurboFect transfection reagent

(ThermoScientific, Lithuania)

Cells were seeded as a seeding density of 3x105 cells/tube and 1x105 cells/well in 2ml and 1ml

growth media respectively. They were then incubated in a humidified CO2 incubator at 37C,

24hrs prior to transfection.

Table 4: Reaction mixtures prepared for each reaction during the transfection of BHK cells

with TurboFect transfection reagent:

TurboFect

transfection

reagent: pSin-GFP

ratio

TurboFect

transfection

reagent volume

(µl)

pSin-GFP plasmid

volume (µl)

*Dilution media

volume (µl)

2:1 2 1 100

3:1 3 1 100

*Dilution media: serum-free DMEM supplemented with 1% L-glu

The DNA was diluted in a 100µl aliquot of serum-free dilution media. To the dilution, TurboFect

transfection reagent was added and mixed by pipetting. The transfection mixture was then

incubated at room temperature for 20min. The growth media in cells was discarded and

replaced with transfection media. After incubation, transfection complexes were added drop-

wise to the cells and incubated o/n at 37C.

2.9.3.5. Transfection of BHK cells in a 24-well plate with X-tremeGENE HP transfection

reagent (Roche, Mannheim, Germany)

For transfection, cells were seeded at 1x105 cells/well seeding density in 1 ml growth media.

After 24hrs of incubation, transfection was carried out as follows: three reactions were set up

and within each, 1µg of plasmid DNA was added into a 100µl aliquot of Opti-MEM and mixed.

Each reaction coincided with the three ratios of transfection reagent to plasmid DNA used, 1:1,

2:1 and 3:1. As a consequence, 1µl, 2µl and 3µl of X-tremeGENE HP was added to each reaction

respectively, mixed and incubated at room temperature for 15min. the cells media was

27

discarded and replaced with transfection media. After incubation, transfection complexes were

added to the cells and incubated overnight under appropriate conditions.

2.9.4. Transfection of Vero cells with different transfection reagents

Transfection reagents: Lipofectamine™2000

TurboFect

X-tremeGENE HP

2.9.4.1. Transfection of Vero cells with Lipofectamine 2000 transfection reagent

before transfection, cells were trypsinized, counted and plate out in a 24-well plate at a seeding

density of 8 x 104

cells per well in 500µl of Essential Minimum Eagle Medium (EMEM, Lonza,

MD, USA) supplemented with 10% heat-inactivated FBS (Biochrome AG, Berlin, Germany), 1%

NEAA and 1% L-glu, for a 90-95% cell confluence after 24hrs incubation. For each well, 0.8 μg of

DNA was diluted into 50 μl of Opti-MEM. A 2.0μl aliquot of Lipofectamine 2000 (LF2000)

transfection reagent was diluted in 50μl of Opti-MEM I Medium and incubated for 5 min at

room temperature. The two dilutions were combined and incubated for 20 min at room

temperature to allow DNA- Lipofectamine 2000 reagent complexes to form. The growth

medium was then discarded and replaced with 500µl of serum-free EMEM (with NEAA). The

DNA-LF2000 reagent complexes (100 μl) were then directly added to respective wells and mix

gently by rocking the plate back and forth. Thereafter, the cells were incubated at 37°C in a

humidified CO2

incubator for 4-5 h. Then 500µl of EMEM supplemented with 20% FBS for a final

concentration of 10% FBS was added and the cells incubated in a CO2

incubator with protein

expression monitored 48hrs post-transfection.

2.9.4.2. Transfection of Vero cells with TurboFect transfection reagent

Cells were seeded and incubated as in 2.9.4.1 above. Afterwards, transfection reaction mix of

1µl plasmid/DNA and 2µl of TurboFect transfection reagent in 100µl of serum-free EMEM was

prepared and incubated for 20min at room temperature for transfection complexes to form.

Growth media was discarded and replaced with 500µl of 2% heat-inactivated FBS EMEM (with

28

NEAA). The complexes were then directly added to each respective well in a drop-wise manner

and mixed by gently shaking the plate briefly on a shaker. Cells were then maintained as 2.9.4.1

above.

2.10. Cell processing post-transfection

Following cell transfection and incubation for 24hrs, media was discarded and cells were fixed

with methanol-acetone (1:1) fixative solution at room temperature for a minimum of 20min.

After fixing, cells transfected with pSin-GFP a replicon plasmid expressing a green fluorescing

protein (GFP), were air dried and directly mounted up-side down onto glycerol mounting

solution (see appendix I) on microscope slides (Superior, W. Germany). Cells were then

examined for fluorescence under a fluorescent microscope (Nikon fluorescent, Tokyo, Japan).

2.10.1. Immunofluorescence assay (IFA) for confirmation of positive transfection of BHK

cells with pcDNA3.1TOPO-CCHFVNP plasmid

Cells were fixed with methanol: acetone (1:1) fixative solution at room temperature for a

minimum of 20min. Cover slips were placed on microscopic slides and cells blocked with a

blocking solution (see appendix 2) of 10% sucrose (Merck, Darmstadt, Germany) and 0.5%

triton X-100 (Promega, WI, USA) in 1x Phosphate buffered saline (PBS) (Sigma, Steinem,

Germany), for 20min at room temperature. Triton X-100 is a nonionic surfactant whose purpose

is to permeabilize fixed cells and expose the protein. The primary antibody, human-anti CCHFV

diluted 1:10 in the blocking solution was added to the fixed cells. Slides were incubated in a

tightly closed container, under humidified conditions for 90 min at 37°C. After incubation, cells

were washed three times with 1% TWEEN® 20 detergent (Cal Biochem, Darmstadt, Germany) in

1x PBS (see appendix 2). TWEEN® 20 detergent removes all unnecessary particles that would

cause non-specific binding. Cells were then reacted with the fluorescein isothiocyanate (FITC)-

conjugated goat anti-human IgG and incubated under similar conditions for 30 min. Thereafter,

another wash was performed. Following that, cells were briefly air dried, mounted upside-down

onto glycerol mounting solution on microscope slides and examined for green fluorescence

under a fluorescent microscope.

