University of Ghana http://ugspace.ug.edu.gh
UNIVERSITY OF GHANA 
COLLEGE OF BASIC AND APPLIED SCIENCES 
 
 
DETECTION OF CRIMEAN-CONGO HAEMORRHAGIC FEVER VIRUS (CCHFV) 
IN TICKS COLLECTED FROM LIVESTOCK IN GHANA 
 
BY 
CHARLOTTE ADWOA ADDAE 
(10599402) 
 
THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN 
PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL 
MOLECULAR CELL BIOLOGY OF INFECTIOUS DISEASES DEGREE 
 
DEPARTMENT OF BIOCHEMISTRY, CELL AND MOLECULAR BIOLOGY 
 
JULY 2018 
 
 
i 
 
University of Ghana http://ugspace.ug.edu.gh
DECLARATION 
I, Charlotte Adwoa Addae hereby declare that this thesis is the outcome of my own research 
project under the supervision of Dr. Osbourne Quaye of the Department of Biochemistry, 
Cell and Molecular Biology, University of Ghana and Dr. Shirley Nimo-Paintsil, United 
States Naval Medical Research Unit No. 3, Ghana Detachment, Accra, Ghana. To the best of 
my knowledge, this thesis has not been presented for the award of any degree or published 
elsewhere. Any mention of other authors’ works has been duly acknowledged and properly 
referenced. 
 
 
……………………………………..                                                ..………………………….. 
Charlotte Adwoa Addae                                                                                    Date 
(Student) 
 
…………………………………….                                               …………………………….. 
Dr. Osbourne Quaye                                                                                         Date 
(Supervisor) 
 
…………………………………….                                               …………………………….. 
Dr. Shirley Nimo-Paintsil                                                                                 Date  
(Co-Supervisor) 
ii 
 
University of Ghana http://ugspace.ug.edu.gh
ABSTRACT 
Crimean-Congo haemorrhagic fever virus (CCHFV) is an arbovirus which belongs to the 
Crimean-Congo haemorrhagic fever serogroup. It belongs to the viral family, Nairoviridae 
and genus, Orthonairovirus. Crimean-Congo haemorrhagic fever serogroup and Nairobi 
sheep disease serogroup both fall under this genus. Viruses from these two serogroups are 
pathogenic to humans and animals respectively, and therefore have a significant economic 
impact. Tick species of three genera are known to transmit these viruses; Rhipicephalus, 
Amblyomma, and Hyalomma. This study focused on screening field-collected ticks for the 
presence of CCHFV. Ticks were collected from dogs, sheep, cattle and goats in seven sites 
within three regions of Ghana; Greater Accra, Northern and Upper East.  A total of 1,813 
ticks were collected and morphologically identified using the African Ixodidae identification 
keys. Ticks were pooled (by species, gender, the site collected, and animal host), 
homogenized, nucleic acid extracted and screened for CCHFV.  Seven of the pools were 
positive for CCHFV and were further analyzed using United States Army Medical Research 
Institute of Infectious Diseases (USAMRIID) next-generation RNA Access protocol. 
Sequencing performed on all seven pools failed to confirm the presence of CCHFV however, 
the resulting data from an Amblyomma variegatum pool (from Michel camp-Greater Accra) 
showed whole genome sequence of Dugbe virus. Phylogenetic analysis of the complete 
sequence of the L and S segments of the genome using maximum likelihood tree algorithm 
showed a close relationship (bootstrap value of 99%) with the Dugbe strain previously found 
in Ghana. However, there was also a close relationship with the reference Dugbe virus strains 
from Kenya and Nigeria with a bootstrap value of 99%.  
The findings from this surveillance study demonstrate the circulation of Dugbe virus in 
Ghana. Therefore, the need to further investigate to detect the virus prevalence and risk of 
human and veterinary infections in Accra and the country as a whole. 
iii 
 
University of Ghana http://ugspace.ug.edu.gh
DEDICATION 
This thesis is dedicated to my family; Adepa and William for always being there. 
 
 
 
 
 
 
 
 
 
 
 
 
  
iv 
 
University of Ghana http://ugspace.ug.edu.gh
ACKNOWLEDGEMENT 
My utmost appreciation goes to the Almighty God for seeing me through this programme. I 
wish to express my profound gratitude to my supervisors Dr. Osbourne Quaye of the 
Department of Biochemistry, Cell and Molecular Biology, University of Ghana and Dr. 
Shirley Nimo-Paintsil, United States Naval Medical Research Unit No. 3 (NAMRU-3), 
Ghana Detachment, Accra, Ghana for their patience and guidance. I also want to thank the 
West African Center for Cell Biology of Infectious Pathogens (WACCBIP) for paying my 
fees and providing me with a monthly stipend throughout my second year. I also want to 
thank NAMRU-3 Ghana detachment for the opportunity to work on this funded project. 
I am grateful to Dr. Joseph Diclaro, Dr. Dadzie, Colonel Defeamekpor, Catherine Pratt, Dr. 
Michael Wiley, Dr. Randy Schoepp and Dr. Jeff Koehler for their guidance and mentorship. 
My heartfelt appreciation also goes to Danielle Ladzekpo, Isaac Adrah, Seth Offei Addo, 
Karen Ocansey, Bright Agbodzi, Mba-Tihssommah Mosore, Eric Behene the staff of 
NAMRU-3 and Vestergaard Vector labs, Noguchi Memorial Institute for Medical Research 
(NMIMR). A very special appreciation goes to Rebecca Pwalia, Samuel Akporh Sowah, 
Joannita Joannides, Godwin Amlalo, Dominic Acquah-Baidoo, Sampson Gbagba and 
Ibrahim Gyimah for their help and support throughout this programme. I am also grateful to 
the entomology team at the Navrongo Health Research Center and members of the Veterinary 
Department of the Ghana Armed forces for their help in the field. This work would not have 
been successful without their support.  
To Mr. Kofi William, I appreciate your encouragement and guidance. A heartfelt 
appreciation to Raymond Adjei, Abena Kissi-Twum and members of the Virology 
Laboratory, Department of Biochemistry, Cell and Molecular Biology for their help 
throughout this work.  
v 
 
University of Ghana http://ugspace.ug.edu.gh
TABLE OF CONTENTS 
DECLARATION...................................................................................................................... ii 
ABSTRACT ............................................................................................................................ iii 
DEDICATION......................................................................................................................... iv 
ACKNOWLEDGEMENT ....................................................................................................... v 
LIST OF FIGURES ................................................................................................................ xi 
LIST OF TABLES ................................................................................................................. xii 
LIST OF ABBREVIATIONS AND ACRONYMS ........................................................... xiii 
CHAPTER ONE ...................................................................................................................... 1 
1.0 INTRODUCTION .......................................................................................................... 1 
1.1 JUSTIFICATION .......................................................................................................... 4 
1.2 HYPOTHESIS ................................................................................................................ 4 
1.3 AIM .................................................................................................................................. 4 
1.4 SPECIFIC OBJECTIVES ............................................................................................. 5 
CHAPTER TWO ..................................................................................................................... 6 
2.0 LITERATURE REVIEW .............................................................................................. 6 
2.1 HISTORY OF CRIMEAN-CONGO HAEMORRHAGIC FEVER VIRUS AND 
DUGBE VIRUS .................................................................................................................... 6 
2.2 ENDEMIC REGIONS ................................................................................................... 6 
2.2.1 Global distribution of CCHFV .................................................................................. 9 
2.3 TRANSMISSION AND ENDEMIC CYCLE OF CCHFV AND DUGBE VIRUS 11 
2.4 DISEASE PATHOGENESIS OF CCHFV AND DUGBE VIRUS .......................... 12 
vi 
 
University of Ghana http://ugspace.ug.edu.gh
2.5 SIGNS AND SYMPTOMS OF CCHFV AND DUGBE VIRUS INFECTION ...... 13 
2.6 DIAGNOSIS OF CCHFV AND DUGBE VIRUS INFECTION .............................. 14 
2.7 TREATMENT .............................................................................................................. 15 
2.8 CRIMEAN-CONGO HAEMORRHAGIC FEVER VIRUS .................................... 16 
2.9 HOST CELL INVASION AND REPLICATION ..................................................... 18 
2.10 DUGBE VIRUS .......................................................................................................... 20 
2.11 VECTOR OF CCHFV AND DUGBE VIRUS ......................................................... 21 
2.11.1 Distribution of ticks ............................................................................................... 22 
2.11.2 Tick life cycle ........................................................................................................ 25 
2.11.3 Hunting and feeding behaviour of ticks ................................................................ 25 
2.11.4 Life cycle of CCHFV and Dugbe virus in tick vectors ......................................... 27 
2.12 METHODS OF TICK SURVEILLANCE ............................................................... 27 
2.13 TICK IDENTIFICATION ........................................................................................ 29 
2.13.1 Morphological identification ................................................................................. 29 
2.13.2 Molecular identification ........................................................................................ 30 
2.14 DETECTION OF CCHFV AND DUGBE VIRUS IN VECTORS AND HOSTS 31 
2.15 SEQUENCING AND CHARACTERISATION OF CCHFV AND DUGBE 
VIRUS ................................................................................................................................. 32 
CHAPTER THREE ............................................................................................................... 35 
3.0 METHODOLOGY ....................................................................................................... 35 
3.1 CHEMICALS AND REAGENTS............................................................................... 35 
3.2 ETHICAL CONSIDERATIONS ................................................................................ 35 
vii 
 
University of Ghana http://ugspace.ug.edu.gh
3.3 STUDY SITES .............................................................................................................. 35 
3.4 TICK COLLECTION, SORTING AND IDENTIFICATION ................................ 37 
3.5 RNA EXTRACTION FROM TICKS ........................................................................ 38 
3.6 VIRUS DETECTION, REAL-TIME REVERSE TRANSCRIPTION PCR .......... 39 
3.7 SAMPLE PREPARATION FOR NEXT GENERATION SEQUENCING ........... 40 
3.7.1 RNA Fragmentation ................................................................................................ 40 
3.7.2 First strand cDNA synthesis .................................................................................... 40 
3.7.3 Second strand cDNA synthesis................................................................................ 41 
3.7.4 Sample clean-up ...................................................................................................... 41 
3.7.5 3’-ends adenylation .................................................................................................. 42 
3.7.6 Ligation of adapters ................................................................................................. 42 
3.7.7 Sample clean-up ...................................................................................................... 43 
3.7.8 First PCR amplification ........................................................................................... 44 
3.7.9 Sample clean-up ...................................................................................................... 45 
3.7.10 Library validation .................................................................................................. 45 
3.7.11 First hybridization.................................................................................................. 45 
3.7.12 First capture ........................................................................................................... 46 
3.7.13 First Wash .............................................................................................................. 46 
3.7.14 First Elution ........................................................................................................... 47 
3.7.15 Second hybridization ............................................................................................. 47 
3.7.16 Capture sample clean-up ....................................................................................... 48 
viii 
 
University of Ghana http://ugspace.ug.edu.gh
3.7.17 Second PCR amplification ..................................................................................... 48 
3.7.18 Second PCR clean-up ............................................................................................ 49 
3.7.19 Quality assessment of Library ............................................................................... 49 
3.7.20 Library qPCR ......................................................................................................... 49 
3.7.21 Normalization and pooling .................................................................................... 50 
3.7.22 MiSeq loading preparation .................................................................................... 50 
3.8 STATISTICAL ANALYSIS ........................................................................................ 51 
CHAPTER FOUR .................................................................................................................. 52 
4.0 RESULTS ...................................................................................................................... 52 
4.1 TICK COLLECTION ................................................................................................. 52 
4.2 CCHFV DETECTION ................................................................................................. 58 
4.3 CCHFV INFECTION RATE ...................................................................................... 60 
4.4 NEXT GENERATION SEQUENCING AND PHYLOGENETIC ANALYSIS .... 61 
5.0 DISCUSSION ............................................................................................................... 64 
5.1 ECOLOGICAL ZONES AND TICK DISTRIBUTION .......................................... 64 
5.2 VECTOR HOST ........................................................................................................... 66 
5.3 CCHFV DETECTION ................................................................................................. 67 
5.4 CCHFV INFECTION RATE AND VECTOR DISTRIBUTION............................ 68 
5.5 PHYLOGENETIC ANALYSIS OF DUGBE VIRUS ............................................... 70 
5.6 LIMITATIONS ............................................................................................................ 72 
CHAPTER SIX ...................................................................................................................... 73 
ix 
 
University of Ghana http://ugspace.ug.edu.gh
6.0 CONCLUSION AND RECOMMENDATION ......................................................... 73 
6.1 CONCLUSION ............................................................................................................. 73 
6.2 RECOMMENDATION ............................................................................................... 73 
REFERENCES ....................................................................................................................... 74 
APPENDICES ........................................................................................................................ 89 
APPENDIX 1 ...................................................................................................................... 89 
APPENDIX 2 ...................................................................................................................... 90 
APPENDIX 3 ...................................................................................................................... 91 
APPENDIX 4 ...................................................................................................................... 92 
APPENDIX 5.1 ................................................................................................................... 93 
APPENDIX 5.2 ................................................................................................................... 94 
 
  
x 
 
University of Ghana http://ugspace.ug.edu.gh
LIST OF FIGURES 
Figure 2.1: Annual global distribution of CCHFV .................................................................... 8 
Figure 2.2: A diagram of CCHFV showing its various components ....................................... 17 
Figure 2.3: Life cycle of CCHFV inside a host cell ................................................................ 19 
Figure 2.4: The ecological zones of Ghana ............................................................................. 24 
Figure 2.5: An ixodid tick feeding on its host ......................................................................... 26 
Figure 3. 1: Map of Ghana showing the various study sites ………………………………...36 
Figure 4. 1: Distribution of ticks from the two ecological zones, blue bars show the number of 
ticks collected from the ecological zones. ............................................................................... 52 
Figure 4. 2: Tick species distribution across the seven study sites. ......................................... 55 
Figure 4. 3: Tick species distribution across the seven study sites. ......................................... 56 
Figure 4. 4: Distribution of animal source of ticks from the seven study sites........................ 57 
Figure 4. 5: Positive curves for pools 1, 2, 3 and 4 from the real-time RT-PCR. ................... 59 
Figure 4. 6: Positive curves for pools 5, 6 and 7 from the real-time RT-PCR.. ...................... 60 
Figure 4. 7: Phylogenetic tree illustrating the relationship between the L segments of the new 
Dugbe virus detected in Ghana, other Dugbe virus strains and other viruses in the 
Nairoviridae family. ................................................................................................................. 62 
Figure 4. 8: Phylogenetic tree illustrating the relationship between the S segments of new 
Dugbe virus detected in Ghana, other Dugbe virus strains and other viruses in the 
Nairoviridae family.. ................................................................................................................ 63 
 
 
 
xi 
 
University of Ghana http://ugspace.ug.edu.gh
LIST OF TABLES 
Table 4. 1: Distribution of tick sex across the two ecological zones ....................................... 53 
Table 4. 2: Distribution of tick species across the ecological zones. ....................................... 54 
Table 4. 3: Distribution of animal sources of ticks across the two ecological zones. ............. 57 
Table 4. 4: Details of CCHFV positive pools .......................................................................... 58 
Table 4. 5: Prevalence of CCHFV among different tick species collected from Greater Accra, 
Northern and Upper East region .............................................................................................. 61 
 
  
xii 
 
University of Ghana http://ugspace.ug.edu.gh
LIST OF ABBREVIATIONS AND ACRONYMS 
Act D - Actinomycin D   
AVE – Elution buffer 
AVL – Lysis buffer 
AW1 – Wash buffer 1 
AW2 – Wash buffer 2 
BLAST – Basic Local Alignment Search Tool 
CCHFV - Crimean-Congo Haemorrhagic Fever Virus 
CDC – Center for Disease Control 
cDNA – complementary Deoxyribonucleic acid 
CT3 - Capture Target Buffer 3  
DD - Decimal Degrees 
DMS - Degrees Minutes and Seconds  
dTTP - deoxythymidine triphosphate  
dUTP - deoxyuridine triphosphate  
EE1 - Enrichment Elution Buffer 1 (EE1)  
ELISA - Enzyme-linked immunosorbent assay  
EPF - Elute Prime Fragment  
EWS - Enrichment Wash Solution  
FSA - First Strand Synthesis Act D  
xiii 
 