29

2.10.2. Protein extraction and expression via sodium dodecyl sulphate polyacrylaminde

gel electrophoresis (SDS-PAGE) gel

To confirm expression of CCHFV NP, cell lysates were separated using an SDS-PAGE. SDS-PAGE

separates proteins in accordance to size and in order to attain that, all proteins have to

configure a similar charge (negative). The SDS therefore disrupts all protein structures resulting

with a linearized polypeptide chain covered with negatively charged SDS molecules.

Cells were seeded at 1x105cells/well in 24-well plates, transfected and incubated for 24 hrs (see

2.9.3.) before harvesting. Cells were then harvested and protein extracted as follows:

transfection media was discarded and cells briefly rinsed with 1ml PBS without Ca2+/Mg2+. PBS

was discarded and cells trypsinized with a 500µl aliquot of trypsin and incubated for 5min at

37°C to allow cell detach. Thereafter, 100µl of DMEM supplemented with 5%FBS, 1%L-glu and

1%NEAAs’ was added and mixed to inactivate trypsin. The suspension was transferred into a

1.5ml eppendorf tube and then centrifuged for 5min at 6,000g, at 4°C. Afterwards, the

supernatant was discarded, pellet re-suspended in 50µl NET/BSA (see appendix 2)

complemented with complete protease inhibitor cocktail (Roche, Mannheim, Germany) and

then incubated on ice with shaking for 30min and centrifuged for 15min at 10,000g at 4°C. The

supernatant was transferred to a clean pre-chilled (-20C) eppendorf tube. The pellet was re-

suspended in another 50µl of Net/BSA/protease inhibitor cocktail solution and the protein

extract sample thereafter stored at -20°C.

Transfection reactions were set up for BHK cells with pSin-GFP and pcDNA3.1TOPO-CCHFVNP

using Lipofectamine 2000 and TurboFect transfection reagents. Untransfected BHK cells were

used as a negative control.

For an SDS-PAGE gel, 10% resolving gel and 4% SDS-PAGE stacking gel were prepared (Tables 5

and 6 respectively). A 15µl aliquot of each protein extract sample (both the pellet and

supernatant), was added to 6µl of 5x SDS-PAGE protein loading buffer (Thermo Scientific, USA)

with 0.313 M Tris-HCl, pH 6.8, 10% SDS, 0.05% bromophenol blue and 50% glycerol, and heated

at 95°C for not more than 10min. Aliquots of 20µl of each prepared sample were then loaded

into respective wells along with a Spectra Broad Range Pre-stained protein marker (Fermentas,

Illinois, USA) encompassing proteins of size between 10 and 260 kDa. The gel was run at 140V

30

for 1hr in 10x bath buffer (Biorad, California, USA) on the mini-PROTEAN Tetra Cell (Biorad,

California, USA). The gel was then stained with Coomassie Brilliant blue stain (see appendix 2)

o/n with shaking and distained with a gel destaining solution (see appendix 2). Thereafter, the

gel was examined for bands corresponding to CCHFV NP, ≈53kDa and GFP, ≈26.9kDa. The gel

was also executed with a higher protein extract volume (35µl in 10µl loading dye). To dry the

gel, it was soaked briefly in a solution of 30% methanol and 3% glycerol (30ml and 3ml

methanol and glycerol respectively, toped up to a total volume of 100ml with dH2O).

Thereafter, the gel was placed on a piece of filter paper, covered with glad-wrap and eventually

on a dryer at 70°C with applied vacuum for 3hrs.

Table 5: Preparing a 10% resolving gel for an SDS-PAGE:

Reagents Volume per reaction (ml)

30% acryl amide solution/8% bisacrylamide stock

solution (Merck, USA)

2.667

3M Tris-HCl pH 8.8 1.065

dH2O 4.267

10% SDS 0.20

freshly prepared 10% APS 0.05

TEMED (tetramethylethylene diamine) 0.01

Table 6: Preparing a 4% stacking gel for an SDS-PAGE:

Reagents Volume per reaction (ml)

30% acryl amide solution/8% bisacrylamide

stock solution (Merck, USA)

0. 40

0.5M Tris-HCl pH 6.8 0.210

dH2O 2.37

10% SDS 0.03

freshly prepared 1% *Ammonium persulphate

(APS)

0.06

*TEMED 0.006

*TEMED (Merck, Darmstadt, Germany) is added just before addition of each gel because it

promotes gel polymerization. *1% APS (Promega, WI, USA): 0.05g APS in 500µl of dH2O.

31

2.11. Preparation and use of different mounting solutions to solve problems

encountered during visualization of fluorescent cells under fluorescent electron

microscope

During IFA assays, certain difficulties were encountered during cells examination under a

fluorescent microscope which included bubble formation and a blur that inhibited cell focus.

The standard glycerol mounting solution was re-prepared as a precaution and a glucose syrup

mounting solution was also prepared.

2.11.1. Preparation a glycerol mounting solution

A 1:10 dilution of 1XPBS and glycerol was prepared and mixed well. The pH of the solution was

adjusted to 8.6 through a drop-wise addition of 0.1M sodium hydroxide (NaOH) and stored at

4C.

2.11.2. Preparation of “glucose syrup” mounting solution

15 g of glucose powder was added in 5ml of water and left in a water bath at 62°C till glucose

had completely dissolved forming clear syrup. Sticky syrup was formed and kept at room

temperature.

2.12. Titration of Neomycin (G418) (Life technologies, NY, USA) in a 96-well plate (Nunc,

Denmark)

One of the features of the pcDNA3.1TOPO is a neomycin resistance gene, the G418 conveyed

by geneticin (Life technologies, NY, USA). Prior to transfection, the concentration of the

selective drug (G418) required to kill untransfected cells but allow neomycin resistance

expressing cells to proliferate, was determined. BHK cells stably transfected with cpDNA

3.1TOPO-CCHFVNP plasmid thereby express the resistance gene. On the other hand, cell

susceptibility to antibiotics such as G418 differs and as a consequence, their survival rate

depends on the amount of G418 in the medium and at a certain concentration, cells can stop

proliferating and die. This concentration is vital during the selection of a stably transfected cell

line. The G418 concentration just above (0.1-0.5mg/ml) that which shows complete cell death

was considered for use.