University of Ghana http://ugspace.ug.edu.gh
GC – Glycoprotein C 
GenBank – Genome bank 
GN – Glycoprotein N 
GPS - Global Positioning System  
HT1 – High target 1 
ICAM-1 – intercellular adhesion molecule 1 
IgG – Immunoglobulin G 
IL-6 – Interleukin 6 
IL-8 – Interleukin 8 
IL-10 – Interleukin 10 
IgM – Immunoglobulin M 
L segment – Large segment 
MEGA6 – Molecular Evolutionary Genetics Analysis 6 
mRNA - Messenger RNA   
NAMRU3 – Navy medical research unit 3 
NGS - Next generation sequencing 
NSDV - Nairobi Sheep Disease Virus  
PCR – Polymerase chain reaction 
qPCR – quantitative (real time) Polymerase Chain Reaction 
RdRp - RNA dependent RNA polymerase  
RNA – Ribonucleic acid 
ROX - 6-Carboxyl-X-Rhodamine (dye) 
xiv 
 
University of Ghana http://ugspace.ug.edu.gh
rpm – runs per minute 
RSB - Resuspension Buffer  
RT – Reverse Transcriptase  
S segment – Small segment 
TNF-a – Tumour Necrosis Factor-alpha 
USAMRIID – United States Army Medical Research Institute of Infectious Diseases 
VCAM – Vascular Cell Adhesion Molecule 1 
VHF - Viral Haemorrhagic Fever   
WHO - World Health Organization  
  
xv 
 
University of Ghana http://ugspace.ug.edu.gh
CHAPTER ONE 
1.0 INTRODUCTION 
The virus that causes Crimean-Congo haemorrhagic fever (CCHF) was first detected 
in Crimea (1944) (Grashchenkov, 1945) and Congo (1956) (Hoogstraal, 1979). The virus was 
named after these two places where it was first discovered.  
The CCHF virus belongs to the viral family, Nairoviridae and genus Orthonairovirus. 
It is in the same CCHF serogroup with Hazara, Tofla and Artashat viruses. Crimean-Congo 
haemorrhagic fever virus (CCHFV) is a negative-sense enveloped virus with a single-
stranded ribonucleic acid (RNA) genome which is divided into three segments; small (S), 
medium (M) and large (L) segments (Bente et al., 2013). The S segment is the smallest RNA 
segment, and it codes for nucleoproteins that encapsulate the three RNA segments of the 
virus. The M segment is the second largest RNA segment in the virus. This RNA segment 
codes for the glycoproteins GN and GC found on the surface of the virus. These glycoproteins 
bind to host cell surface receptors leading to the invasion of the host cell by the virus. The L 
(large) segment codes for RNA-dependent RNA polymerase enzyme, the virus uses this 
enzyme to synthesize its RNA genome (Bente et al., 2013). 
The CCHF virus has been found in arthropods such as ixodid ticks Rhipicephalus spp. 
and Hyalomma spp. (Keshtkar-Jahromi et al., 2013). Household pets, wild animals, and farm 
animals serve as reservoirs and hosts for viral amplification in which case the virus has been 
shown to have very low pathogenicity (Al-Abri et al., 2017). The virus circulates between the 
parasitic ticks and animals when virus-infected ticks feed on the animals and transfer the 
virus into the bloodstream of the animals or take up the virus from infected animals (Bray, 
2005). Infected parasitic female ticks are also known to pass on the virus transovarially. 
Infected male ticks also pass it on sexually to uninfected female ticks. Also, an infected tick 
1 
 
University of Ghana http://ugspace.ug.edu.gh
feeding at the same location as other ticks can pass on the virus through its saliva to other 
ticks (Walker et al., 2014). 
Tick bites can cause human CCHFV infection or exposure to infected blood from 
infected animals or persons. Another way by which a person can be infected with the virus is 
from hospitals or health centers when there is an outbreak (Leblebicioglu, 2010). 
Veterinarians, animal farm workers, abattoir workers and people living in households with 
livestock are all at risk of CCHFV infection due to their constant exposure to animals or 
preferred tick hosts (CDC, 2014). The incubation period after exposure to the virus for 
humans is typically between five to six days. However, up to thirteen days have been 
reported (CDC, 2014). Like other viral haemorrhagic fever (VHF) viruses, CCHFV causes 
sporadic outbreaks with symptoms such as headache, severe fever, back pains, vomiting, and 
joint pains (Ergönül, 2006). Even though it falls under the class of haemorrhagic fevers, 
infected people do not necessarily bleed through their mouths, nostrils, and other orifices. 
Instead, in severe cases, patients often develop large patterns of ecchymoses resulting from 
internal organ bleeding which may eventually lead to death (Ergönül, 2008).  
There is no cure for the disease; symptoms are managed as they appear. Therefore, it 
is essential that people who live in areas where the vectors are present or people who work 
with and handle animals take the necessary precautions to prevent an infection (Gunes et al., 
2009). Veterinarians, abattoir workers, and farm workers are advised to wear the proper 
clothing to protect them from any tick bites. When coming into contact with bodily fluids 
from the animals, they are encouraged to take extra care and wear the necessary personal 
protection equipment for safety (Fajs et al., 2014).  
Detection of the virus is done using Real-time reverse transcriptase polymerase chain 
reaction (real-time RT-PCR) to detect the small (S) RNA segment of the viral genome. The 
Enzyme-linked immunosorbent assay (ELISA) is also another technique for identifying viral 
2 
 
University of Ghana http://ugspace.ug.edu.gh
antigens or host antibodies to the viral antigens (CDC, 2014). However, if an individual dies 
due to viral infection, immuno-histochemical staining can be used to identify the virus 
antigens in the person’s tissues fixed in formalin (Vanhomwegen et al., 2012).  
The CCHF virus is found in hot and semiarid areas such as sub-Saharan Africa and 
some regions in the Middle East and Europe. Current global distribution models predict a 
relatively high occurrence of the virus in West Africa (Messina et al., 2015) due to the 
abundance of the vector in the sub-region (Walker et al., 2014). Typically, CCHFV infections 
are not seasonal but somewhat sporadic, and so they occur at any given time. In the temperate 
regions, CCHFV human infections are known to occur during the summer when the weather 
is favourable for the vector to move about, feed, and reproduce (Bente et al., 2013).  
According to Al-Abri et al., (2017), due to the spread of CCHFV infected vectors by 
migratory birds and livestock trade across various countries, CCHFV is gradually spreading 
and should, therefore, be of significant concern to the World Health Organization (WHO). 
Also, ecological models indicate that temperature rise and rainfall reduction is gradually 
increasing the habitats for the viral vector (Estrada-Pena and Venzal, 2007). An increase in 
the vector habitat means more ticks will emerge over time resulting in the spread of the virus 
among animals (Williams et al., 1972) which may eventually increase the risk of the spread 
of the virus to human populations. It is therefore vital that policies be put in place for 
prevention and control of the virus infection. Sporadic outbreaks of the disease and 
distribution of likely infected vectors by migratory birds or exported livestock have led to the 
question of the transmission of strains specific to one region of the globe being found in other 
areas of the world (Estrada-Pena and Venzal, 2007). Phylogenetic analysis of CCHFV strains 
shows the presence of West African strains in China (Papa et al., 2002). Middle Eastern and 
European strains have also been found in Africa (Burt and Swanepoel, 2005). 
3 
 
University of Ghana http://ugspace.ug.edu.gh
1.1 JUSTIFICATION 
Even though CCHFV case-fatalities of up to about 30% have been reported in some 
West African countries (Bente et al., 2013; Sang et al., 2011), there is little epidemiological 
information on the disease in Ghana. Previously collected tick samples from farm animals in 
Ghana have tested positive for CCHFV (Akuffo et al., 2016). Also, CCHFV antibodies have 
been detected in animal handlers and animal farmers in areas where there has been no case of 
CCHFV infection. This information is another area of concern about the incidence and 
prevalence of the virus (Akuffo et al., 2016). 
There is, therefore, the need to characterize the strains of the virus that are found in 
the country to be better informed and prepared for any eventual future outbreaks. The 
information can also influence the development of various testing platforms for diagnosis that 
will be used to detect CCHFV isolates found in Ghana. Early detection will help curb the 
threats posed by the virus. 
 
1.2 HYPOTHESIS 
The CCHFV and other orthonairoviruses are prevalent in Ghana, but these viruses may have 
been imported from other endemic regions in Africa.  
 
1.3 AIM 
To detect and characterize CCHFVs and other orthonairoviruses in field-collected ticks.  
 
 
  
4 
 
University of Ghana http://ugspace.ug.edu.gh
1.4 SPECIFIC OBJECTIVES 
1. To determine the different species of ticks collected from livestock in Ghana.  
 
2. To determine the presence of CCHFV using real-time Reverse Transcriptase 
Polymerase Chain Reaction (real-time RT-PCR).  
 
3. To determine the phylogenetic relationships between the various strains of 
orthonairoviruses circulating in Ghana. 
 
 
 
 
 
 
 
  
5 
 
University of Ghana http://ugspace.ug.edu.gh
CHAPTER TWO 
2.0 LITERATURE REVIEW 
2.1 HISTORY OF CRIMEAN-CONGO HAEMORRHAGIC FEVER VIRUS AND 
DUGBE VIRUS 
Crimean-Congo Haemorrhagic Fever Virus (CCHFV) causes Crimean-Congo 
haemorrhagic fever (CCHF). It was first discovered in Crimea after a group of Russian 
soldiers got sick with an unknown disease in 1944 (Grashchenkov, 1945). Years later in 
1956, a disease with similar symptoms was detected in a group of people in the Congo 
(Hoogstraal, 1979).  The virus was therefore named after the two places it was discovered. 
Since its first discovery, the disease has been reported in various countries and has a fatality 
rate of 3 – 30% (Sang et al., 2011). 
 Dugbe virus was first isolated at the Virus research laboratory, faculty of medicine, 
University of Ibadan, Nigeria. It was first detected in Amblyomma variegatum ticks and has 
since been detected in livestock, mosquitoes, and humans. The virus was named after the 
town it was discovered in, Dugbe in Ibadan Nigeria (David-West, 1974). 
 
2.2 ENDEMIC REGIONS 
The CCHFV is found in hot and semiarid areas such as sub-Saharan Africa and some 
regions in the Middle East and Europe (Messina et al., 2015). Due to the epidemic potential 
and high fatality rate, CCHF is a threat to public health (WHO, 2013).  Some of the regions 
that are endemic for CCHF are considered as conflict zones with the potential of military or 
civil conflicts.  Historically, more casualties of war are caused by arthropod-borne diseases 
than the enemy (Tucker, 2009).  Besides the potential negative impact on public health or the 
6 
 
University of Ghana http://ugspace.ug.edu.gh
population during wars, there is also the concern that CCHFV can be used as a biological 
weapon (Mertens et al., 2013).   
Current global distribution models predict a relatively high potential for the 
occurrence of CCHF in West Africa (Messina et al., 2015). Previous prediction modeling 
techniques for CCHFV concentrated on environmental factors such as temperature and 
precipitation, but current modelling also incorporates land cover type. Land cover type is 
defined as the observed physical cover of the earth surface. This consists of the vegetation 
and man-made features of a geographical location (Comber et al., 2005). Land cover type is 
critical to identifying ecological niches for wild or domestic animals that would serve as 
potential hosts.   
As of 2014, the United States Centre for Disease Control (CDC) reported that 47 
countries are endemic for CCHF (CDC, 2014). In Africa, CCHF has been recorded in over 30 
countries (Morikawa et al., 2007).  It has been documented that CCHF case-fatalities of up to 
approximately 30% or more have been reported in the various endemic African countries. 
According to current CCHF distribution maps (Figure 2.1), it is uncertain if CCHF is 
prevalent in Ghana, Sierra Leone, Togo, Liberia and Cote d’Ivoire (Leblebicioglu, 2010). 
7 
 
University of Ghana http://ugspace.ug.edu.gh
 
Figure 2.1: Annual global distribution of CCHFV. In regions marked yellow, there is the presence of the 
CCHFV vector and also virological or serological evidence indicating the presence of CCHFV. Areas marked 
orange record 5-49 CCHF cases per year. Areas marked red record 50 or more cases per year (Bente et al., 
2013). 
 
Dugbe virus just like CCHFV is distributed in regions where its vector is found. So 
far it has been detected in East Africa (Kenya and Uganda), West Africa (Ghana and 
Nigeria), Central Africa and Asia (India, Sri Lanka, and China) (Coates, 1990). Serological 
evidence also suggests the presence of the virus in Mozambique, Botswana (Health, 2016). 
Variant strains of Dugbe virus have been detected in East Africa, (Kenya), Central 
Africa and India. In Central Africa, the virus was detected in an infected laboratory worker 
(Digoutte, 1971). A strain of the virus known as the Nairobi sheep disease virus has also been 
identified in Uganda and Kenya (East Africa) from ticks ((Tukei, 1970, Coates, 1990). In 
8 
 
University of Ghana http://ugspace.ug.edu.gh
India, Ganjam virus which is also another strain of the virus has been detected (Coates, 
1990).  
2.2.1 Global distribution of CCHFV 
The CCHF virus has been shown to have a long distance geographical genetic linkage 
from country to country and continent to continent.  The global occurrence of CCHFV has 
been predicted using niche modelling with the goal of providing critical information to assist 
in prevention (Messina et al., 2015). The modelling does not take genetic relationships into 
account even though that may explain possible virus migration.  In recent serological studies 
in Africa, CCHFV was first documented in Mozambique (Muianga et al., 2017).  
Over 1000 serum samples from cows across Mali were tested with CCHFV IgG 
ELISA that yielded 66% CCHFV positive samples (Maiga et al., 2017).  When the positive 
samples were compared by region, there was a direct correlation between high densities of 
the cattle with a higher concentration of positives.  In Sudan, there was a high prevalence of 
CCHFV found in sampled camels. Although CCHFV has been reported in Sudan previously, 
it has not been well characterized (Suliman et al., 2017). 
Ghana, Mozambique, Mali, and Sudan have little information on the prevalence of 
CCHFV, and no sequence information has been published regarding the virus to date. Ticks 
sampled from farm animals in Ghana tested positive for CCHFV (Akuffo et al., 2016), but no 
sequencing data was obtained from those samples.   
In 1999, different clusters of CCHFV was detected in Iran; this initiated intense 
research to identify the genetic diversity of CCHFV in the country (Keshtkar-Jahromi et al., 
2013).  According to Mild et al., (2010), the first CCHFV isolate collected in Iran in 1979 
shared its genotype with an African strain.  This initial strain was in the same clade as those 
from Mauritania, South Africa, and Senegal.   
9 
 
University of Ghana http://ugspace.ug.edu.gh
In 2003, the initial case of CCHF in human was recorded in 38 patients in 
Nouakchott, Mauritania (Nabeth et al., 2004).  Utilizing basic local alignment search tool 
(BLAST), the S-segment was identified as the CCHFV from Mauritania from 1988.  The 
initial 2003 outbreak is thought to have started by a young woman who had slaughtered an 
infected goat.  Tick and animal sampling conducted suggest that the CCHFV responsible for 
this outbreak originated from a different region of the country and may have been imported 
through animal trade.   
Previous phylogenetic analyses of the S-segment of the virus have demonstrated that 
viruses sharing the same genotype can be spread over distances (Mild et al., 2010).  Besides 
the same genotype circulating in West Africa and Iran; the same genotype found in the 
Middle East was also found in Madagascar (Burt and Swanepoel, 2005). Different genotypes 
can circulate in one country and share separate genetic linkages to other countries; the S-
segment from South Africa, Burkina Faso, Nigeria, Namibia, Senegal, and Mauritania have 
been shown to be in the same genotype. A CCHFV was found in Hyalomma spp. from 
camels and cattle in Kenya (Sang et al., 2011). Although there were only two other reports of 
CCHFV in Kenya before these ticks being collected, these samples were not sequenced. A 
previous study comparing the global diversity of CCHFV phylogenetic relationships does not 
list any East African countries but did demonstrate multiple RNA segment re-assortment 
events (Deyde et al., 2006).  A CCHFV positive human blood sample collected in 1956 from 
the Democratic Republic of the Congo shared a genetic relationship with CCHFV samples in 
a tick in 1969 and one goat in 1972 from Senegal on the M-segment. However, the S segment 
and L segment of the Congo strain were closely related to European strains (Leblebicioglu, 
2010).  
 