32

In the process, a 100µl aliquot of growth media (DMEM) was added in each well. An aliquot of

100µl cell suspension of 1x105 cells was then added to each well of the first column. Thereafter,

a 2 fold dilution was made across the plate by pipetting 100µl from each well into the next

(next column). From the last well (column 12) of each row, a 100µl aliquot was discarded. To

the first raw (A) a 100µl aliquot of G418 containing media (5.6µl of G418 in 72.8µl media) was

added, to the final G418 concentration of 1.4mg/ml. In the following rows (B-G), G418

concentration decreasing in steps of 0.2mg/ml (1.2, 1.0, 0.8, 0.6, 0.4 and 0.2mg/ml) (see

appendix 6) was added and in the last row (H), was set up as a negative control without G418

(fig 7). By gently tapping the plate on the sides, the cells were mixed with neomycin, the plate

silt well and incubated at 37°C in a 5% CO2 incubator with saturated humidity -95-99%. Cell

death was monitored and recorded daily over a period of four days by observing the plate

under a microscope. The procedure was done in duplicate.

Figure 8: The matrix titration of neomycin (G418) concentration and cell number to determine the

concentration resulting with complete cell death within a number of days (4) per cell number. This

concentration was used for selection of stable transfectants.

33

2.13. 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 and incubated as explained earlier.

After 24 hours of incubation, the 2% FBS growth medium was discarded and replaced with 2%

FBS growth media supplemented with G418 (1.2mg/ml) (selection media). After six days, BHK

cells transiently/stably expressing NP (that remained attached) were trypsinized (20µl) and

transferred into six well plates with 2ml selection media and incubated accordingly. Four days

later, cells were trypsinzed, cultured in T25 flasks and an IFA performed in 8-well multitest slides

(flow tissue culture glassware, flow laboratories, UK) to determine protein expression. Selection

media was changed every 48hrs. Stably transfected BHK cells were then maintained in growth

media supplemented with 1.2mg/ml G418.

34

Chapter 3

3. Results

3.1. Transformation of competent OverExpress C43 (DE3) E. coli cells using a heat-

shock method

The transformation efficiency refers to the effectiveness of the bacterial cells to take up a

foreign DNA plasmid and express its proteins. The transformation efficiency of OverExpress

C43 (DE3) cells was determined with a control plasmid, pUC19 and was calculated to be 4.5x108

cfu/µg of DNA.

3.2. Plasmid purification and DNA concentration

The plasmid was purified more than once and with different plasmid purification kits (Table7).

Both pcDNA3.1TOPO-CCHFVNP and pSin-GFP plasmids were purified.

Table 7: The number of times within which the plasmid was purified and the nucleic acid

concentration at each occasion

Plasmid Plasmid purification kit Nucleic acid concentration

Comment

pcDNA3.1TOPO-CCHFVNP Colony I

Pure Yield™ Plasmid Miniprep System

270.1 ng/µl

Good yield indicative of positive transformation but not appropriate for mammalian cell transfection (not endotoxin free).

QIAGEN Plasmid Mini Kit (25) 22.6 ng/µl Very low for mammalian cell transfection.

QIAGEN Plasmid Mini Kit (25) 4.7 ng/µl Very low for mammalian cell transfection.

QIAGEN Plasmid Mini Kit (25)

17.2 ng/µl

Very low for mammalian cell transfection.

pcDNA3.1TOPO-CCHFVNP Colony II

Pure Yield™ Plasmid Miniprep System

298.7 ng/µl

Good yield indicative of positive transformation.

QIAGEN Plasmid Mini Kit (25)

997.4 ng/µl

Excellent yield indicative of positive transformation.

QIAGEN® Plasmid Plus Midi Kits

390.1ng/µl Average yield

QIAGEN® Plasmid Plus Midi Kits

966.0ng/µl

Excellent yield and good for mammalian cell transfection.

QIAGEN® Plasmid Plus Midi Kits

2021ng/µl

Excellent yield good for mammalian cell transfection.

pSin-GFP QIAGEN® Plasmid Plus Midi Kits

1029 ng/µl Good yield

Plasmid purified with QIAGEN Plasmid Mini Kit (25) from colony I glycerol stocks always

resulted with a very low concentration and could not be used for mammalian cell transfection

studies. However, restriction enzyme digestions of Pure Yield™ Plasmid Miniprep System

purified plasmid (fig 7) showed positive transformation with pcDNA3.1TOPO-CCHFVNP (3.3).

After this was repeated a number of times, colony I glycerol stock was discarded and colony II

cultured and plasmid purified similarly. After a high yield with Pure Yield™ Plasmid Miniprep

System (table 7), a double digestion was performed, which proved positive transformation and

insertion of the NP. Plasmid was then purified with QIAGEN Plasmid Mini Kit (25) and QIAGEN®

Plasmid Plus Midi Kits for a small scale and large scale plasmid purification procedures. In both

occasions, the nucleic acid concentration was high and recommended for mammalian cell

transfection studies. Figure 7 is an illustration of how pure plasmid curve should be.

36

Figure 9: Plasmid purification results with QIAGEN® Plasmid Mini Kit (25). Sample ID is pcDNA3.1TOPO-

CCHFVNP, sample 3 water: to clear out bubbles that may cause discrepancies on results. Sample 3 and 4:

nucleic acid concentration of the purified plasmid measured in duplicate. The table shows nucleic acid

concentration and purity at A260/280.