 
10 
 
University of Ghana http://ugspace.ug.edu.gh
2.3 TRANSMISSION AND ENDEMIC CYCLE OF CCHFV AND DUGBE VIRUS 
 According to the WHO (2013) and CDC (2014), CCHFV can infect humans when 
they come in contact with blood from an infected human or animal. Humans can also be 
infected with CCHFV through bites from infected ixodid ticks.  Household pets, wild and 
farm animals serve as reservoirs and amplifying hosts for the virus since it has very low 
pathogenicity in animals (Leblebicioglu, 2010). There is a high prevalence of CCHFV in wild 
birds in endemic areas, but like other animals, these birds show resistance to the virus.  This 
was demonstrated in South Africa where a CCHF outbreak occurred at an ostrich abattoir; the 
ostriches did not show any symptoms of infection (WHO, 2013).   
Dugbe virus is also transmitted through tick bites. Infected adult ticks can transmit the 
virus for up to 2 years. Dugbe virus cannot be transmitted among animals through contact 
with an infected animal. However, humans can get infected when they come in contact with 
an infected human or animal (Health, 2016).  
 Generally, wild and domestic animals are infected through the bite of an ixodid tick 
(CDC, 2014).  Although CCHFV and Dugbe virus only remain in the animal’s bloodstream 
for approximately a week, the presence of multiple vectors taking blood meals from the 
infected animal keeps the viruses in circulation (CDC, 2014).   
Animal to human transmission mostly occurs among individuals that come in contact 
with blood or tissues of infected animals, such as veterinarians or slaughterhouse employees.  
Akuffo and his colleagues reported that abattoir workers in the Ashanti region tested positive 
for CCHF virus antibodies, this may be because of exposure to the virus during animal 
handling and slaughtering (Akuffo et al., 2016). Transmission of viruses from one human to 
the other can occur, through direct contact with infected body fluids or organs.  Infection can 
also occur through contaminated medical equipment or needles (CDC, 2014). 
11 
 
University of Ghana http://ugspace.ug.edu.gh
Reservoirs of the viruses; wild animals and livestock, can assist in keeping them 
endemic in a region (Wilson et al., 1991).  The tick vector also serves to maintain these 
because they are transmitted in the vector population transovarially and transtadially.  It is not 
common for humans to be infected directly from a tick bite, but it is possible (Hoogstraal, 
1979).  To assist in the migration of CCHFV and Dugbe virus, it has been shown that 
migrating birds can carry virus positive ticks to other geographical regions (Palomar et al., 
2013). Ticks on these birds can drop off from the birds and get onto other animals which may 
eventually get infected with any of the viruses. This explains why viruses of the same or 
similar strain found in one country have been found in other countries as well (Palomar et al., 
2013). Livestock animals transported from one region to the other may also carry infected 
ticks with them to their destination (Williams et al., 1972). 
 
2.4 DISEASE PATHOGENESIS OF CCHFV AND DUGBE VIRUS 
In 1945, Grashchenkov’s work described CCHF disease pathogenesis as blood 
circulatory disturbances in the patient’s organs mainly in the capillaries and small blood 
vessels.  The actual disease pathogenesis is not very clear, but a study was done on lab mice 
infected with Ebola haemorrhagic virus which has many characteristics that are the same as 
as CCHFV. This study suggested that the host immune response induces a change in the 
host’s vascular functions; induction of proinflammatory cytokines, platelet degranulation and 
aggregation, leukocyte adhesion and activation of the intrinsic coagulation cascade (Mahanty 
and Bray, 2004; Schnittler and Feldmann, 2003). Further studies have also shown elevated 
levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 
(VCAM-1) with the secretion of high levels of interleukin 6 (IL-6) and interleukin 8 (IL-8) in 
CCHF patients. Also, tumour necrosis factor-alpha (TNF-a) is associated with critical cases 
since IL-6 and IL-10 have been detected in critical and fatal cases (Saksida et al., 2010). 
12 
 
University of Ghana http://ugspace.ug.edu.gh
Studies have found that CCHFV replicates in monocyte-derived dendritic cells causing 
proinflammatory cytokines to be released (Connolly-Andersen et al., 2009). Nitric oxide and 
natural killer cells have been implicated in host immune response mechanisms to the virus 
(Yilmaz et al., 2008; Simon et al., 2006). 
Dugbe virus is a neuro-invasive virus (Boyd, 2008). The virus first infects cells of the 
major organs of the host; liver and lung (Coates, 1990). It then targets neuronal cells in the 
brain and eventually spreads to other cells in the brain. It is known that the type1 IFN system 
plays a role in host immune response, but the exact mechanism of action and types of 
proteins involved in this process is not clearly understood (Health, 2016). Young animals are 
most susceptible to the virus and have a higher mortality rate of 30% – 40% than adult 
animals (Coates, 1990). 
 
2.5 SIGNS AND SYMPTOMS OF CCHFV AND DUGBE VIRUS INFECTION 
The incubation period after exposure to CCHFV in humans depends on how the virus 
was acquired (Hoogstraal, 1979). The incubation period after viral transmission through tick 
bite is between one to five days. After contact with infected blood, incubation period is 
typically between five to seven days, however, up to thirteen days have been reported (CDC, 
2014). Like other viral haemorrhagic fevers (VHFs), CCHFV has been shown to cause 
sporadic outbreaks with flu-like symptoms such as severe fever, headache, red eyes, 
vomiting, back and joint pains. In severe cases, patients often develop large patterns of 
ecchymoses, which can lead to death (Leblebicioglu, 2010). Patients may also die from 
multiple organ failure as the disease progresses with no medical intervention (Bente et al., 
2010). Haemorrhage rarely occurs, but when they do, they may occur at injection sites, severe 
nose bleeds and severe bruising occurs as the illness progresses (CDC, 2014).  
13 
 
University of Ghana http://ugspace.ug.edu.gh
The incubation period for Dugbe virus infection in humans may range from one to 
seven days. In animals, it may vary from one to five days. The virus has been known to cause 
mild fever and, in some cases, thrombocytopenia in people (Moore et al., 1975) but it is more 
pathogenic in animals (Crabtree et al., 2009). The virus causes Nairobi sheep disease in 
animals, with symptoms such as diarrhoea accompanied with blood and mucus, difficulty in 
breathing, conjunctivitis, and discharge of bloody mucus from the nose (Coates and Sweet, 
1990). Young animals infected with the virus may die within 12 hours of onset of fever 
(Health, 2016). Pregnant female animals may have still-births. Loss of livestock due to this 
disease can become a financial burden to farmers (Crabtree et al., 2009). 
 
2.6 DIAGNOSIS OF CCHFV AND DUGBE VIRUS INFECTION 
Due to the colour of the typical West African’s skin, ecchymoses from CCHFV 
infection can sometimes go unnoticed as a symptom. When a key symptom like ecchymoses 
(Bente et al., 2013), is not noticed, the patient may not be appropriately diagnosed and their 
condition could become fatal.   Misdiagnosis is also a significant factor of high mortality in 
CCHF virus-infected patients and Dugbe virus-infected animals. Real-time RT-PCR and 
ELISA are techniques that are used in diagnosis (Vanhomwegen et al., 2012), but these types 
of diagnostic techniques may not be available in the rural areas of Ghana. Real-time RT-PCR 
detects the small RNA segment of the viral genome whereas ELISA detects the presence of 
viral antigen or host antibodies to the viral antigens (CDC, 2014). Another technique which 
can be used in identifying the viruses is Immuno-histochemical staining. This technique 
detects the presence of viral antigens in tissues fixed in formalin. This is typically done post-
mortem (Vanhomwegen et al., 2012).  
 
14 
 
University of Ghana http://ugspace.ug.edu.gh
2.7 TREATMENT 
Symptoms of the disease are like other febrile illnesses. They initially present like flu, 
often a patient who presents such symptoms may be mis-treated for a more common fever 
illness such as malaria, especially in Africa (CDC, 2014). The primary form of treatment for 
CCHF is supportive care; symptoms are treated as they manifest (WHO, 2013). Patients who 
lose a lot of blood are given blood transfusion. Body fluids that are lost can be replaced by 
intravenous fluids administered to the patient (Ergönül, 2008). 
For about two decades, Ribavirin, a drug used to treat hepatitis C and respiratory virus 
infections has been used to treat CCHFV infected patients. The drug inhibits CCHFV 
replication in patients (Bergeron et al., 2010). Ribavirin is a guanosine or adenosine analog 
depending on its rotation. The carboxamide group attached to molecule gives it this unique 
feature (Graci and Cameron, 2006). This analog binds to either cytosine or uracil. This causes 
mutation in the viral RNA and viral proteins which are eventually fatal to the virus (Ortega-
Prieto et al., 2013).  The drug has shown success in treating patients in South Africa (van de 
Wal et al., 1985), Iran and Turkey ( Jabbari et al., 2006; Smego et al., 2004; Midilli et al., 
2007) and Pakistan (Fisher-Hoch et al., 1992).  Ribavirin is believed to be more effective if 
administered in the early stage of illness (Mardani et al., 2003; Alavi-Naini et al., 2006).  
In Bulgaria, immune globulin therapy was used to treat CCHF patients (Papa et al., 
2004; Vassilev et al., 1991; Christova et al., 2009). This therapy involves the intramuscular 
injection of anti-CCHF immunoglobulin into the patient. In South Africa (van Eeden et al., 
1985b) and Turkey (Kubar et al., 2011) hyperimmune serum developed from CCHF 
survivors’ blood has also been used to treat patients. 
15 
 
University of Ghana http://ugspace.ug.edu.gh
In Dugbe virus infected humans, symptoms are treated as they appear. Adult animals 
may survive the disease if supportive treatment, improved sanitation and quality feed are 
given (Health, 2016). There are no known drugs for treating Nairobi sheep disease. 
 
2.8 CRIMEAN-CONGO HAEMORRHAGIC FEVER VIRUS 
The CCHFV is an arbovirus (viruses transmitted by arthropods) from the family 
Nairoviridae and genus Orthonairovirus (Adams et al., 2017). Nairoviruses can be 
distinguished by their large segments (Morikawa et al., 2007). Within the Orthonairovirus 
genus, there are seven serogroups. Viruses in the various serogroups are found or associated 
with different animals; Hughes, Dera Ghazi Khan and Sakhalin serogroups - birds, Crimean-
Congo Haemorrhagic Fever and Nairobi Sheep Disease (NSD) serogroups - birds, rodents, 
ungulates and humans, Thiafora and Qalyub serogroups - shrews and rodents (Adams et al., 
2017). 
Viruses within CCHF serogroup and NSDV serogroups are of significant concern 
because they are pathogenic in humans and animals, respectively. The CCHF serogroup is 
made up of Hazara, Tofla Artashat and CCCHF viruses. These viruses are pathogenic in 
humans but not animals even though they can be detected in animals (Adams et al., 2017). 
Nairobi sheep disease, Dugbe and Kupe viruses which fall within the NSD serogroup cause 
abortions in infected pregnant female livestock. This, therefore, causes a major economic 
burden on animal farmers (Coates and Sweet, 1990). These viruses are also geographically 
distributed as CCHFV since they share the same vector.  
Crimean-Congo haemorrhagic fever virus is spherical and is made up of a lipid 
envelope and three RNA segments covered with nucleocapsid proteins. The envelop is made 
up of two lipid layers and contains glycoprotein spikes (Morikawa et al., 2007). The single-
stranded RNA genome is negative-sense and is divided into three segments namely, small, 
16 
 
University of Ghana http://ugspace.ug.edu.gh
medium and large segments (Kinsella et al., 2004). The small (S) segment (1,700–2,100 
nucleotides) encodes nucleocapsid proteins that encapsulate the three RNA segments. The 
medium (M) segment which is 4,400–6,300 nucleotides long encodes glycoproteins GC and 
GN. These glycoproteins interact with host cell surface receptors during host cell invasion. 
The 11,000–14, 000 nucleotides long large (L) segment encodes RNA-dependent RNA 
polymerase which is required for RNA synthesis, Figure 2.2 shows the various components 
of CCHFV (Bente et al., 2013).  
 
 
Figure 2.2: A diagram of CCHFV showing its various components (Bente et al., 2013). 
 
 
17 
 
University of Ghana http://ugspace.ug.edu.gh
2.9 HOST CELL INVASION AND REPLICATION 
The virus targets macrophages, hepatocytes, and endothelial cells when it infects a 
host (Simon et al., 2009). Once a human host is infected by direct contact to CCHFV, the 
virus interacts with host cell surface receptors using the glycoprotein spikes on the surface of 
the viral envelop. The virus enters the cell through clathrin-dependent receptor-mediated 
endocytosis (Bente et al., 2013).  The viral lipid envelope fuses with the endosomal 
membrane of the host cell resulting in the RNA genome being transferred into the host cell 
cytoplasm. The nucleocapsid is dissociated and complementary RNA is made from the viral 
negative sense RNA. This complementary RNA is used to produce more negative sense RNA 
for new viral particles to be made (Simon et al., 2009).  
Messenger RNA (mRNA) is transcribed from the viral negative sense RNA and 
translated to essential proteins for the assembly of new viral particles. Complementary RNA 
and mRNA are generated by RNA-dependent RNA-polymerase (RdRp) found as part of the 
viral genome (Simon et al., 2009). New viral RNA-dependent RNA-polymerase, viral RNA 
and capsid proteins assemble to form new nucleocapsids. Viral mRNAs are translated into 
precursor proteins in the endoplasmic reticulum and further processed into glycoproteins in 
the Golgi body. Finally, new particles bud out of the cell through exocytosis as shown in 
Figure 2.3 (Bente et al., 2013). 
18 
 
University of Ghana http://ugspace.ug.edu.gh
 
Figure 2.3: Life cycle of CCHFV inside a host cell (Bente et al., 2013). Virions bind to host cell surface 
receptors (A) and through clathrin-dependent, receptor-mediated endocytosis, they enter the host cell (B). 
nucleocapsids are released into the cytosol after viral envelope and endosomal membranes fuse (C). 
19 
 
University of Ghana http://ugspace.ug.edu.gh
Nucleocapsids are dissociated (D) leading to the generation of mRNA and cRNA by RdRp. New viral proteins 
are assembled from translated mRNA. The cRNA is used as template for new viral RNA (vRNA) genome (E). 
Viral glycoproteins are translated in the endoplasmic reticulum (F) and are transported to the Golgi complex (G) 
for further processing (H). Matured glycoproteins are then used to form new virions (I). These new virions are 
transported to the plasma membrane and released (J). 
 
2.10 DUGBE VIRUS 
Dugbe virus has the same structural and genomic features as CCHFV and other 
viruses of the Nairovirus family (Marriott and Nuttall, 1996). Its negative sense RNA genome 
encodes glycoproteins, RNA dependent RNA polymerase and nucleocapsid (Bente et al., 
2013). Genetically, Dugbe virus is different from CCHFV through its RNA genome 
nucleotide composition (Clerex-Van et al., 1982). This variability in nucleotide composition 
results in the translation of different viral proteins unique to each virus. Genetic diversity 
among viruses in the Nairovirus family is as a result of genome recombination and 
reassortment in the tick vector (Marriott and Nuttall, 1996). This virus also uses the 
glycoproteins spikes on its envelope to invade host cells just like CCHFV (Figure 2.2) and 
also replicates in the same manner (Figure 2.3).  
Dugbe virus is mostly found in Central and East Africa but was first detected in 
Nairobi, Kenya. It has since been detected in other countries in Asia, other regions of Africa 
and the Middle East (Hotez and Kamath, 2009; David-West and Porterfield, 1974). Like 
CCHFV, the vector for Dugbe virus transmission is the ixodid tick. Ticks of the genera; 
Rhipicephalus, Amblyomma, Haemaphysalis and Hyalomma (Honig et al., 2004) are 
implicated in its transmission.  
 
20 
 
University of Ghana http://ugspace.ug.edu.gh
2.11 VECTOR OF CCHFV AND DUGBE VIRUS 
The primary vector for CCHFV and Dugbe virus includes many species of ixodid 
ticks. There are 866 identified species of ticks in the world (Madder et al., 2013b).  Ticks 
belong to the phylum Arthropoda just as insects but are in the class Arachnida with mites and 
spiders.  Ticks and mites share the same order; Acari, but there is a suborder of Ixodida that 
ticks are generally classified.  Within the order Ixodida there are two families for ticks: 
Argasidae for soft ticks and Ixodidae for hard ticks.  The dominant vector species for CCHFV 
and Dugbe virus tend to be Ixodidae (Walker et al., 2014).  
Crimean-Congo haemorrhagic fever virus has been detected in Ixodidae or hard ticks 
Hyalomma spp. and Rhipicephalus spp. (Bente et al., 2013).  The genus Rhipicephalus 
contains 80 species; they are easily identified by their hexagonal shaped capituli from the 
dorsal angle.  The genus Hyalomma are Old World ticks that are comprised of 32 species.  
However, there have been 25 tick species that have been previously reported as vectors of 
CCHFV (Hoogstraal, 1979). Tick species implicated as vectors of CCHFV in different 
regions of the world can vary, in Southern and West Africa; Hyalomma rufipes, Hyalomma 
marginatum and Hyalomma turanicum, in Madagascar; Boophilus microplus, in China and 
Uzbekistan; Hyalomma asiaticum, in Tajikisstan; Dermacentor niveus, in Pakistan; 
Hyalomma anatolicum, in Russia Balkan; Hyalomma marginatum marginaum, in Turkey; 
Hyalomma marginatum marginaum, Rhipicephalus bursa, in Greece; Rhipicephalus bursa 
(Morikawa et al., 2007).  
Dugbe virus, on the other hand, has been detected in mostly Rhipicephalus, 
Amblyomma, Haemaphysalis and Hyalomma ticks (Hotez and Kamath, 2009). In Ghana, the 
virus was detected in Amblyomma variegatum, however, in Kenya and Central Africa, the 
virus has been detected in Rhipicephalus appendiculatus. Dugbe virus has also been detected 
21 
 
University of Ghana http://ugspace.ug.edu.gh
in China from Haemaphysalis longicornis. The main vector for Dugbe virus in Asia (Sri 
Lanka and India) is the Haemaphysalis spp. (Health, 2016). 
These ticks are known to parasitize a variety of small and medium-sized wild 
mammals and are a major ectoparasite of livestock. Ticks are associated with a variety of 
hosts, which include birds, snakes, lizards, mammals and turtle species (Walker et al., 2014). 
2.11.1 Distribution of ticks  
The geographical distribution of CCHFV and Dugbe virus is a direct representation of 
the vector abundance (Morikawa et al., 2007).  Ticks maintain the virus in endemic areas 
through transovarial transmission of CCHFV and Dugbe virus; this explains the high rate of 
infected adult ticks (Garrison et al., 2013).  Dependant on the species of tick, a single female 
can oviposit a few hundred to over 20,000 eggs in one large batch (Walker et al., 2014). 
Females lay their eggs during the rainy season when the soil is moist.  
Various tick species are distributed across warmer climates around the world. This is 
because ticks require habitats that have three essential elements; high humidity, warm 
temperature and viable hosts (Sonenshine, 2018). Since ticks do not drink water, they require 
an environment that has enough moisture in the atmosphere that their bodies can absorb. 
High temperatures are required for easy movement in their environment when questing for 
hosts. Viable hosts are needed for easy access to blood, the host’s body should also make it 
easy for the ticks to firmly anchor onto it (Walker et al., 2014). The rain forest climate around 
central Africa and coastal regions and the savanna climate of the northern parts of Africa fit 
this criteria and are suitable places for ticks to survive (Walker et al., 2014). The climate 
differences across the continent also influences tick distribution as certain tick species may 
require more or less of these essential elements. Ticks found in locations that have viable 
hosts but moderately low humidity and temperatures have adapted to these habitats. Ticks can 
move to different locations geographically due to the mobility of their hosts. An example is 
22 
 
University of Ghana http://ugspace.ug.edu.gh
the detection of CCHFV infected ticks on migratory birds from northern Spain that were 
identified in Morocco (Palomar et al., 2013). 
Ghana has a good climate across its six ecological zones (Figure 2.4) which makes it 
suitable for ticks to survive. There are five genera of ticks distributed across Ghana; 
Rhipicephalus, Amblyomma, Haemaphysalis, Hyalomma, and Ixodes (Ntiamoa-Baidu et al., 
2004). Four genera within these five genera; Rhipicephalus, Amblyomma, Haemaphysalis, 
and Hyalomma have been implicated in CCHFV and Dugbe virus transmission. 
23 
 
University of Ghana http://ugspace.ug.edu.gh
 
Figure 2.4: The ecological zones of Ghana shown in different shades of purple;  Sudan Savannah, 
Guinea Savannah,  Forest Savannah Transition,  Semi-Decideous Rainforest,  High Rainforest 
and  Coastal Savannah.  show the various towns. UW – Upper West region, UE – Upper East region, NR 
– Northern region, BA – Brong Ahafo region, ASH – Ashanti region, ER – Eastern region, VR – Volta region, 
GA – Greater Accra region, CR – Central region, WR – Western region (Ntiamoa-Baidu et al., 2004). 
24 
 
University of Ghana http://ugspace.ug.edu.gh
2.11.2 Tick life cycle  
There are four stages in a tick’s life cycle: egg, larvae (looks like the adult but has six 
legs), nymph (looks like the adult but smaller), and adult.  Apart from the egg, all stages of 
the tick are parasitic. When a tick hatches from an egg into a six-legged larva, it undergoes at 
least one or more nymph instars until it is an adult (Vesco et al., 2011). Depending on the 
species, ticks can feed on one or multiple hosts to acquire a blood meal. Ixodid ticks 
essentially feed on one, two or three hosts during their lifetime whereas argasids and other 
species of ticks feed on multiple hosts. Female ticks come off their host to lay eggs on the 
ground. Unlike ixodid ticks which lay eggs once in their lifetime and die afterward, argasid 
ticks lay eggs intermittently (Estrada-Peña and Salman, 2013). The one-host tick will undergo 
all its developmental stages on the same host. It remains on the same host while it moults to 
the next stage of its lifecycle. One-host female ticks drop off their host to lay eggs on the 
ground. Two-host ticks spend their immature life stages (larva and nymph stages) on one host 
and then drop off that host onto another host at their adult stage.  Three-host ticks feed on 
three separate hosts at the three life stages; larva, nymph, and adult (Walker et al., 2014).  
2.11.3 Hunting and feeding behaviour of ticks 
Ticks will quest for a potential host in vegetation with their odour detecting sensory 
organ located on the dorsal surface of the tarsus found on their legs called Haller’s organ 
(Williams et al., 1972). The Haller’s organ is on all stages of ticks’ life cycle.  Ticks detect 
hosts through their odour, heat, and visual cues.  They gain access to the host by crawling on 
to the body of the host when the animal brushes the leaf blade or object they are hanging 
(Walker et al., 2014).  
Male and female ticks are obligate parasites and must take blood meals for 
spermatogenesis and oogenesis, respectively. The tick penetrates the host’s skin with its 
mouthparts made up of the palps, chelicerae, and the hypostome. The chelicerae are used to 
25 
 
University of Ghana http://ugspace.ug.edu.gh
cut through the host’s dermis (Figure 2.5).  Once attached to a host, a tick can feed for 3 to 7 
days (Walker et al., 2014). To initiate a blood meal, a tick inserts its hypostome into the host 
tissue to assist in anchoring the tick to the host by its curved teeth.  During feeding, the tick 
secretes a cement-like substance in its saliva which reinforces its attachment to the host.  The 
saliva also contains antihistamines and anticoagulants that decrease the host immune 
response and promote blood flow (Francischetti et al., 2010). In the process of feeding, the 
tick continuously alternates between injecting its salvia into and sucking blood from the host.  
If the tick is infected with a tick-borne pathogen, it will infect the host via the saliva (de la 
Fuente et al., 2017). A fully engorged tick will fall off its host after feeding. 
 
Figure 2.5: An ixodid tick feeding on its host (Walker et al., 2014). The tick penetrates the host’s skin 
with its chelicerae. The palp and a cement-like secretion reinforce its attachment to the host. the exchange of 
saliva from the tick and blood from the host may result in the transfer of a virus from one to the other. 
26 
 
University of Ghana http://ugspace.ug.edu.gh
2.11.4 Life cycle of CCHFV and Dugbe virus in tick vectors 
Infected adult female ixodid ticks will transovarially pass on the virus to their 
offspring when they lay eggs. Six-legged larvae hatch from the eggs and climb onto their 
hosts, blood feed and moult into nymphs (Walker et al., 2014). These nymphs also blood feed 
and moult into adult male or female ticks. At each blood feeding stage, the arthropod will 
infect its host with the virus. In the case where more than one tick feed at the same location 
on the host at the same time, the virus can be transmitted to the uninfected ticks when they 
pick up saliva of an infected tick. Uninfected ticks can also pick up the virus from infected 
hosts (CDC, 2014).  
 
2.12 METHODS OF TICK SURVEILLANCE 
Vector surveillance consists of methodical monitoring of medically important 
arthropods that are associated with the transmission of pathogens (Madder et al., 2013b).  
Surveillance can assist in understanding vector ecology: vector abundance and distribution, 
species diversity, seasonality of targeted vector.  Vector surveillance is also essential to detect 
medically important arthropod species (de la Fuente et al., 2017).  This could provide critical 
information if a potential disease-causing vector was not previously detected in the area.  
Continuous vector population monitoring and surveillance can also be helpful to determine 
the efficacy of control strategies by adding insecticide resistance assays (Madder et al., 
2013a). A systematic surveillance approach that incorporates laboratory assays can be used to 
evaluate the risk or presence of vector-borne pathogens.  Vectors collected from sentinel 
surveillance sites can be used as bio-indicators to assist in early or real-time detection of 
pathogens and evaluate infected vector abundance that can serve as possible predictor for an 
epidemic event (Estrada-Pena, 1999.).   
27 
 
University of Ghana http://ugspace.ug.edu.gh
Ticks are collected from harbouring vegetation and from host species such as goats, 
cattle, and dogs (Bryson et al., 2000).  Collecting Global Positioning System (GPS) and 
climate data during collection can associate the point of occurrence with the geographical 
location to evaluate the abundance of ticks seasonally for a single or more host species.  
Collecting free-living adult and immature ticks can be done by tick flagging or dragging 
(Sprickett et al., 1991). Using flannel strips of cloth attached to a long wooden bar, a person 
can collect ticks by pulling the bar and flannel strips by a string or twine harness for 
approximately 250 m over vegetation areas.  After reaching a set distance, the flannel strips 
are flipped to remove attached ticks that were questing in the vegetation (Ginsberg and 
Ewing, 1989). Attached ticks can also be removed with forceps and stored in vials of 70% 
ethanol. Upon reaching the lab, ticks are stored appropriately based on the assay they will be 
used for. Ticks that will be used for viral detection studies or RNA studies are stored in 
RNAlater right from the point of collection. This is to preserve the viral RNA in them 
(Madder et al., 2013a). 
Tick drags can be conducted multiple times as needed in woodland and grassland.  
Tick drags are not done over wet or early morning dew grass.  When wet, the flannel strips 
decrease in their efficacy (Sprickett et al., 1991). Adult ticks are also collected by hand from 
vegetation.  Sampling ticks from vegetation and tick drag methods are typically used to 
collect host questing exophilic ticks.  Other free-living tick sampling methods can be 
conducted using vacuum systems in nest and burrows of host animals as well as carbon-
dioxide or lure baited traps (Norval et al., 1989). Ticks collection from the live host can also 
be performed using forceps to remove them. Collecting ticks from the host will result in 
collecting mostly engorged blood fed ticks, therefore, care must be taken to not crush them 
(Koffi et al., 2012). Passive surveillance is another method of tick surveillance where 
28 
 
University of Ghana http://ugspace.ug.edu.gh
residents in an area voluntarily submit ticks they find to a lab or health center (Koffi et al., 
2012). 
 
2.13 TICK IDENTIFICATION 
2.13.1 Morphological identification 
In tick identification, the most important features to determine genus are length of 
mouthparts, the presence of eyes, conscutum (ornate or inornate), leg colour, existence or 
nonexistence of festoons, and existence or nonexistence of anal plates (Walker et al., 2014). 
For this reason, it is important that ticks collected with forceps be handled with care, so they 
do not loose vital body parts needed for accurate morphological identification. For example; 
characteristics of the genus Amblyomma include: very long mouthparts with an elongated 
second segment of palps; the conscutum and scutum ornate; the presence of eyes; the 
presence of festoons; on males the adanal plates are absent or reduced; the legs are banded  
(Walker et al., 2014).   
The genus Rhipicephalus, which was previously named Boophilus, are identified by: 
very short mouthparts with sclerotized palp segments II and III; the conscutum may be 
sclerotized and have a dark pattern of caeca that can be visualized from above; eyes are 
inconspicuous but present; festoons are absent; on males adenal plates are well defined, males 
may also have caudal processes (Madder et al., 2013a).   
The Hyalomma genus are identified by their long mouthparts with the second segment 
of palps that is elongated; the scutum are pale to dark brown; there are also convex eyes; 
festoons are visible; the adenal and accessory anal plates are sub-anal and present on males; 
legs are banded and coxae of first pair of legs long prominent posteriorly directed spurs 
(Walker et al., 2014). 
29 
 
University of Ghana http://ugspace.ug.edu.gh
Another indicator of identification is the geographical location of ticks (Madder et al., 
2013a). Certain species of ticks are restricted to certain locations geographically, this helps to 
narrow down the possible genus or species of tick being identified.  
2.13.2 Molecular identification 
Molecular identification of ticks can also be done. This identification method is more 
accurate than morphological identification. In this type of method, only a small body part of 
the tick is needed for DNA extraction (Adama et al., 2017). Primers specific for different 
species are used to determine the species of an unknown tick (Wodecka et al., 2010). This 
method can also be used with the morphological identification method. In that, when the 
genus of a tick has been determined morphologically, molecular method can be used to 
determine the species. In recent times, matrix-assisted laser desorption/ionization time-of-
flight mass spectrometry (MALDI-TOF MS) has used to identify ticks based on protein 
analysis of the ticks (Adama et al., 2017).  
Sequencing has in recent times been used to identify ticks. Even though it is 
expensive as compared to the other methods mentioned above, it is a sure way of acquiring 
the accurate information of a detected tick species (Wilhelmsson et al., 2010). Ribosomal 
subunits such as 12S, 16S or 18S genes have been established as a reliable tool for ixodid tick 
identification. Sequences from these genes are compared to online databases to help identify 
the tick specimen (Adama et al., 2017). 
 
 
 
 
 
30 
 
University of Ghana http://ugspace.ug.edu.gh
2.14 DETECTION OF CCHFV AND DUGBE VIRUS IN VECTORS AND HOSTS 
Genetic diversity across CCHFV and Dugbe virus strains is quite extensive within the 
three RNA segments, L, M and S segment (Whitehouse, 2004; Hewson et al., 2004). This 
diversity therefore requires a detection assay that will detect a broad range of strains (Deyde 
et al., 2006). Due to the similarity of CCHF symptoms to other febrile illnesses, misdiagnosis 
is very common (Vanhomwegen et al., 2012). Clinical observation, patient information and 
history are some of the useful indicators of human diagnosis of CCHF (Whitehouse, 2004). 
These diagnosis methods are not conclusive and are not helpful in distinguishing the specific 
haemorrhagic fever (Drosten et al., 2003). In the case of Dugbe virus, it is quite difficult to 
diagnose infected livestock with the virus. Cell culture, ELISA and real-time RT-PCR are the 
tools used in CCHFV and Dugbe virus detection and infection diagnosis (Burt et al., 1994). 
In most cases, ELISA is used in serological analysis; this is where antibodies for 
CCHFV and Dugbe virus, IgM and IgG, are found in infected animal or human blood 
samples. Antigens from the viruses are exposed to isolated viral antibodies from the blood 
samples (Sas et al., 2018). Detection of IgG and IgM is limited until after about seven days 
from the disease onset. At this point, a person infected with CCHFV may die since the 
disease may become fatal at this point (Kalvatchev and Christova, 2008). These antibodies 
are also rarely detectable after death (Shayan et al., 2015). There is also the issue of cross 
reactivity with antigens of viruses from the same family of Nairoviruses (Spengler et al., 
2016). Cell culture is the way of propagating the virus by injecting into tick cell lines 
(Ferraris et al., 2015, Bell-Sakyi et al., 2012 ).  
In real-time RT-PCR detection method, primers that are specific to the S segment of 
the virus are used (Atkinson et al., 2012). The use of primer sequences that target conserved 
regions on the viral S segment is important. This is because other viruses in the Nairoviridae 
family have similar RNA genome (Khurshida et al., 2015). Consensus sequence of the virus 
31 
 
University of Ghana http://ugspace.ug.edu.gh
isolates from GenBank were used to design the primers that were used in this study (Garrison 
et al., 2007). Positive results from the real time PCR show cycle threshold (Ct) values of less 
than 40 (Afonina et al., 2002).  
 