3.3. Restriction enzyme digestion reaction for confirmation of positive transformation

Positive transformation of OverExpress C43 (DE3) cells with pcDNA3.1TOPO-CCHFVNP was

confirmed via a double restriction enzyme digestion of plasmid purified from the overnight

bacterial cultures using two restriction enzymes that flank the clonal insertion site of

pcDNA3.1D/V5-His-TOPO; Not1 and BamH1. After the gel was run and observed under UV

transilluminator, results analyzed. As seen on fig 8, lane two and three are representative of

positive transformation and insertion of the correct gene of interest into the plasmid. In both

lanes, the first and second bands represent CCHFV NP encoding gene of ≈1.5 kb and

pcDNA3.1D/V5-His-TOPO of ≈ 5.5 kb respectively. Lane four and five represent linearized

plasmid, product of single digestion of plasmid with Not1 and BamH1 only respectively, ≈ 7020

Sample 1 (H2O)

Sample 2

Sample 3

37

kb long. The results showed that both restriction enzymes are functional and the correct gene

has been inserted in the correct plasmid. Lane one is the molecular marker, DNA ladder -

O’GeneRuler DNA ladder mix # SM1173 used to determine the sizes of the bands.

Figure 10: Gel electrophoresis analysis of double restriction enzyme digestion of pcDNA3.1TOPO-

CCHFVNP with Not1 and Bamh1 restriction enzymes for confirmation of OverExpress C43 (DE3) cells

positive transformation with the correct plasmid.

Figure 11 below shows a double restriction enzyme digestion of large scale plasmid purification

with QIAGEN Plasmid Plus Midi kit done in duplicate. The concentration of the plasmid was

966.0ng/µl represented by the first three lanes and 2021ng/µl represented by the last three

lanes respectively. Lane 1 is the DNA ladder, lane 2,3 and 4, purified plasmid of concentration

966.0ng/µl cut with both BamH1 and Not1, with Not1 and then with BamH1 respectively. Lanes

5, 6 and 7 represent the 2021ng/µl plasmid treated similarly as the former. Lanes 2 and 5 show

two bands one of 1.5kb and the other 5.5kb representative of CCHFV NP and pcDNA3.1TOPO

respectively which therefore confirm positive insertion of the gene of interest. Lanes 3, 4, 6 and

7 represent linearized plasmid with CCHFV NP inserted.

38

Figure 11: Gel electrophoresis analysis of double restriction enzyme digestion of pcDNA3.1TOPO-

CCHFVNP with Not1 and Bamh1 restriction enzymes to confirm protein expression by E. coli cells in the

glycerol stock.

3.4. Determining the appropriate seeding densities for transfection

Appropriate seeding densities for transfection of BHK mammalian cells were determined with

cells propagated in growth media in flat-bottomed tubes and 24-well plates at different

proposed seeding densities. Cell confluence was examined in 24hrs. Flat-bottomed tubes were

seeded at 2.5x105 and 3.0x105 cells/tube. After 24hrs, tubes seeded at 2.5x105 cells had reached

a 50%to 60% confluence instead of 70%-90% confluence. For most transfection reagents, a

50%-60% confluence is low for transfection. However, after 48hrs more than half of the cells

that had attached had died and detached. Cells that remained attached had distorted cell shape

showing distress, therefore not ideal for transfection either. Consequently, transfection carried

39

out 48hrs post transfection resulted with loss of cells during cell fixation with 1:1 methanol-

acetone solution. As a result, an extremely low transfection efficiency (5%) was obtained or

none at all. On the contrary, when the seeding density was increased to 3x105cells/tube, an

80%-90% cell confluence was attained within 24hrs of incubation and as a result, less cell loss

was encountered during fixation which in turn resulted with increased transfection efficiency.

With 24-well plates, only a seeding rate of 1x105cells/well resulted with cells 70-80% confluent

and not over- or under seeded wells post 24hrs of seeding.

3.5. Transfection of BHK and Vero cells using different transfection reagents

Successful integration of pcDNA 3.1TOPO-CCHFVNP and expression of the NP by BHK cells was

confirmed by and indirect immunofluorescence assay using human anti-CCHFV antibodies and

an FITC conjugated goat anti-human IgG antibodies. With each transfection reagent, the

transfection was performed on more than one occasion (Table 8) to monitor the toxicity levels,

transfection efficiency and transfection reagent to DNA ratios at which each reagent is more

efficient for reagent for transfection of BHK cells (Table 8).

3.5.1. Transfection of BHK cells with FuGene®6 transfection reagent

In all transfection experiments with FuGene®6 transfection reagent, 0% transfection efficiency

was attained except for experiment 3 (≤40%), ratio 3:2 with CCHFV NP (Table 8). In experiment

1, cells had been incubated for 48hrs before transfection and almost 80% of cells washed off

during fixation. The transfection reaction was repeated (experiment 2) with a higher seeding

density (3x105cells/tube) that allowed transfection in 24hrs. However there was no

fluorescence. In experiment 3, 24-well plates were used instead of plat-bottomed tubes

and≤40% transfection efficiency was obtained. Experiment 5 was performed to confirm exp. 4

results and was exposed to similar conditions throughout, but no transfection was observed. In

all negative results, cells showed a yellow appearance.

40

3.5.2. Transfection of BHK cells with Lipofectamine™ 2000, TurboFect and X-tremeGENE

HP transfection reagents

Out of 12 and 10 experiments, a positive transfection with pSin-GFP was seen in six and five

with the highest transfection efficiency of 70% (exp. 19) and 80% (exp. 5) for Lipofectamine

2000 and TurboFect respectively (Table 8). The first two experiments with Lipofectamine were

carried out independent of TurboFect. At first, 24-well plates seeding density resulted with over

seeded cells which let to high cell death. Cells transfected with both reagents were exposed to

similar incubation conditions. From exp. 3 to 5 and Exp.1 for Lipofectamine 2000 and TurboFect

transfection reagent respectively, both ratios of transfection reagent: DNA were used to

configure the ratio with the best efficiency and less toxicity to cells. With the former, 2:1 ratio

had better transfection efficiency in all 3 experiments. As a consequence, duplicate transfection

reactions at 2:1 ratio were done from exp. 6 to 12 with positive transfection with NP in exp. 11

and 12. With the latter, the 3:1 ratio was made by correlation it with the recommended 2:1

ratio. When it failed, all the experiments were done with the recommended ratio. When proper

incubation conditions were not maintained (Exp. 8 to 9 Lipofectamine 2000 and 6 to 7,

TurboFect), slow cell propagation was experienced which meant slow metabolism hence low

protein expression or none at all. Following the above experiments, a vial of BHK cell at a low

passage level (P6) was used instead of passage level 42 (Exp.10-12 and 8-10).

With the X-tremeGENE, positive transfection with very low transfection efficiency (≤5%) was

observed with pSin-GFP at the 2:1 transfection reagent: DNA ratio only.

3.5.3. Transfection of Vero cells with three transfection reagents

Veros are known to be slow growing. As a result they express protein very slowly. Positive

transfection was attained only with Lipofectamine 2000 transfection reagent. The transfection

efficiency was not high either, (20% GFP and 5% NP).

41

Table 8: Transfection efficiencies (in %) of different transfection reagents at different

transfection reagent: DNA ratios with BHK and Vero cell lines

Cell line Transfection Reagent

Experiment number

*Transfection reagent: DNA Ratio

Transfection Efficiency

in percentage (%)

GFP CCHFV-NP

BHK

FuGENE®6 Exp. 1 3:1 3:2 6:1

0 0 0

0 0 0

Exp. 2 3:1 3:2 6:1

0 0 0

0 0 0

Exp. 3 3:1 3:2 6:1

0 0 0

0 ≤40 0

Exp. 4 3:1 3:2 6:1

0 0 0

0 0 0

Lipofectamine 2000

Exp. 1 2:1 3:1

0 0

0 0

Exp. 2 2:1 3:1

0 0

0 0

Exp. 3 2:1 3:1

≤10 0

0 0

Exp. 4 2:1 3:1

30 0

0 0

Exp. 5 2:1 3:1

≥50 0

0 0

Exp. 6 2:1 2:1

0 0

0 0

Exp. 7 2:1 2:1

≤5 0

0 0

Exp. 8 2:1 2:1

0 0

0 0

Exp. 9 2:1 2:1

0 0

0 0

Exp. 10 2:1 2:1

70 0

0 0

Exp. 11 2:1 20 ≥10

Exp. 12 2:1 ≥10 ≥20

TurboFect

Exp. 1 2:1 3:1

0 0

0 0

Exp. 2 2:1 2:1

0 0

0 0

Exp. 3 2:1 2:1

40 0

0 0

Exp. 4 2:1 2:1

65 ≤2

0 0

Exp. 5 2:1 2:1

80 20

0 0

Exp. 6 2:1 2:1

0 0

0 0

Exp. 7 2:1 2:1

0 0

0 0

Exp. 8 2:1 2:1

0 0

0 0

Exp. 9 2:1 50 0

Exp. 10 2:1 ≤10 0

X-tremeGENE HP

Exp. 1 1:1 2:1 3:1

0 ≤5 0

0 0 0

Veros

Lipofectamine 2000

Exp. 1 2:1 2:1

20

10

5

0

TurboFect Exp. 1 2:1 2:1

0 0

0 0

*Transfection reagent: DNA: µl: µg

3.6. Immunofluorescence assay (IFA) for confirmation of positive transfection of BHK

cells with pcDNA3.1TOPO-CCHFV plasmid

Expression of the nucleocapsid protein of CCHFV by BHK cells positively transfected with pcDNA

3.1TOPO-CCHFVNP plasmid was monitored with an immunofluorescence staining 24 hours post

transfection. The optimization of transfection experiments was performed using pSin-GFP.

Positive transfectants were attained with both TurboFect and Lipofectamine transfection

43

reagents hence a positive fluorescence was observed (fig 12 (A and B). With BHK cells positively

transfected with CCHFV NP attained with the Lipofectamine transfection reagent at a 2:1 ratio,

a positive immunofluorescence staining was observed when reacted with an IFTC conjugated

goat anti-human IgG antibody at a dilution of 1:10 (Fig 12 C). This showed that the protein was

been expressed.

Figure 12: Positive transfection with pSin-GFP and IFA staining of BHK cells expressing CCHFV

nucleocapsid protein (NP). A: BHK cells expressing GFP (transfected with TurboFect) at x40

magnification. B: Lipofectamine 2000 transfection reagent. B (i): GFP expressing cells observed under

Light microscope at x40 (UV at 450nm) using 0.60 N/A (Eclipse TE 2000-E, Nikon, Tokyo, Japan) and B (ii)

using confocal microscopy using Argon-ion at 454-676nm laser (spectra physics, USA). C: CCHFV NP

expressing cells viewed under a confocal microscope (see B (i) and C (ii) under a fluorescent microscope

at a magnification of x40 C (ii).

44

3.7. Protein expression (CCHFV NP) by BHK cells post transfection monitored through

an SDS-PAGE

Apart from an IFA, protein expression was also monitored through an SDS-PAGE. Protein was

harvested from cells transfected with pcDNA3.1TOPO-CCHFVNP using both TurboFect and

Lipofectamine 2000 transfection reagents and pSin-GFP using TurboFect transfection reagent as

well as the negative control. Both the pellet and the supernatant were loaded on the gel and

expected were bands of sizes ≈53kDa and 26.9kDa for NP and GFP respectively. However; no

band corresponding to both GFP and NP were detected from cells transfected with either

transfection reagent (fig 13) regardless of positive immunofluorescence staining. That could

have been attributed to by the possibility of too little protein been expressed by the cells as

well as low transfection efficiency for detection using an SDS-PAGE. The cell cultures will be

scaled up in later experiments or after generation of a stable cell line.

Figure 13: SDS-PAGE analysis of protein expressed in total protein extract from BHK cells transfected

with pSin-GFP and pcDNA3.1TOPO-CCHFVNP plus a negative control.