2.15 SEQUENCING AND CHARACTERISATION OF CCHFV AND DUGBE VIRUS 
DNA and RNA sequencing involve determining the composition and order of 
nucleotides of a DNA fragment (Sengupta and Cookson, 2010, Victoria et al., 2008). Several 
techniques have been developed for this process; Sanger sequencing, Pyro sequencing and 
next generation sequencing. Whereas DNA sequencing involves the sequencing of 
nucleotides in a genome, RNA sequencing shows the portion of the genome that has been 
actively expressed in the cell. RNA sequencing also gives information about the functions of 
the cell by determining the types of genes that are transcribed in the cell (Djikeng et al., 
2009). 
In the case of RNA viruses, RNA sequencing does not necessarily tell the story of the 
transcriptome but also gives an insight into the genetic makeup of the virus (Sengupta and 
Cookson, 2010). Viruses have diverse genomes and this diversity aids in proper identification 
of viruses (Edwards and Rohwer, 2005). In recent times, viruses are mainly identified using 
sequencing methods (Willner et al., 2009; Djikeng et al., 2009; Riesenfeld et al., 2004; 
Victoria et al., 2008; Edwards and Rohwer, 2005), either using Sanger sequencing or next 
generation sequencing (NGS).  
Prior to sequencing a viral genome, the sample containing the virus may or may not 
go through a lot of sample/library preparation. The aim is to minimize sample contamination 
as much as possible. However, some samples may contain small amount of viral nucleic acids 
and therefore they have to be enriched. Depending on the sequencing method, viral nucleic 
acid content and presence of carrier RNA or other contaminants in sample, some samples 
32 
 
University of Ghana http://ugspace.ug.edu.gh
may go through stringent manipulation steps before sequencing (Li et al., 2015). Sanger 
sequencing is preferred when only a target sequence is required, whereas NGS can sequence 
multiple sequence targets simultaneously. Also, there is more accuracy with NGS (Sengupta 
and Cookson, 2010). 
Sequenced data are mostly used for phylogenetic analysis this analysis involves the 
construction of phylogenetic trees that show evolutionary relationship between sequenced 
data and already known sequences in online databases. This evolutionary relationship is 
based on the similarities or differences of their genetic makeup. Bioinformatics tools are used 
for phylogenetic analysis (Ciccarelli et al., 2006). This analysis has been used over the years 
to acquire knowledge on the relationships among the viruses detected globally. 
Previous phylogenetic analysis of CCHFV has been generated using S-segment and 
M-segment. However, there are few CCHFV sequences in GenBank utilizing all three 
segments. In recent times, phylogenetic analysis is focused on the S and L segments of the 
virus. This is because, these two segments are not prone to mutations as much as the M 
segment. The M segment mutates to produce glycoproteins that will evade host immune 
defence mechanisms (Bente et al., 2013). 
The CCHFV demonstrates the greatest degree of diversity when compared to other 
arboviruses with divergence of 20, 22 and 31% among the virus isolates compared in Bente et 
al., (2013). The CCHFV has shown ancestral linkages in specific and distant geographical 
regions.  Previous phylogenetic trees have shown that there is a genetic relationship between 
the S-segment of CCHFVs circulating in Congo and Uganda while the S-segment in Senegal 
falls into its own separate clade. There is also evidence showing a linkage between the S-
segment circulating in Mauritania with those in Uganda and South Africa (Anagnostou and 
Papa, 2009).   
33 
 
University of Ghana http://ugspace.ug.edu.gh
The S-segment of viruses in Sudan, South Africa and Nigeria is shown to have direct 
genetic relationship. However, high bootstrap values have been shown to demonstrate a close 
genetic relationship between Nigerian and South African M-segment strains. This has also 
been documented in Turkey and several African countries. The Turkey-kelkit06 CCHFV 
strain was found in patient blood samples collected in 2006 in a CCHFV endemic area of 
Turkey (Ozdarendeli et al., 2010).  Phylogenetic tree created on the complete genome 
sequence of Turkey-kelkit06 strain demonstrated a close relationship to both European and 
African strains of the virus.  Phylogenetic analysis of two CCHFV strains found in the 
Xinjiang Province, China, revealed three subtypes of the virus with one cluster containing  
known sequences from West African strains (Nigeria) (Papa et al., 2002). 
34 
 
University of Ghana http://ugspace.ug.edu.gh
CHAPTER THREE 
3.0 METHODOLOGY 
3.1 CHEMICALS AND REAGENTS 
The reagents for the homogenisation and RNA extraction which included QIAamp 
Viral RNA Kit were obtained from QIAGEN, Valencia, CA. Superscript CCHFV master mix 
and reverse transcriptase Platinum Taq Mix were obtained from the immunodiagnostics and 
biologics department of the United States Army Medical Research Institute, (USAMRIID), 
Fort Dettrick, MD. Reagents used in the library preparation for next generation sequencing 
were obtained from Illumina Inc, San Diego CA. Standard chemicals and consumables were 
obtained from various commercial sources. 
 
3.2 ETHICAL CONSIDERATIONS 
Although live animals were included in the study, animals have been raised as 
livestock, not as research animals. Therefore, review by the Institutional Animal Care and 
Use Committee was not required. Informed verbal consent was obtained from the livestock 
owners, herdsmen and farm managers. Protocols for tick collection were developed as part of 
a larger study under the GS-115 project. Institutional review board (IRB) approval was sort 
from the Noguchi Memorial Institute for Medical Research (NMIMR). 
 
3.3 STUDY SITES  
Ticks were collected from seven study sites within two ecological zones in Ghana 
(guinea savannah and coastal savannah (Figure 3. 1). The seven study sites were, Navrongo 
in the Upper East region; 6th Battalion Infantry (Kamina Barracks), Air Borne Force (Barwah 
Barracks) and Airforce Base all in Tamale in the Northern region; 5th Battalion Infantry 
35 
 
University of Ghana http://ugspace.ug.edu.gh
(Burma camp), 1st Battalion Infantry (Michel camp) and Army Recruit Training School (Shai 
hills) in the Greater Accra region.  
 
 
Figure 3. 1: Map of Ghana showing the various study sites. Navrongo (grey), Airforce Base, Tamale (green), 6th 
Battalion Infantry, Tamale brown (brown) Air Borne Force, Tamale (purple), Army Recruit Training School, 
Shai hills (yellow), 1st Battalion Infantry, Michel camp (red) and 5th Battalion Infantry, Burma camp (blue). 
 
 
36 
 
University of Ghana http://ugspace.ug.edu.gh
At the Accra sites, ticks were collected monthly from December 2017 to March 2018.  
Ticks from the Northern and Upper East regions were collected twice in August 2017 and 
twice in March 2018. All the collection sites except Navrongo are Ghana Armed Forces 
military camps. These military camps have kraals where cattle are kept and sent out for 
grazing daily. Cattle are treated every other month with fipronil, a type of acaricide to rid 
them of pests especially ticks. On the other hand, the sheep, goats and dogs included in the 
study are kept in households.  
 
3.4 TICK COLLECTION, SORTING AND IDENTIFICATION  
Ticks were collected from sheep, cattle, goats and dogs and transported in RNAlater 
to the Naval Medical Research Unit No. 3 (NAMRU3) laboratory of NMIMR for further 
analysis.  The collected ticks were sorted and identified morphologically using the African 
Ixodidae identification keys (Walker et al., 2014). This involved identification based on 
unique features such as the adenal plates, mouthparts and patterns on scotum on the tick. 
Identified ticks were pooled based on species, gender, developmental stage, and animal 
source. A pool either consisted of three males or two females. Some pools comprised of 
either two males or one female depending on the size of the ticks. Pooled samples were kept 
at -80 °C until further analysis. 
 
 
 
 
37 
 
University of Ghana http://ugspace.ug.edu.gh
3.5 RNA EXTRACTION FROM TICKS 
According to the reference protocol, (Crowder et al., 2010), each tick pool was 
transferred into clean 1.5 mL tubes containing 0.15 gm of 0.1 mm beads and 0.75 gm of 2.0 
mm beads. A volume of 560 µL lysis buffer (Buffer AVL), was added to the tubes. The tick 
pools were homogenized for 2 minutes in the Mini-Beadbeater-96 and later centrifuged at 
8000 rpm for 2 minutes. Clear supernatant was transferred into clean 1.5 mL microcentrifuge 
tubes. The tubes were incubated at room temperature for 10 minutes and briefly centrifuged 
to remove drops from the lid. To each of the samples, 560 µL 96% ethanol was added and 
mixed using a vortex. The tubes were again briefly centrifuged to remove drops from inside 
the lid. RNA was extracted from the crushed ticks following the QIAamp Viral RNA Kit 
(Qiagen, Valencia, CA). QIAamp Mini spin columns were placed in a 2 mL collection tube 
and 600 µL of supernatant was carefully transferred into them without wetting the rim. With 
the caps tightly closed, the tubes were centrifuged at 8000 rpm for 1 minute. The QIAamp 
Mini spin columns were placed into clean 2 mL collection tubes and the tube containing the 
filtrate was discarded. The QIAamp Mini spin columns in new collection tubes were again 
centrifuged at 8000 rpm for 1 minute, filtrate discarded and the QIAamp Mini spin columns 
placed into clean 2 mL collection tubes. 
A volume of 500 µL wash buffer 1 (Buffer AW1) was added to the QIAamp mini 
column and centrifuged for 8000 rpm for 1 minute. This buffer washed the extract by 
removing proteins and other biomolecules aside RNA. The filtrate was discarded and the 
QIAamp mini column placed in a clean 2 mL collection tube. A volume of 500 µL wash 
buffer 2 (Buffer AW2) was added to the QIAamp mini column and centrifuged for 14000 
rpm for 3 minutes. The filtrate was discarded and the QIAamp mini column placed in a clean 
2 mL collection tube. The QIAamp mini column was placed in a clean 1.5 mL 
microcentrifuge tube and the old collection tube containing the filtrate was discarded. A 
38 
 
University of Ghana http://ugspace.ug.edu.gh
volume of 80 µL elution buffer (Buffer AVE) was carefully added to the QIAamp mini 
column and left to incubate at room temperature for 1 minute. It was then centrifuged at 8000 
rpm for 1 minute. The 1.5 mL microcentrifuge was removed, cap closed and stored at -80 oC. 
 
3.6 VIRUS DETECTION, REAL-TIME REVERSE TRANSCRIPTION PCR 
The virus was detected from the extracted RNA following the protocol by Crowder et 
al., (2010). Master mix containing, 14.6 µL Superscript CCHFV master mix, 0.4 µl reverse 
transcriptase/Platinum Taq enzyme mix was added to each well in a 96 well plate. Five 
microliters RNA extract was added to each well and the plate sealed with a micro seal B 
adhesive. The real-time reverse transcription reaction and amplification of the viral RNA S 
segment was set up on the Applied Biosystems 7300 Real-Time PCR System. The cycling 
conditions included an initial melting temperature at 50 °C for 15 minutes followed by a 
cycle at 95 °C for 5 minutes and 45 cycles of amplification (5 seconds at 95 °C, 30 seconds at 
60 °C). A final 30 second extension step at 40 °C. The superscript master mix consisted of 
2X reaction mix, 1.0 mM forward primer and reverse primer and MGB probe (0.2 mM) and 
nuclease free water. The set of primers had less diversity; the forward primer which is made 
up of 20 nucleotides binds to nucleotide region from nucleotide 649 (5` GGA VTG GTG 
VAG GGA RTT TG 3`), and the reverse primer which is also 17 nucleotides long, (5` CAG 
GGT GGR TTG AAR GC 3`), binds to nucleotide region from nucleotide 705 on the S 
segment. They also, accounted for a significant amount of diversity within the CCHFV 
strains by the presence of the nonspecific letters (ambiguous nucleotides) included in the 
primer sequences (Koehler et al., 2017). The probe for the reaction was 6FAM-
CAARGGCAARTACATMAT. 
The results were then analysed. Pools that had cycle threshold (Ct) values of less than 
29 were strong positives, an indicator of high amount amplified RNA content. Pools with Ct 
39 
 
University of Ghana http://ugspace.ug.edu.gh
values from 30 to 37 had moderate amount of amplified RNA. Pools with Ct values from 38 
to 40 had very small amount of amplified RNA. Pools within all categories were identified as 
positive.  
 
3.7 SAMPLE PREPARATION FOR NEXT GENERATION SEQUENCING 
To further characterise the isolated CCHFV genome, next generation sequencing was 
done using the USAMRIID optimised RNA-Access protocol. 
3.7.1 RNA Fragmentation 
RNA quality was evaluated using nanodrop and fragmented for cDNA synthesis. 
C6/36 cells were added to samples with lower RNA concentration. Prior to addition of cells 
to the test samples, the cells were fragmented for 7 minutes. This was done by adding 0.5 µL 
of Elute Prime Fragment Mix (EPF mix) to 0.5 µL of cells RNA (C6/36) at 94 oC. The RNA 
was diluted with nuclease-free water to a final volume of 8.5 µL in a 96-well plate. Elute 
Prime Fragment High Mix of volume, 8.5 µL was added to the RNA. The contents in the 
plate was sealed and mixed thoroughly by shaking on a microplate shaker continuously at 
1600 rpm for 20 seconds.  The sealed plate was placed on the pre-programmed thermal cycler 
and incubated at 94 oC for 0-2 minutes to fragment and prime the RNA with random 
hexamers in the EPF mix. Prior to the incubation, the thermocycler was preheated to 100 oC. 
Degraded samples were not incubated, but rather moved immediately to the next stage which 
was to Synthesize First Strand cDNA 
3.7.2 First strand cDNA synthesis  
The adhesive seal was removed from the plate and 50 µL of SuperScript IV was 
added to 450 µL of thawed First Strand Synthesis Act D Mix tube. The mixture was mixed 
by gently swirling the tube and centrifuged briefly. First Strand Synthesis Mix Act D and 
40 
 
University of Ghana http://ugspace.ug.edu.gh
SuperScript IV mix of 8 µL was added to each well, the plate was sealed with a Micro Seal B 
and mixed thoroughly by shaking the plate on a microplate shaker continuously at 1600 rpm 
for 20 seconds. The sealed plate was incubated in a pre-programmed thermal cycler with the 
following program: pre-heat lid option set to 100 oC, 25 oC for 10 minutes, 42 oC for 15 
minutes, 70 oC for 15 minutes, hold at 4 oC. The next procedure was commenced 
immediately after the thermocycler reached 4 oC. 
3.7.3 Second strand cDNA synthesis 
To each well, 5 µL resuspension buffer was added, 20 µL of thawed Second Strand 
Marking Master Mix was also added after the tube had been centrifuged. The plate was 
sealed with a Micro-Seal B and its contents thoroughly mixed by shaking it on a microplate 
shaker continuously at 1600 rpm for 20 seconds. The plate was incubated on a pre-heated 
thermal cycler at 16 oC for 1 hour. The seal was removed, and the plate was left to cool for 2 
minutes. 
3.7.4 Sample clean-up 
The tube containing the thawed AMPure XP beads was thoroughly mixed using a 
vortex to evenly disperse them. An amount of 90 µL of the beads were added to each well in 
the plate. The plate was sealed and its contents, thoroughly mixed by shaking on a micro 
plate shaker at 1800 rpm for 2 minutes. The plate was incubated at room temperature for 5 
minutes and later moved onto a magnetic stand for 2 minutes. An amount of 135 µL of clear 
supernatant was discarded removed from each well. With the plate still on the stand, 200 µL 
of freshly prepared 80% ethanol was added to each well gently without disturbing the beads. 
The plate was left to stand for 30 seconds after which the 80% ethanol was discarded. 
Ethanol was added one more time and discarded as indicated earlier. Using a 10 µL 
multichannel pipette, remaining ethanol was removed from each well without disturbing the 
beads. 
41 
 
University of Ghana http://ugspace.ug.edu.gh
The plate was left to stand at room temperature for 5 minutes to dry on the magnetic 
stand. The plate was removed from the magnetic stand and 17.5 µL resuspension buffer was 
added to each well. The plate was sealed and shaken on the micro plate shaker at 1800 rpm 
for 2 minutes. The plate was incubated at room temperature for 2 minutes and centrifuged at 
280 g for 1 minute. The seal was removed and the plate placed on the magnetic stand for 1 
minute. A volume of 15 µL of clear supernatant was transferred from each well of the plate to 
a new plate.  
3.7.5 3’-ends adenylation 
A volume of 2.5 µL resuspension buffer was added to each well of the plate. Thawed 
A-Tailing Mix tube was centrifuged at 600 g for 5 seconds and 12.5 µL added to each well of 
the plate. Contents in the sealed plate were thoroughly mixed by shaking the plate on a 
microplate shaker at 1800 rpm for 2 minutes. The plate was centrifuged at 280 g for 1 minute. 
The plate was incubated in a thermocycler at 37 oC for 30 minutes. The plate was removed 
immediately and placed in another thermocycler to incubate at 70 oC for 5 minutes. The plate 
was immediately removed from the thermocycler after incubation and placed on ice for 1 
minute. 
3.7.6 Ligation of adapters 
TruSeq dual index (TruSeq RNA Adapter Index) was diluted at a ratio of 1:1 with 
nuclease free water, 2.5 µL each of resuspension buffer and Ligation Mix was added to each 
well after which 2.5 µL of the diluted TruSeq RNA Adapter Index was also added. The plate 
was sealed with Micro-Seal ‘B’ and its contents thoroughly mixed by shaking on a microplate 
shaker at 1800 rpm for 2 minutes. The plate was centrifuged at 280 g for 1 minute and 
incubated in a thermocycler at 30 oC for 10 minutes. 
42 
 