45

3.8. Effects of different mounting solutions in visualization of fluorescent cells under

fluorescent electron microscope

When examining cells under fluorescent microscope post IFA, blurredness, bubbles, fluorescent

color quenching and unclear cell shapes were some of the encountered problems. The glycerol

mounting solutions that was used was old as a result; a fresh mounting solution was prepared

and kept under correct conditions (4°C). This solved the problems and it was realized also that

the pH of the two old mounting solutions was higher or lower (≈10 and ≈6) than recommended

which is 8.6. With the “glucose syrup”, the vision of fluorescence cells was clearer with no film

inhibiting it and no bubbles. The only problem with the “glucose syrup” mounting solution was

the fact that slides could not be stored for long term examination because the glucose syrup

crystallized when kept in the fridge. Applying nail polish along the edges of a cover slip after

mounting it and keeping them in a moist container on a bench reduced the crystallization and

allowed for longer slide preservation. However, eventually the two were used depending on

the time frame within which the results were to be used.

3.9. Titration of Neomycin (G418) (Life technologies, NY, USA) in a 96-well plate

In this study, selection of a stable transfectants was based on antibiotic resistance. BHK cells

stably transfected with pcDNA3.1D/V5-His TOPO express the neomycin resistance gene carried

by the expression vector. Consequently, stable transfectants of pcDNA 3.1TOPO-CCHFVNP have

the ability to grow in media supplemented with neomycin. However, the neomycin

concentrations to which non-transfected BHK cells are susceptible had to be predetermined via

a titration in a 96-well plate, prior to use for selection of a stable transfection.

Table 10 documents the estimated percentage of cells that were still viable three days since

they were propagated in growth media supplemented with G418. The higher the G418

concentration, the quicker the cells die (1.4mg/ml-1.2mg/ml). However that was also

dependent on the amount of cells per G418 concentration (fig 14). Regardless of G418

concentration, a quick cell death was observed with both high and a low cell density. BHK cells

grow fast therefore, with the former, high cell density results with over seeded cells which in

turn escalated cell death. With the latter, it seemed there was a higher G418 concentration per

46

cell therefore leading to increased cell death. A high percentage of cells that remained viable

for as long as five days was recorded in cell densities ranging from 6.25x103cells/well –

1.563x103cells/well at G148 concentration of 0.4mg/ml – 0.6mg/ml. At this point, cell

concentration and G418 concentration “balanced” each other. The negative control maintained

a high viable cell percentage. In both occasions, results showed minor differences. At ht end,

1.2mg/ml was the G418 concentration considered appropriate for selection of positive

transfectants.

Table 9: Symbols used to interpret results on Table 10

Symbol Meaning: percentage of cells alive (%)/well

xx +/- 25

x +/- 75

x +/- 50

xx 0

+/- 100

x. +/- 80

.xx +/- 10

47

Table 10: Results from the third day of neomycin titration on a 96-well plate. Cell death

increases with increasing G418 concentration

48

Figure 14: Estimated percentage of cells that remained viable after four days since they were

propagated in growth media supplemented with decreasing concentrations of G418. A correlation of

Table 9 data, which shows a decline in cell growth with increasing G418 concentration.

3.10. Stable transfection and expression of CCHFV NP

Stable transfection of BHK cells was determined with an IFA using human sera from a

convalescent patient. A positive green fluorescence was observed (Fig 15) confirming a stable

expression of CCHFV nucleocapsid protein by the cells.

Figure 15: Positive immunofluorescence staining of CCHFV nucleocapsid protein-

expressing BHK cells with a 1:10 dilution of human anti-CCHFV antibodies from a

convalescent patient serum (from separate wells of different “clones”).

0

10

20

30

40

50

60

70

80

1.4 1.2 1 0.8 0.6 0.4 0.2 0 Esti

mat

ed

% o

f vi

able

/we

ll

Decreasing neomycin (G418) concerntration (mg/ml)

A bar chart representing estimated percentages of viable cells out of the total number of cells at a given concentration

of neomycin (G418) on the third day of trial 1

Series1

100,000 cells/well

50,000 cells/well

25,000 cells/well

12,500 cells/well

6,250 cells/well

3,125 cell/well

1,563 cells/well

49

Chapter 4

4. Discussion

Since the first outbreak of CCHF virus infection in 1944, the virus geographic distribution has

spread around the world with an increasing prevalence (Hoogstraal et al., 1979) especially in

CCHFV endemic such as Turkey (Bente et al., 2013). The virus prevalence coincides with the

distribution of its principal vector, H. marginatum marginatum ticks (Hoogstraal et al., 1979).

Despite all research on the virus since the first outbreak, there is no vaccine developed against

CCHFV and given its high mortality rate (30%) (Grard et al., 2011) and its ability to cause

nosocomial infections, quick, specific and sensitive diagnostic approaches are needed.

However, all diagnostic methods have to be safe and limit the need for bio-safety level four

laboratory facilities as are very scares even in regions where CCHF is endemic. In all CCHFV

strains nucleocapsid protein (NP) is the most antigenic and highly conserved CCHFV protein and

therefore the target protein for preparation of recombinant laboratory diagnostic reagents (Yu

et al., 2012; Burt et al., 2011).

This study was aimed at preparing a construct that can be used to prepare a stable cell line

constitutively expressing the nucleocapsid protein of CCHFV. This was attained by exploiting the

ability of mammalian cells to take up plasmid and express proteins inserted in them as well as

that of expression vectors themselves. In the previous study, CCHFV NP was amplified and

cloned into pcDNA3.1D/V5-His TOPO and its expression confirmed in this study.