University of Ghana http://ugspace.ug.edu.gh
After removing the adhesive seal covering the plate, 5 µL of Stop Ligation Buffer was 
added to each well to inactivate the ligation mix. The contents in the plate were thoroughly 
mixed by shaking on a microplate shaker at 1800 rpm for 2 minutes after the plate had been 
sealed. The plate was centrifuged at 280 g for 1 minute. 
3.7.7 Sample clean-up  
The tube containing the thawed AMPure XP beads was thoroughly mixed using a 
vortex to evenly disperse them. To each well in the plate, 42 µL of the beads were added, the 
plate was sealed. The contents were thoroughly mixed by shaking on a micro plate shaker at 
1800 rpm for 2 minutes. The plate was incubated at room temperature for 5 minutes and 
moved onto a magnetic stand for 2 minutes. An amount of 79.5 µL of the supernatant was 
removed from each well and discarded. With the plate still on the stand, 200 µL of freshly 
prepared 80% ethanol was added to each well gently without disturbing the beads. The plate 
was left to stand for 30 seconds after which the 80% ethanol was removed and discarded. 
Ethanol was added one more time and discarded as indicated above. Using a 10 µL 
multichannel pipette, remaining ethanol was removed from each well without disturbing the 
beads. 
The plate was left to dry at room temperature for 5 minutes on the magnetic stand. 
The plate was removed from the magnetic stand and 52.5 µL resuspension buffer added to 
each well. The plate was sealed and shaken on the micro plate shaker at 1800 rpm for 2 
minutes. The plate incubated at room temperature for 2 minutes, centrifuged at 280 g for 1 
minute. The seal was removed and the plate placed on the magnetic stand for 1 minute. A 
clear supernatant of 50 µL was transferred from each well of the plate to a new plate. 
To each well in the plate, 50 µL of thawed and well mixed AMPure XP beads were 
added. The plate was sealed and its contents, thoroughly mixed by shaking on a micro plate 
43 
 
University of Ghana http://ugspace.ug.edu.gh
shaker at 1800 rpm for 2 minutes. The plate was incubated at room temperature for 5 
minutes, moved onto a magnetic stand for 2 minutes. An amount of 95 µL of the supernatant 
was removed from each well and discarded. With the plate still on the stand, 200 µL of 
freshly prepared 80% ethanol was added to each well gently without disturbing the beads. 
The plate was left to stand for 30 seconds after which the ethanol was removed and 
discarded. Ethanol was added one more time and removed as indicated above. Using a 10 µL 
multichannel pipette, remaining ethanol was removed from each well without disturbing the 
beads. 
The plate was left to dry on the magnetic stand at room temperature for 5 minutes. 
The plate was removed from the magnetic stand and 22.5 µL resuspension buffer was added 
to each well. It was sealed and shaken on the micro plate shaker at 1800 rpm for 2 minutes. 
The plate was incubated at room temperature for 2 minutes and centrifuged at 280 g for 1 
minute. The seal was removed, and the plate placed on the magnetic stand for 1minute. A 
clear supernatant of 20 µL was transferred from each well of the plate to a new plate.  
3.7.8 First PCR amplification 
To each well in the PCR plate, 5 µL and 25 µL of thawed PCR Primer Cocktail and 
PCR Master Mix were added respectively. The plate was sealed and shaken on the micro 
plate shaker at 1600 rpm for 20 seconds. The plate was incubated at room temperature for 2 
minutes, centrifuged at 280 g for 1 minute. The plate was placed in a thermocycler and 
amplification was done under the following conditions. The pre-heat lid option was selected 
and set to 100 oC, 98 oC for 30 seconds, 20 cycles of: (98 oC for 10 seconds, 60 oC for 30 
seconds), 72 oC for 30 seconds, 72 oC for 5 minutes and hold at 4 oC. 
44 
 
University of Ghana http://ugspace.ug.edu.gh
3.7.9 Sample clean-up 
To each well in the plate, 50 µL of the thawed and well mixed AMPure XP beads 
were added. The plate was sealed and its contents, thoroughly mixed by shaking on a micro 
plate shaker at 1800 rpm for 2 minutes. It was then incubated at room temperature for 5 
minutes moved onto a magnetic stand for 2 minutes. An amount of 95 µL of the supernatant 
was removed from each well and discarded. With the plate still on the stand, 200 µL of 
freshly prepared 80% ethanol was added to each well gently without disturbing the beads. 
The plate was left to stand for 30 seconds after which the 80% ethanol was removed and 
discarded. Ethanol was added one more time and removed as indicated above. Using a 10 µL 
multichannel pipette, remaining ethanol was removed from each well without disturbing the 
beads. 
The plate was left to stand at room temperature for 5 minutes to dry on the magnetic 
stand. It was removed from the magnetic stand and 17.5 µL resuspension buffer was added to 
each well. The plate was sealed and shaken on the micro plate shaker at 1800 rpm for 2 
minutes. It was then incubated at room temperature for 2 minutes, centrifuged at 280 g for 1 
minute. The seal was removed, and the plate placed on the magnetic stand for 1 minute. A 
clear supernatant of 15 µL was transferred from each well of the plate to a new plate. 
3.7.10 Library validation 
The concentration of the DNA library was measured in nanogram using a Qubit. DNA 
libraries with standard concentrations were first measured and the concentration of the DNA 
libraries of the samples were measured against those of the standards. 
3.7.11 First hybridization 
The volume was calculated to a final concentration of 200 ng in a final volume of 
11.25 µL. The maximum volume required for a sample was 11.25 µL. Therefore, for samples 
45 
 
University of Ghana http://ugspace.ug.edu.gh
with volumes less than 11.25 µL, the volume was brought up to 11.25 µL with nuclease-free 
water. Also, samples whose final concentration could not reach 200 ng, their initial 
concentrations were used with a volume 11.25 µL was used. The reagents were added to each 
well in the 96 well plate in the following order to make a total volume of 25 µL. Library 
sample (DNA) - 11.25 µL, resuspended Capture Target Buffer 3 (CT3) - 12.50 µL and 
Capture Oligos 1X Stock - 1.25 µL. The plate was sealed with a Micro-seal ‘B’ adhesive seal 
and its contents thoroughly mixed on a microplate shaker at 1200 rpm for 1 minute and 
centrifuged at 280 g for 1 minute. The sealed plate was placed on a pre-programmed thermal 
cycler under the following conditions: Preheat lid at 100 oC, 10 minutes at 95 oC, 18 cycles of 
1-minute incubation, starting at 94 oC, then decreasing 2 oC per cycle, 95 minutes at 58 oC. 
3.7.12 First capture 
The plate was centrifuged at 280 g for 1 minute. To each well in the plate, 62.5 µL of 
well mixed Streptavidin Magnetic Beads were added. Contents in the sealed plate were 
thoroughly mixed on a microplate shaker at 1200 rpm for 5 minutes. The plate was left to 
stand at room temperature for 25 minutes. The plate was centrifuged at 280 g for 1 minute, 
the adhesive tape removed, and the plate placed on the magnetic stand for 1 minute at room 
temperature. The supernatant was carefully removed and discarded from each well in the 
plate without disturbing the beads. The plate was removed from the magnetic stand. 
3.7.13 First Wash 
To each well in the plate, 50 µL of thoroughly mixed Enrichment Wash Solution was 
added. The entire contents in each well was gently pipetted up and down three times to 
ensure complete resuspension of the sample. The plate was sealed and its contents thoroughly 
mixed on a microplate shaker at 1800 rpm for 4 minutes. The sealed plate was incubated at 
50 oC for 20 minutes in a thermocycler. The adhesive tape was removed, and the plate placed 
on the magnetic stand for 2 minutes at room temperature. Clear supernatant was carefully 
46 
 
University of Ghana http://ugspace.ug.edu.gh
removed and discarded from each well of the plate without disturbing the beads. The plate 
was removed from the magnetic stand. This process was repeated one more time to make a 
total of two washes. 
3.7.14 First Elution 
To make an elution pre-mix, the reagents were added to a new 1.5 mL 
microcentrifuge tube in the following order 7.12 µL Enrichment Elution Buffer 1 (EE1), 0.38 
µL 2 M NaOH to make a total volume of 7.5 µL. Six microliters of elution pre-mix was 
added to each well. The plate was sealed and its contents thoroughly mixed on a microplate 
shaker at 1800 rpm for 2 minutes. The sealed plate was left to stand at room temperature for 2 
minutes. The plate was centrifuged at 280 g for 1 minute. The adhesive tape was removed 
and the plate placed on the magnetic stand for 2 minutes at room temperature. Clear 
supernatant of about 5.25 µL was transferred from well into a new plate. The plate was 
removed from the magnetic stand. To neutralize the elution, 1 µL Elute Target Buffer 2 was 
added to each well of the new plate containing the samples. The plate was sealed and its 
contents thoroughly mixed on a microplate shaker at 1200 rpm for 1 minute. The plate was 
centrifuged at 280 g for 1 minute. 
3.7.15 Second hybridization 
Resuspension buffer of volume, 5 µL, 12.5 µL Capture Target Buffer 3 (CT3) and 
1.25 µL Capture Oligos 1 X Stock were added to each well in the 96 well plate. The plate 
was sealed with a Micro-seal ‘B’ adhesive seal and its contents thoroughly mixed on a 
microplate shaker at 1200 rpm for 1 minute and centrifuged at 280 g for 1 minute. The sealed 
plate was placed on a pre-programmed thermal cycler under the following conditions: Preheat 
lid at 100 oC, 10 min at 95 oC, 18 cycles of 1-minute incubations, starting at 94 oC, then 
decreasing 2 oC per cycle, 95 minutes at 58 oC. 
47 
 
University of Ghana http://ugspace.ug.edu.gh
A second capture, second wash, and second elution were done following the same 
steps as the first capture, wash and elution procedures. 
3.7.16 Capture sample clean-up 
To each well of the plate, 11.25 µL of well-mixed AMPure XP beads was added. The 
plate was sealed with a Micro-seal ‘B’ adhesive seal, its contents thoroughly mixed by 
shaking it on a microplate shaker at 1800 rpm for 1 minute. It was then incubated at room 
temperature for 5 minutes and placed on the magnetic stand for 2 minutes. All the supernatant 
was removed from each well and discarded. With the plate on the magnetic stand, 200 µL 
freshly made 80% ethanol was slowly added to each well without disturbing the beads. The 
plate was left to stand at room temperature for 30 seconds. The 80% ethanol was carefully 
removed from each well and discarded. The 80% ethanol was added to the wells and removed 
again as stated above. This made up two washes. Using a 10 µL multichannel pipette, 
remaining ethanol was removed from each well without disturbing the beads. The plate was 
left on the magnetic stand to stand at room temperature for 5 minutes to dry. The plate was 
removed from the magnetic stand and 7 µL resuspension buffer was added to each well. The 
plate was sealed with a Micro-seal ‘B’ adhesive seal and its contents thoroughly mixed on a 
microplate shaker at 1800 rpm for 2 minutes. The plate was left to stand at room temperature 
for 2 minutes. The plate was centrifuged at 280 g for 1 minute, seal removed and left on the 
magnetic stand for 2 minutes at room temperature. Clear supernatant of 6.25 µL was 
carefully removed from each well without disturbing the beads and transferred into a new 
plate.  
3.7.17 Second PCR amplification 
In this process, the DNA library was enriched through PCR amplification for 
sequencing. To each well in the plate, 1.25 µL PCR Primer Cocktail and 5 µL Enhanced PCR 
Mix were added in that order. The plate was sealed with a Micro-seal ‘B’ adhesive seal and 
48 
 
University of Ghana http://ugspace.ug.edu.gh
its contents thoroughly mixed on a microplate shaker at 1200 rpm for 1 minute and 
centrifuged at 280 g for 1 minute. The seal was removed, and the plate placed on a pre-
programmed thermal cycler under the following conditions: Preheat lid at 100 oC, 98 oC for 
30 seconds, 17 cycles of: (98 oC for 10 seconds, 60 oC for 30 seconds, 72 oC for 30 seconds), 
72 oC for 5 minutes. Hold at 10 oC. 
3.7.18 Second PCR clean-up 
This process used AMPure XP beads to purify the enriched library and remove 
unwanted products. The same procedure used in the first PCR clean-up (section 3.7.19) was 
used here with a few changes to the volumes of AMPure XP beads (22.5 µL), and 
Resuspension buffer (11 µL). At the end of the clean-up, 10 µL clear supernatant was 
carefully removed from each well without disturbing the beads and transferred into a new 
plate. 
3.7.19 Quality assessment of Library 
To assess the quality of the enriched library, 1µL of the library was loaded on Agilent 
High Sensitivity DNA Chip. The size of the library was checked for distribution of DNA 
fragments with a size range from approximately 200 bp -1kb. All samples were normalized to 
2 nm. Concentrations of the samples were measured in pg/µl using the bioanalyzer. 
3.7.20 Library qPCR 
To ensure that an accurate concentration of the samples was loaded for sequencing, 
each library was also quantified by quantitative PCR using the KAPA SYBER FAST 
Universal qPCR kit (Illumina) (KAPA Biosystems, KK4824). For a 10 µL reaction volume, 
1.8 µL nuclease-free water, 6 µL 2X KAPA SYBR FAST qPCR Master Mix with primers, 2 
µL diluted 1:1000/1:5000 pooled libraries and 0.2 µL 50X ROX Low were added to each 
49 
 
University of Ghana http://ugspace.ug.edu.gh
well on 1 cycle initial denaturation at 95 °C for 3 minutes, 40 cycles denaturation at 95 °C for 
1-3 seconds and annealing/extension/data acquisition at 60 °C for 30 seconds 
3.7.21 Normalization and pooling 
All samples were normalized to the same concentration of 1 nm to have an equal 
representation of each sample. Sample concentrations were changed from pico-mole since 
that was the concentration of samples from qPCR to nano-mole and calculated as described 
below. Sample concentrations were normalized with the required volume of resuspension 
buffer. 
Calculation with the dilution factor: (sample concentration) (dilution factor: 1000)/1000 
(2.99 pmol) (1000 dilution factor)/1000 = 2.99 nm 
In a 1.5 mL microfuge tube, the total volume of resuspension buffer needed to 
normalize each sample to 1 nm was added and the corresponding volume for the individual 
samples was added. The tube was briefly vortexed and centrifuged. This tube was labelled as 
tube 1. 
3.7.22 MiSeq loading preparation 
PhiX (product of bacteriophage Phi X174) was diluted from 10 nM to 2 nM PhiX 
using resuspension buffer with volumes, 1 µL 10 nM PhiX + 9 µL resuspension buffer. The 
tube was labelled 2; it was briefly vortexed and centrifuged. Using resuspension buffer, 2 M 
NaOH was diluted to 0.2 M NaOH using the volumes 2 µL of 2 M NaOH and 18 µL 
resuspension buffer. The tube was labelled as 3 and briefly vortexed and centrifuged.  
In a new 1.5 mL tube labelled 4, 2 µL of diluted PhiX from tube 2 and 18 µL pool 
library from tube 1 was added, the content of the tube was mixed using a vortex and 
centrifuged. To tube 4, 20 µL of 0.2 M NaOH was added. The content of the tube was mixed 
using a vortex, centrifuged and incubated at room temperature for 5 minutes. To the same 
50 
 
University of Ghana http://ugspace.ug.edu.gh
tube labelled 4, 20 µL resuspension buffer was added. Also, 940 µL pre-chilled HT1 buffer 
was added to make up 1000 µL. The content of the tube was mixed using a vortex, 
centrifuged and kept on ice. To dilute the library to a final concentration of 20 pM, 400 µL 
pre-chilled HT1 was added to 600 µL of library from tube 4 into a new tube labelled 5. The 
diluted library from tube 5 was loaded into the sample compartment of the thawed MiSeq 
cartridge. The cartridge was loaded onto the MiSeq desktop sequencer (Illumina). 
 
3.8 STATISTICAL ANALYSIS 
For categorical variables, chi-square was used to determine if there was any statistical 
association between the various sites. The statistical significance level was set at p-value 
<0.05 with a degree of freedom of 2. Bar charts were used to describe the tick species 
distribution and animal sources of ticks across the seven study sites. Bar chart was also used 
to compare the number of ticks collected from the two ecological zones. Tables were used to 
describe tick species distribution, tick sex and animal source of ticks between the two 
ecological zones.  
The infection rate of the virus in pools of different species was detected using 
PoolScreen 2.0. Software Version 2.0.1 (Katholi and Unnasch, 2006). This calculation was 
done at 95% confidence interval and it included the pool size used, the number of pools 
studied and the number of negative pools. 
 
 
  
51 
 
University of Ghana http://ugspace.ug.edu.gh
CHAPTER FOUR 
4.0 RESULTS 
4.1 TICK COLLECTION  
A total of 1,813 ticks were collected and grouped into 813 pools. Ticks collected from 
the Guinea savannah ecological zone made up 70% of the total number whereas, ticks 
collected from the Coastal savannah made up 30%. The difference in the proportion of ticks 
from the two ecological zones was statistically significant (P<0.01), this difference shows 
that there is an association between tick abundance and ecological zones Figure 4. 1 gives an 
overview of the total number of tick distribution across the two ecological zones. 
 