Using competent C43 (DE3) OverExpress cells, it was confirmed that the construct was still

expressed via transformation, plasmid purification and double restriction enzyme digestion

using Not1 and BamH1. Results were then confirmed via a 1% agarose gel electrophoresis, with

a band of 1500bp corresponding the NP. Transfection of mammalian BHK cell line with the

plasmid using four different transfection reagents; FuGene6, Lipofectamine™ 2000, TurboFect

and X-tremeGENE showed different transfection efficiencies at different transfection reagent:

DNA ratios. However, it was also noticed that high transfection efficiency was consistent with

specific ratio for each reagent, 2:1 for both TurboFect and Lipofectamine 2000. TurboFect had

50

the highest efficiency with pSin-GFP and Lipofectamine with NP, with FuGene6 being the least

efficient in both proteins.

Nonetheless, a number of factors influence cell conditions which in turn determine their

survival as well as their seeding rate. A major factor is handling cells well especially during

passaging, avoiding any factors that could lead to their increased cell death. This could have

been the case with the 2.5 x105 cell/ml seeding density however; the 3x105 cell/ml seeding

density resulted with 80%-90% confluence in 24hrs hence considered the final seeding density.

On the other hand, use of less than recommended amount of DNA could also result in low

transfection efficiency as well as high toxicity level in a transfection reagent (Lipofectamine

2000). The amount of cells still attached to the cover slip post fixing which can be influenced by

both the seeding density and the incubation period in turn influences the transfection efficiency

of a transfection reagent. In all the transfection reactions, human error which can be as a result

of mainly lack of experience in the process may have played a role in the results obtained.

Given transfection results with BHK cells observed via an IFA that is either negative or with a

very low transfection efficiency for a number of trials with either transfection reagent, a

number of influencing factors were considered. Those included the use of BHK cells at a very

low passage number (P6) and use of a different cell line, Vero cells which unlike BHK cells can

be maintained for longer without dying, detaching or losing cell shape. Veros were also

advantageous for protein expression post transfection because they could be incubated for

three days post transfection as opposed to 24hrs with BHK, which meant an extended time for

protein expression and therefore higher transfection efficiency was expected. No increased

protein expression was observed.

Post transfection, cells were harvested, lysed and protein expression determined via an SDS-

PAGE gel within which a 53kDa band and a 26.9kDa band were expected for CCHFV NP and GFP

respectively. No protein was 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. Due to insufficient time, protein expression could not be

confirmed with SDS-PAGE. Nonetheless, this will be confirmed in later experiments. However,

protein expression was also determined with an IFA using human sera-based antibodies against

51

CCHFV and fluorescein isothiocyanate (FITC) conjugated goat anti-human IgG. Positive

transfection was observed through green fluorescing cells under a fluorescent and/or confocal

microscope. Of the three mountants, “glucose syrup” was mainly used because it resulted with

the clearest vision without bubbles.

For selection of a stably transfected cells, a neomycin concentration titration had to be carried

out prior to selection determine the inhibitory concentration to untransfected BKH cells. During

the titration, the decrease in cell death seemed to progress starting from C4-C7 up to G4-G7

square and then shrinking within that square with daily examination till the last concentration.

Even in this case, it is a matter of an individual’s opinion to decide the state of cell life within

each well hence why it was done in repeat. The cells were diluted across the plate while

neomycin concentration decreased going down the plate. In the first two rows (A and B) the

neomycin concentration is still too high regardless of the number of cells per well hence why

mostly are dead. Going down the columns 1, 2, 3 and then 8, 9, 10, 11, 12; different

observations were made. With the first set, there is less neomycin concentration per cell

because its concentration is decreasing yet the number of cells remains the same. On the

contrary, with the second set there is a high concentration of neomycin per cell in each well

because the cell number is at this highly diluted resulting with more cells dying due to

neomycin toxification regardless of their resistance. In the middle of the plate, the amount of

neomycin and that of cells seemed to balance each other out. Two days post selection with

1.2mg/ml G418, all untransfected BHK cell died similarly with 1.4mg/ml. The former was at the

end, selected as the appropriate final concentration of neomycin that could be used for

selection of positive transfectants. After selection of stable transfectants, high cell loss was

encountered during an IFA using the 8-well multitest slides. However, a positive fluorescence

was observed with attached cells. A careful attention had to be given to cell during a stable cell

line developments and factors such as the amount of trypsin to use and when to change media

had to be considered. Mainly, avoiding contamination was the main factor to always

remember.

52

Conclusion

At the end of this study, it could be deduced that BHK cells were capable of taking up plasmid

using a selected transfection reagent (Lipofectamine 2000) and in turn express the protein

encoded by the gene cloned into the expression vector. BHK positive transfectants expressed

CCHFV NP, picked up by human raised anti-CCHFV antibodies through an IFA. This means a

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, a stable cell line; which can in turn be used in the development of diagnostic assay

such as antigen slides, which could be used for diagnosis of CCHFV infection without the

requirement of bio-safety level 4 facilities.

53

Appendix

Preparations:

1. Media

1.1. SOC Media (1L)

I. Add 20g Bacto-tryptone, 5g Bacto-yeast Extract, 2ml 5M NaCl, 2.5ml 1M KCl, 10ml 1M

mgCl2, 10ml 1M MgSO4 and 20ml 1M glucose in 900ul of dH2O.

II. Adjusted final volume of the solution to 1 litre with water

III. Mix thoroughly, autoclaved and store at 4°C.

1.2. Luria Broth Media (LB) with ampicillin (L) and plates

Broth:

I. Dissolve 5g Bacto-yeast extract 10g Bacto-tryptone and 10g NaCl in 900ml of water

II. Adjust the pH to neutral (7.0) with 1M NaOH.

III. Adjust volume to 1L with distilled water, autoclave and allow cooling to 50°C before

adding ampicillin to the final concentration of 100µg/ml

IV. Store at 4°C till use.

Plates:

I. Dissolve 5g Bacto-yeast extract 10g Bacto-tryptone and 10g NaCl in 900ml of water

II. Adjust the pH to neutral (7.0) with 1M NaOH.

III. Adjust volume to 1L with distilled water, add 15g Bacto-gar and mix thoroughly and

autoclave

IV. Allow cooling to 50°C before adding ampicillin to the final concentration of 100µg/ml

V. Pour to plates and allow solidifying

VI. Store at 4°C until use.

1.3. Dulbecco's Modified Eagle Medium (DMEM) (Lonza, Verviers, Belgium)

I. Add: 50ml (5%) FBS (Delta Bio-products, Johannesburg, SA), 5ml (1%) pen/strep (10,000 U/µg

per ml, Sigma, USA), 5ml (1%)200mM L-glutamine (Lonza, Verviers, Belgium) and 5ml (1%) 100x

NEAA's (Lonza, Verviers, Belgium) into a bottle of 500ml DMEM (Lonza, Verviers, Belgium)

II. Mix and keep at 4°C.

54

1.4. 2x TY broth with kanamycin (1L)

II. Dissolve Bacto-tryptone 16g, Bacto-yeast extract 10g, NaCl 5g in distilled H2O to make a

final volume of 1L.