 
Figure 4. 1: Distribution of ticks from the two ecological zones, blue bars show the number of ticks collected 
from the ecological zones. Comparison of tick distribution using Chi square at a p value of <0.05. 
 
 
52 
 
University of Ghana http://ugspace.ug.edu.gh
Male ticks accounted for 72% of the total number of ticks collected whereas females 
recorded 28% (Table 4. 1). Across the seven study sites, more male ticks were collected than 
female ticks. The highest number of males and females were collected from the Tamale 
Airborne force barracks (16%) and Navrongo (10%) respectively. Michel camp (3%) and 
Kamina barracks (1%) recorded the lowest number of males and females respectively. Since 
both male and female ticks can transmit both CCHFV and Dugbe virus, the difference in the 
number of male and female ticks collected is not statistically significant in CCHFV infection 
rate. The number of male ticks collected was statistically significant (p<0.01). 
 
Table 4. 1: Distribution of tick sex across the two ecological zones 
Vector sex Guinea savannah Coastal savannah Total 
Female 298 211 509 
23.39 39.15 28.08 
Male 976 328 1,304 
76.61 60.85 71.92 
Total 1274 539 1,813 
70.27 29.73 100 
           
 
At 95% confidence interval and p value of less than 1, there was a significant 
difference in the tick species collected from each ecological zone. Amblyomma variegatum 
represented about 66% of the tick species collected from all the ecological zones making it 
the highest number of tick species collected and Rhipicephalus boophilus, the least number of 
tick species collected. Ticks of the Rhipicephalus genus, represented about 24% of total 
53 
 
University of Ghana http://ugspace.ug.edu.gh
number of ticks collected. Most of the ticks in this genus were in the sanguineus species. The 
genus Hyalomma made up about 10% of the total ticks collected making them the least 
number of ticks collected (Table 4. 2).  
 
Table 4. 2: Distribution of tick species across the ecological zones. 
Morphological ID Guinea savannah Coastal savannah Total 
Amblyomma variegatum 751 449 1,200 
58.95 83.3 66.19 
Hyalomma rufipes 12 40 52 
0.94 7.42 2.87 
Hyalomma truncatum 126 1 127 
9.89 0.19 7 
Rhipicephalus sanguineus 372 41 413 
29.2 7.61 22.78 
Rhipicephalus boophilus 0 1 1 
0 0.19 0.06 
Rhipicephalus evertsi 13 7 20 
1.02 1.3 1.1 
Total 1274 539 1813 
70.27 29.73 100 
  
 
 
54 
 
University of Ghana http://ugspace.ug.edu.gh
From all the seven study sites, Amblyomma variegatum was the highest number of 
tick species collected except for Navrongo where 95.14% of the ticks collected were of the 
Rhipicephalus genus.  Ticks from the Amblyomma variegatum species and the Hyalomma 
genus were detected in all seven study sites whereas the Rhipicephalus genus was detected in 
only four study sites namely, Navrongo, Burma camp, Michel camp and Shai hills (Figure 4. 
2 and Figure 4. 3). The difference in the proportion of the various species of ticks from the 
seven sites was statistically very significant (P<0.01). This is an indication that there is an 
association between the study sites/location and the tick species. This also correlates to the 
ecological zones and tick species. 
 
 
Figure 4. 2: Tick species distribution across the seven study sites.  Amblyomma variegatum,   Hyalomma 
rufipes,  Hyalomma truncatum. Comparison of tick species distribution using Chi square at a p value of <0.05.  
 
 
 
55 
 
University of Ghana http://ugspace.ug.edu.gh
 
Figure 4. 3: Tick species distribution across the seven study sites.  Rhipicephalus sanguineus,  Rhipicephalus 
boophilus,  Rhipicephalus evertsi. Comparison of tick species distribution using Chi square at a p value of 
<0.05. 
 
The animal sources of ticks in this study were sheep, cattle, dogs and goats. In all, 
cattle were the highest source of ticks (82%), dogs were the second highest (17%), and the 
least number of ticks collected were from goats and sheep, 0.4% and 0.6%, respectively 
Table 4. 3. Ticks were collected from only cattle in all the six military sites; however, in 
Navrongo, ticks were collected from all four animal sources (Figure 4. 4). The highest 
number of ticks collected in Navrongo were from dogs (80%) and this made up 17% of the 
total number of ticks collected. Navrongo also recorded a much lower number of ticks from 
cattle (n=60). 
 
 
56 
 
University of Ghana http://ugspace.ug.edu.gh
Table 4. 3: Distribution of animal sources of ticks across the two ecological zones. 
Animal source Guinea savannah Coastal savannah Total 
Cattle 943 539 1,482 
74.02 100 81.74 
Dog 314 0 314 
24.65 0 17.32 
Goat 7 0 7 
0.55 0 0.39 
Sheep 10 0 10 
0.78 0 0.55 
Total 1274 539 1,813 
70.27 29.73 100 
              
 
 
Figure 4. 4: Distribution of animal source of ticks from the seven study sites.    Cattle,  Dog,  Goat,  Sheep. 
Comparison of tick hosts using Chi square at 95% confidence interval.  
57 
 
University of Ghana http://ugspace.ug.edu.gh
4.2 CCHFV DETECTION 
Of the total number of ticks collected (1,813) ~1% (n=15) tested positive for CCHFV. 
Seven pools (0.9%) of the 813 pools tested positive for the presence of the CCHFV. Table 4. 
4 shows the positive pools, the site from which they were collected, animal source, tick 
species and vector sex. The cycle threshold (Ct) values for the 7 positive pools ranged from 
strong positives to weak positives are indicated in Figure 4. 5 and Figure 4. 6 below. 
 
Table 4. 4: Details of CCHFV positive pools 
 Region Study site Host Tick species Vector sex Pool size 
1. Greater Shai hills Cattle Hyalomma rufipes Female 1 
2**. Accra Michel camp Cattle Amblyomma variegatum Male 2 
3.  Airforce base Cattle Amblyomma variegatum Male 2 
4. Northern Airforce base Cattle Amblyomma variegatum Male 3 
5.  Navrongo Sheep Rhiphicephalus sanguineus Male  2 
6. Upper East Navrongo Dog Rhiphicephalus sanguineus Male 2 
7. Region Navrongo Dog Rhiphicephalus sanguineus Male 3 
** Even though sample two initially tested positive for CCHFV, in a second assay which had more specific 
CCHFV primers showed ct values that gave an indication of low CCHFV RNA content. Also, full genome 
sequence showed reads of Dugbe virus sequence. This could mean that the pool was coinfected. 
 
 
 
58 
 
University of Ghana http://ugspace.ug.edu.gh
Positive control (1:10) - Ct 27.1973
Pool 1 - Ct 36.552
Positive control (1:100) - 32.1363
Pool 2 -Ct 37.6351
Positive control (1:10) - Ct 30.023
Positive control (1:100) - Ct 36.016
Pool 3 - Ct 32.425
Pool 4 -  Ct 26.179
Positive control (1:10) - Ct 32.023
Positive control (1:100) - Ct 37.983
 
Figure 4. 5: Positive curves for pools 1, 2, 3 and 4 from the real-time RT-PCR. CCHFV detection assay 
targeting the S segment of genome (Koehler et al, 2017). 
 
59 
 
University of Ghana http://ugspace.ug.edu.gh
Positive control (1:10) - Ct 32.015
Pool 5 - Ct 28.673
Positive control (1:100) - Ct 38.859
Positive control (1:10) - Ct 26.623
Pool 7 - Ct 31.325
Positive control (1:100) - Ct 37.431
 
Figure 4. 6: Positive curves for pools 5, 6 and 7 from the real-time RT-PCR. CCHFV detection assay targeting 
the S segment of genome (Koehler et al, 2017). 
 
4.3 CCHFV INFECTION RATE  
Hyalomma rufipes recorded the highest rate of infection among the three species that tested 
positive for CCHFV. Even though Rhipicephalus sanguineus and Amblyomma variegatum 
were the highest number of CCHFV infected ticks, they had the lower infection rates of 
0.70% and 0.25%, respectively (Table 4. 5). 
60 
 
University of Ghana http://ugspace.ug.edu.gh
Table 4. 5: Prevalence of CCHFV among different tick species collected from Greater 
Accra, Northern and Upper East region 
Tick species Male (%) Female (%) Overall infection rate (%) 
Amblyomma variegatum 0.30  0 0.25 
(0.06a-0.88b) (0.05a-0.72b) 
Hyalomma rufipes 0 4.17 1.87 
(0.13a-19.68b) (0.06a-9.34b) 
Rhipicephalus sanguineus 1.55  0 0.70  
(0.30a-4.45b) (0.14a-2.02b) 
a, b Minimum and maximum likelihood of infection respectively 
 
4.4 NEXT GENERATION SEQUENCING AND PHYLOGENETIC ANALYSIS 
To further confirm and identify the isolated viral genome as that of CCHFV, next 
generation sequencing was done using the USAMRIID RNA-Access protocol. This protocol 
aimed at targeting unknown RNA viral genome with a variety of viral probes. Analysis of the 
sequencing data from the PCR positive CCHFV pools showed reads from one pool (sample 
2) aligning to the full genome sequence of each segment of Dugbe virus as shown in Figure 
4. 7 and Figure 4. 8. No reads specific for CCHFV or Dugbe virus were detected in the 
remaining six PCR-positive pools. The six pools tested positive for CCHFV after a second 
run. The detection of Dugbe virus genome in one of the pools confirmed a theory of CCHFV 
and Dugbe virus coinfection. 
Sequence alignment using muscle and phylogenetic analysis were done on the 
sequenced data using MEGA6 software. The L and S segments of the Dugbe virus was 
phylogenetically analysed with Dugbe virus strains and reference strains from the 
61 
 
University of Ghana http://ugspace.ug.edu.gh
Orthonairovirus genus and Nairoviridae family. Using maximum likelihood tree with 
bootstrap value of 1000, the L and S segments of the detected Dugbe virus had closer 
relationships with Dugbe virus strains from Kenya and Nigeria. 
 
Figure 4. 7: Phylogenetic tree illustrating the relationship between the L segments of the new Dugbe virus 
detected in Ghana, other Dugbe virus strains and other viruses in the Nairoviridae family. The tree was obtained 
using MEGA 6 software from MUSCLE sequence alignment.  The new Dugbe virus detected in the red box. 
The first virus detected in Pokuase blue box. Bunyamwera virus is the root of the tree. Strains that are closely 
related are highlighted in the yellow box. 
62 
 
University of Ghana http://ugspace.ug.edu.gh
 
Figure 4. 8: Phylogenetic tree illustrating the relationship between the S segments of new Dugbe virus detected 
in Ghana, other Dugbe virus strains and other viruses in the Nairoviridae family. The tree was obtained using 
MEGA 6 software from MUSCLE sequence alignment. The new Dugbe virus detected in red box. Bunyamwera 
virus is the root of the tree. Strains that are closely related are highlighted in the yellow box.  
63 
 
University of Ghana http://ugspace.ug.edu.gh
CHAPTER FIVE 
5.0 DISCUSSION 
It is well documented that CCHFV distribution is directly proportional to the 
abundance or presence of the tick vector (Sang et al., 2011). However, there is an uncertainty 
of the actual global distribution of CCHFV.  There is little information in literature that can 
map out the true distribution of CCHF, possibly due to a lack of cases reported. This project 
aimed to acquire a better understanding of CCHFV circulating in Ghana and assess its genetic 
lineage.  The detection of CCHFV in Ghana, 2016 contributed in filling in a knowledge gap 
since CCHF was previously listed as undetermined in literature (Bente et al., 2013).  The 
field collected ticks that tested positive for CCHFV with real-time RT-PCR in this 
surveillance effort contributed more data points that agreed with the findings in Akuffo et al., 
(2016).  
 
5.1 ECOLOGICAL ZONES AND TICK DISTRIBUTION 
During this study, three tick genera were detected in the country across the two 
ecological zones sampled. The ecological zones sampled were; coastal savanna which covers 
the Greater Accra region and guinea savanna which covers the Northern and parts of the 
Upper East regions. The coastal savanna records an annual rainfall of 600 mm. The guinea 
savanna records an annual rainfall of 1000 mm.  
Previously reported by Ntiamoa-Baidu et al., (2004); five genera of ticks are 
distributed across the ecological zones of Ghana. The five genera are, Amblyomma, Ixodes, 
Haemaphysalis, Hyalomma, and Rhipicephalus. The high abundance of Amblyomma and 
Rhipicephalus genera found in both ecological zones supports what is already described by 
Ntiamoa-Baidu et al., 2004. The study also indicated that the most commonly found tick 
genera in the guinea savannah zone; Rhipicephalus, Amblyomma, Ixodes, Hyalomma. 
64 
 
University of Ghana http://ugspace.ug.edu.gh
Rhipicephalus, Amblyomma, Haemaphysalis, and Ixodes genera are commonly found in the 
coastal savannah. Again, Haemaphysalis and Ixodes were not found in the guinea savannah 
zone. The high abundance of Amblyomma variegatum in all seven study sites is an indication 
of how widely distributed this species is across the two ecological zones. 
In the rainy season, the soil is moist and provides a suitable environment for female 
ticks to oviposit.  Environmental conditions such as high temperatures that occur in the dry 
season provide ideal conditions to promote transitions from larval stages to adult and 
provides optimal breeding habitat.  This was one of the reasons ticks were collected in the dry 
season (December) to the beginning of the rainy season (March) in Accra.  Collections in the 
Northern and Upper East regions were done from August before the second rainy season 
through the dry season in December to March the following year, right before the rains 
began, to capitalize on the high abundance of the tick population. The guinea savannah zone 
experiences longer dry season than rainy season, whereas the coastal savannah experiences 
longer rainy seasons than dry seasons. The seasonality difference in the guinea savannah and 
coastal savannah ecological zones of the country may be one of the major factors why more 
ticks were collected from the Northern and Upper East regions than the Greater Accra region.  
The species from Amblyomma, Hyalomma, and Rhipicephalus genera have the highest 
impact on human and animal health. These three genera are among the five genera previously 
documented as CCHFV vectors and implicated for CCHFV transmission.  This study 
confirms what is reported in Sang et al., (2011); that the presence or abundance of these 
vectors in Ghana could mean that the virus is also present. This could also lead to the 
assertion that the CCHFV vector can also be carrying other disease-causing pathogens that 
can affect both humans and animals.  If the vector burden is too high, it could cause major 
economic loss to a nation by affecting human and animal health and productivity (Rajput et 
al., 2006; Vesco et al., 2011). 
65 
 
University of Ghana http://ugspace.ug.edu.gh
5.2 VECTOR HOST  
At the six military sites, ticks were not found on household animals or livestock (sheep, 
goats, dogs). Most of the collected ticks were from cattle because most of the other smaller 
and household animals screened for ticks did not have any ectoparasites. The absence of ticks 
on the animals was because they do not graze or sleep outside of the household compounds.    
Owners explained that the animals are always kept in confined spaces and they do not 
wonder around to pick up ectoparasites. Also, animals are routinely treated with acaricides to 
prevent ticks, fleas and other pests. Veterinarians from the Ghana Army that were assisting 
with the tick collections routinely visit various households in the military barracks to examine 
and treat animals. These routine check-ups help maintain healthy livestock. Acaricide 
treatment of cattle in the military kraals were frequent (twice in a month) in the Greater Accra 
region, but once a month in the Northern region, this could explain why more ticks were 
sampled from the Northern region than the Greater Accra region.  
In the more rural part of Navrongo, cattle and dogs were the highest source of ticks 
than any other animal source. For the security of the cattle herds, dogs join the herd when 
they are grazing from one area to another.  Since the dogs are roaming in the same areas as 
the cattle, they are exposed to the same potential ectoparasites that the cattle encounter. This 
could most likely be the reason the Navrongo dogs had ticks. Livestock in Navrongo were 
mostly owned by civilians and therefore not regularly treated for ectoparasites and a 
veterinarian may not be called unless an animal is sick. Cattle in the Navrongo area were also 
not kept in kraals like on the military camps.  The military kraals tend to be far away from 
households, but in Navrongo the common practice is to have cattle either staked in a field or 
allowed to graze freely during the day and then kept in pens that are directly adjacent to the 
households of the cattle caretakers or owners at night. These practices in Navrongo do not 
only increase the exposure of the cattle to ticks, but also increase the potential risk of tick-
66 
 
University of Ghana http://ugspace.ug.edu.gh
borne illnesses to the families and other household animals since they all sleep in close 
proximity (Camitas et al., 1990).  
This project focused on domestic animals that were in close proximity to humans.  
However, based on the finding of this surveillance effort the hope is that a further 
investigation is conducted that will include the screening of wild animals.   
 