III. Adjust pH to 7.0 with 5 N NaOH

IV. Autoclave and store at 4C.

2. Solutions

2.1. IFA blocking solution

I. Prepare a 1x PBS by adding 10ml PBS (Sigma, Steinem, Germany) into 90ml distilled

water add 10 g (10%) sucrose (Merck, Darmstadt, Germany) and 500ml (0.5%) triton X-

100 (Promega, WI, USA)

II. Mix thoroughly till completely dissolved

III. Close tightly and store at room temperature.

2.2. IFA wash solution

I. Add 1ml (1%) TWEEN® 20 detergent (Cal Biochem, Darmstadt, Germany) to 100ml of 1x

PBS

II. Mix and store at room temperature.

2.3. SDS-PAGE gel staining solution (1L)

I. Add 400ml methanol, 100ml glacial acetic acid and 1g Coomassie Brilliant blue to a clean

2L Schott bottle and fill up to 2L with 500ml dH2O

II. Store at room temperature

III. This is a 40% methanol, 10% glacial acetic acid and 0.1% Coomassie Brilliant blue

staining solution.

2.4. Destaining solution (2L)

I. Add 700ml dH2O, 100ml glacial acetic acid and 200ml methanol to a 2L Schott bottle and

fill up to 2L with dH2O

55

II. Mix and store at room temperature

III. This is 20% methanol and 10% acetic acid.

2.5. NET/BSA (500ml)

I. Add 4.3g NaCl (150mM), 1.04g EDTA (5mM), 3.02g Tris pH 7.4 (50mM), 500mg (0.5g)

BSA (1mg/ml) and 2.5ml NP40 (0.5%) in dH2O

II. Bring up to the 500ml mark and store at room temperature.

3. Buffers

3.1. 50X TAE buffer (1L)

I. Dissolve 242ml of Tris Base in 500ml water

II. Add 57.1ml glacial acetic acid and 100ml 0.5M EDTA (pH 8.0).

III. Mix and adjust volume to 1L with distilled water

IV. Store at room temperature.

V. Prepare a 1X TAE buffer by diluting this solution 1:50.

3.2. 10X Bath buffer (1L)

I. Dissolve 25ml Tris base and 192mM glycine in dH2O

II. Add 0.01% SDS and mix

III. Bring to a final volume of 1L with dH2O

IV. Store at room temperature

V. Prepare a 1X working solution by diluting 1:10 with dH2O.

3.3. 6x blue loading dye (10 ml)

I. Add 25 mg (0.25% (w/v)) of bromophenol blue dye to 6.7 ml of ddH2O and mix

II. Add 25 mg (0.25% (w/v)) of xylene cyanol FF and mix again (for visual tracking of DNA

migration during electrophoresis).

III. Then add 3.3 ml (30% (v/v) glycerol) of glycerol to ensure that the DNA in the ladder and

sample forms a layer at the bottom of the well

56

IV. Mix thoroughly and store aliquots at 4°C but at -20 °C for long-term storage.

4. Gel Preparation

4.1. Agarose gel (1%)

I. Add 1g agarose powder was dissolve in 100ml of TAE buffer and bring to boil for a while

in a microwave. Cool down to room temperature

II. Add 6ul of Ethidium-Bromide and mix completely.

5. Passaging of cells

5.1. Passaging of BHK cells

Baby hamster kidney cells (BHK) which have been maintained in the laboratory and kept as

aliquots of passage number six/seven in liquid nitrogen were cultured according to appropriate

conditions in T75 flasks (75 cm2 cell culture Corning® flasks). First media in the cells was

discarded and 5ml aliquot of PBS added into the flask and rinsed properly to get reed of any

excess media as it inactivates trypsin. A 500ul aliquot of trypsin was added into the flask and

incubated at 37C for 5min to allow the cells to detach. Detached cells were then re-suspended

completely in 5ml DMEM prepared as above and into a new T75 flask with 30ml of media, 1ml

of the re-suspended suspension was added, mixed properly and incubated at 37C for 48 hrs.

After every 48 hrs, cells were passaged again with the passage number increasing with each.

Passaging Vero cells is the same as that of BHK. The only difference is with the incubation time

during trypsinization. Veros are incubated for much longer (≥40min) because of their strong

adherence to the surface

57

6. Calculations

6.1. Calculating the transfection efficiency

5.2. Preparation of geneticin dilutions to give a certain neomycin G418 concentration

.

Calculations: how much (in volume) of geneticin (Life technologies, NY, USA) should be

added to give a certain concentration in a total volume of 200µl per well?

For the first raw (A1-A12): Neomycin concentration required (C2) = 1.4mg/ml

Neomycin stock concentration (C1) = 50mg/ml

Final volume (V2) = 200µl

Initial neomycin volume (V1) = x µl

C1V1=C2V2: x µl= 1.4mg/ml x 200ul

50mg/ml

X µl = 5.6µl of neomycin

To top it up to 100µl, 94.6µl of media was added to the tube and to prepared a dilution

for the entire 12 well raw, both the media and the neomycin volume were multiplied by

thirteen and mixed properly. NB: for every G418 concentration.

58

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