5.3 CCHFV DETECTION  
Sample 2 may have been co infected with CCHFV and Dugbe virus.  There may have 
been a higher amount of Dugbe virus genomic material in the pool that may have resulted in 
the amplification of only Dugbe virus during the sequencing process. This may explain why 
the sample tested positive for CCHFV after repeated tests. The similarity in genome 
sequences of orthonairoviruses may have resulted in the first assays reading as positive. This 
could be attributed by the fact that Dugbe virus and CCHFV belong to the same genus, 
Orthonairovirus (Bente et al., 2013). The set of primers used in the detection assay reduced 
the possibility of false positives due to the similarities between the two viruses. The benefit of 
the false positives was the incidental detection of Dugbe virus in sample 2. The remaining six 
pools not showing any reads from the sequencing may have resulted from the low viral load 
within the ticks.  
In library preparation for next generation sequencing, nucleic acid concentrations 
were detected for all positive samples. For samples with low nucleic acid concentration, their 
concentrations were enriched with C6/36 mosquito cells. This was to help increase the 
nucleic acid content after PCR amplification. The DNA library was validated at different 
stages of the process to ensure that there was ample concentration of DNA in the library and 
to also ensure that the DNA has not been fragmented to smaller fragments. During the entire 
67 
 
University of Ghana http://ugspace.ug.edu.gh
sample preparation process, all data analyzed showed that the library was not compromised. 
Also, the PhiX control added to the library pool provided 100% diversity because, it contains 
25% each of AGT and C.  PhiX was also used in troubleshooting problems with cluster 
generation to ensure that the library preparation was correct. These procedures were to 
increase the confidence that all the samples were properly sequenced (Illumina, 2017). 
Having both confidence in the library preparation and the modified real-time RT-PCR 
CCHFV assays it is speculated that the positive CCHFV ticks could not be sequenced 
because there simply was not enough virus material present in the ticks to sequence but was 
enough to detect. 
The non-detection of reads from the CCHFV positive samples could be attributed to 
viral RNA degradation or fragmentation (smaller fragments) making it impossible for the 
sequencer to pick up reads. Although high concentrations of nucleic acids were detected 
during the various library validation stages in the protocol, the concentrations recorded may 
not have been from the CCHFV genome but rather some other nucleic acid from the ticks 
(Giorgi et al., 2013). The RNA extract may have degraded due to power fluctuations. Also, 
viral detection assays were done a week after RNA had been extracted.  Sample 2 may have 
been coinfected with CCHFV and Dugbe virus, but only Dugbe virus genome was detected 
for the same reason as the other samples. 
 
5.4 CCHFV INFECTION RATE AND VECTOR DISTRIBUTION 
The favourable weather conditions and presence of the tick species in the country 
(Ntiamoa-Baidu et al., 2004) is a cause of concern for public health and safety (Chinikar et 
al., 2012; Shayan et al., 2015).  The global distribution of CCHFV is closely similar to the 
global distribution of ixodid ticks especially Hyalomma ticks (Ergönül, 2006). The high 
68 
 
University of Ghana http://ugspace.ug.edu.gh
infection rate (1.87%) recorded by Hyalomma rufipes in this study which is similar to 2% 
infection rate in Kenya, supports what is known in literature that Hyalomma ticks have a high 
infection rate (Sang et al., 2011). Even though Amblyomma and Rhipicephalus genera have 
also been implicated in CCHFV distribution, (Messina et al., 2015; Akuffo et al., 2016), 
Hyalomma rufipes stands as the tick species with the highest potential of infecting both 
humans and animals (Bente et al., 2013). This surveillance effort detected CCHFV in ~1% of 
1,813 field collected ticks across two ecological zones demonstrating that there is a 
dispersion of the virus across the guinea savannah and coastal savannah zones.  
Comparing the infection rates of CCHFV in both males and females from 
Amblyomma variegatum, Rhipicephalus sanguineus and Hyalomma rufipes, reflects that even 
though Amblyomma variegatum species were the highest number of ticks; their potential to 
cause infection is less as compared to Hyalomma rufipes. A contributing factor is that species 
with high sample size or a larger pool size, require more positive pools in order to have a 
high infection rate. Species with smaller pool size as a result of a small sample size do not 
require a high number of positives to have a high infection rate. This would conclude that, 
even for a small number, their potential to cause infection is high, and so a person stands the 
risk of infection when exposed to fewer numbers of Hyalomma rufipes bites. In a larger 
population, a person will have to be exposed to a relatively larger number of tick bites from 
Amblyomma variegatum, Rhipicephalus sanguineus before he/she can be infected. 
These CCHFV infected ticks may not have contracted the virus from the animals they 
were collected from, but from other animals that co inhabit with them in their households.  
This may also explain why positive pools were from sheep.  Since Navrongo is close to the 
Ghana-Burkina Faso border, which is an entry for livestock, infected ticks may have been 
transported from Burkina Faso into Navrongo through the cattle trade routes. Based on the 
CCHFV enzootic and epizootic-epidemic life cycle, other ticks in Ghana may be maintaining 
69 
 
University of Ghana http://ugspace.ug.edu.gh
the virus after infection.  To address the possibility of tick-borne diseases in livestock and for 
public health concern, livestock being transported into Ghana through the various border 
towns should be screened for infectious diseases such as CCHF. Also, a program should be 
implemented to examine animals for the presence of ticks with a system to analyze any ticks 
collected for the presence of infectious pathogens such as CCHFV could serve as a bio-
indicator for the early detection of potential health risk. 
 
5.5 PHYLOGENETIC ANALYSIS OF DUGBE VIRUS 
Dugbe virus is in the same Nairovirus family and Orthonairovirus genus with 
CCHFV (Sang et al., 2011). The virus belongs to the Nairobi sheep disease serogroup 
(Crabtree et al., 2009). After its first detection in Amblyomma variegatum ticks in Nigeria in 
1964, it has been detected in other African countries; Chad, Egypt, Cameroon, Kenya, 
Uganda, Central African Republic, Ethiopia, Senegal and Sudan (Hoogstraal, 1979; Darwish 
et al., 1976). This study provided the first full Dugbe virus genome sequenced in Ghana. A 
recent study in Ghana detected Dugbe virus in Amblyomma variegatum ticks in Accra 
(Kobayashi et al., 2017). The L segment, which was the only sequenced segment of the virus 
genome from that study showed a close relationship to the Dugbe virus reference strain 
isolated from Kenya.  
Phylogenetic analysis in this study was based on the large and small segments of the 
virus RNA. This is because, the medium segment encodes glycoproteins that are used by the 
virus to identify host cell surface proteins and also attach to these cell surfaces (Connolly-
Andersen et al., 2009). These proteins are therefore a target for host immune defence 
mechanisms and must therefore be modified from time to time to evade recognition by the 
70 
 
University of Ghana http://ugspace.ug.edu.gh
host immune cells (Garrison et al., 2013). Due to this frequent modification, it is not always 
used in phylogenetic analysis.  
Phylogenetic trees for the two viral sequences were rooted with Bunyamwera virus of 
the Nairovirus family and Bunyamwera serogroup (Gerrard et al., 2004). The trees were 
rooted with this virus because it is seen as the prototype for other viruses in this family 
(Odhiambo et al., 2016). Maximum likelihood trees with 1000 bootstrap replications were 
derived as shown in Figure 4. 7 and Figure 4. 8 . The complete L sequence of the virus 
provided in this study reflects a close relationship (99%) with the Dugbe strain (L segment) 
found in Ghana by Kobayashi’s team (LC193450). The L segment is also found in the same 
clade as LC193450, Nigeria Dugbe virus strain and Kenya Dugbe virus strain (reference 
Dugbe virus strain). The S segment is closer to Dugbe strain from Nigeria, AF434163, which 
is in the same clade with Dugbe virus strain from Senegal, Kenya, and Nigeria. More 
importantly, all two sequences, (L and S segments) reported a close relationship with the 
Dugbe virus reference strains from Kenya and Nigeria. 
Based on the close relationship of the two segments with other East and West African 
strains, we may conclude that the virus may have originated from any of these countries. 
Also, the L segment of the new detected virus was in the same clade as the strain detected 
earlier in Ghana by Kobayashi and his team, (LC193450). Since both strains were detected in 
Ghana, we may conclude they both could have originated from the same strain.  
 
 
 
 
71 
 
University of Ghana http://ugspace.ug.edu.gh
5.6 LIMITATIONS 
The results of the study are not a full representation of the country because the middle 
belt was not covered. Pesticide treatment of the livestock especially in the study sites in the 
Greater Accra region affected the true distribution of ticks collected there. The un-specificity 
of the primers used in the CCHFV detection assay affected the results therefore much 
emphasis must be placed on the primer design. 
  
72 
 
University of Ghana http://ugspace.ug.edu.gh
CHAPTER SIX 
6.0 CONCLUSION AND RECOMMENDATION 
6.1 CONCLUSION  
This study shows that CCHFV and Dugbe viruses are present in the country. 
Furthermore, this is the first time a full Dugbe virus genome has been sequenced in Ghana, 
and this study gave information on the genetic linkage of the Dugbe virus detected in Ghana 
to the other strains detected in Africa and other regions of the world. The detected Dugbe 
virus may have originated from the same virus from which LC193450 was sequenced. Both 
viruses have a close relationship with Kenya and Nigeria Dugbe virus strains. Additionally, 
CCHFV infection rate in Hyalomma rufipes is higher than the infection rate of other tick 
species in the Upper East, Northern and Greater Accra regions of Ghana.  
 
6.2 RECOMMENDATION 
Further investigation into the prevalence of Dugbe virus and CCHFV in Ghana needs 
to be done. Serum of livestock from sites where positive pools were detected have to be 
tested for CCHFV and Dugbe virus. Also, serum from the animal farmers and herdsmen have 
to be tested for CCHFV and Dugbe virus. The study has to be broadened to include other 
households that are not on military barracks and also parts of the middle belt of the country 
especially Kumasi where CCHFV antibodies have been detected in abattoir workers.  
 
 
 
  
73 
 
University of Ghana http://ugspace.ug.edu.gh
REFERENCES 
Adama, Z.D., A. Lionel, L. Maureen, B. Jean-Michel, K.K. Abdoulaye, B. Zakaria, D. 
Abdoulaye, D. Ogobara, R. Didier, and P.  Philippe. 2017. 'Molecular and MALDI-
TOF identification of ticks and tick-associated bacteria in Mali', PLoS Negl Trop Dis, 
11. 
Adams, M.J., E.J. Lefkowitz, A.M.Q. King, B. Harrach, R.L. Harrison, N.J. Knowles, A.M. 
Kropinski, M. Krupovic, J.H. Kuhn, A.R. Mushegian, M. Nibert, S. Sabanadzovic, H. 
Sanfac¸on, S.G. Siddell, P. Simmonds, A. Varsani, F.M. Zerbini, A.E. Gorbalenya, 
and A.J. Davison. 2017. 'Changes to taxonomy and the International Code of Virus 
Classification and Nomenclature ratified by the International Committee on 
Taxonomy of Viruses (2017)', Arch Virol, 162: 34. 
Afonina, I., M. Zivarts, I. Kutyavin, E. Lukhtanov, H. Gamper, and R.B. Meyer. 1997. 
'Efficient priming of PCR with short oligonucleotides conjugated to a minor groove 
binder.', Nucleic Acids Res, 25: 4. 
Afonina, I.A., M.W. Reed, E. Lusby, I.G. Shishkina, and Y.S. Belousov. 2002. 'Minor groove 
binder-conjugated DNA probes for quantitative DNA detection by hybridization-
triggered fluorescence', Biotechniques, 32: 940-4, 46-9. 
Akinci, E., H. Bodur, M. Sunbul, and H. Leblebicioglu. 2016. 'Prognostic factors, 
pathophysiology and novel biomarkers in Crimean-Congo hemorrhagic fever', 
Antiviral Res, 132: 233-43. 
Akuffo, R., J.A. Brandful, A. Zayed, A. Adjei, N. Watany, N.T. Fahmy, R. Hughes, B. 
Doman, S.V. Voegborlo, D. Aziati, D. Pratt, J.A. Awuni, N. Adams, and E. Dueger. 
2016. 'Crimean-Congo hemorrhagic fever virus in livestock ticks and animal handler 
seroprevalence at an abattoir in Ghana', BMC Infect Dis, 16: 324. 
74 
 
University of Ghana http://ugspace.ug.edu.gh
Al-Abri, S.S., I.A. Abaidani, M. Fazlalipour, E. Mostafavi, H. Leblebicioglu, N. 
Pshenichnaya, Z.A. Memish, R. Hewson, E. Petersen, P. Mala, T.M. Nhu Nguyen, 
Rahman M.M., P. Formenty, and R. Jeffries. 2017. 'Current status of Crimean-Congo 
haemorrhagic fever in the World Health Organization Eastern Mediterranean Region: 
issues, challenges, and future directions', Int J Infect Dis, 58: 82-89. 
Alavi-Naini, R., A. Moghtaderi, H.R. Koohpayeh, B. Sharifi-Mood, M. Naderi, M. Metanat, 
and M. Izadi. 2006. 'Crimean–Congo hemorrhagic fever in Southeast of Iran.', J. 
Infect, 52: 5. 
Anagnostou, V., and A. Papa. 2009. 'Evolution of Crimean-Congo Hemorrhagic Fever virus', 
Infect Genet Evol, 9: 948-54. 
Atkinson, B., J. Chamberlain, H.C. Logue, N. Cook, C. Bruce, D.S. Dowall, and R. Hewson. 
2012. 'Development of a Real-Time RT-PCR Assay for the Detection of Crimean-
Congo Hemorrhagic Fever Virus', Vector-Borne and Zoonotic Diseases, 12. 
Bell-Sakyi, L., A. Kohl, D.A. Bente, and J.K. Fazakerley. 2012 'Tick Cell Lines for Study of 
Crimean-Congo Hemorrhagic Fever Virus and Other Arboviruses', Vector Borne 
Zoonotic Dis., 12: 13. 
Bente, D. A., N. L. Forrester, D. M. Watts, A. J. McAuley, C. A. Whitehouse, and M. Bray. 
2013. 'Crimean-Congo hemorrhagic fever: history, epidemiology, pathogenesis, 
clinical syndrome and genetic diversity', Antiviral Res, 100: 159-89. 
Bente, D.A., J. B. Alimonti, W-J. Shieh, G. Camus, U. Stro¨her, S. Zaki, and M.S. Jones. 
2010. 'Pathogenesis and Immune Response of Crimean-Congo Hemorrhagic Fever 
Virus in a STAT-1 Knockout Mouse Model', Journal of Virology, 84: 12. 
Bergeron, E., C.G. Albarino, M.L. Khristova, and S.T. Nichol. 2010. 'Crimean–Congo 
hemorrhagic fever virus-encoded ovarian tumor protease activity is dispensable for 
virus RNA polymerase function. ', J. Virol., 84, : 11. 
75 
 
University of Ghana http://ugspace.ug.edu.gh
Bray, M. 2005. 'Pathogenesis of viral hemorrhagic fever', Current Opinion in Immunology, 
17: 5. 
Bryson, N.R., I.G. Horak, E.H. Venter, and C.E. Yunker. 2000. 'Collection of free-living 
nymphs and adults of Amblyomma hebraeum (Acari: Ixodidae) with 
pheromone/carbon dioxide traps at 5 different ecological sites in heartwater endemic 
regions of South Africa.', Experimental and Applied Acarology,, 24: 12. 
Burt, F.J., P.A. Leman, J.C. Abbott, and R. Swanepoel. 1994. 'Serodiagnosis of Crimean-
Congo haemorrhagic fever.', Epidemiol Infect, 113: 551–62. 
Burt, F.J., and R. Swanepoel. 2005. 'Molecular epidemiology of African and Asian Crimean-
Congo haemorrhagic fever isolates', Epidemiology and Infection, 133: 659-66. 
Camitas, J.L., M.L. Wilson, J.P. Cornet, J.P. Digoutte, M.A. Calvo, F. Adam, and J.P. 
Gonzalez. 1990. 'Ecology of ticks as potential vectors of Crimean-Congo hemorrhagic 
fever virus in Senegal: epidemiological implications', Arch Virol: 20. 
CDC. 2014. 'Crimean-Congo Haemorrhagic Fever'. 
Chinikar, S., R. Mirahmadi, M. Moradi, M. S. Ghiasi, and Khakifirouz, S. 2012. 'Crimean-
Congo Hemorrhagic Fever (CCHF),' Zoonosis: 21. 
Christova, I., A. DiCaro, A. Papa, C. Castilletti, L. Andonova, N. Kalvatchev, E. 
Papadimitriou, F. Carletti, E. Mohareb, M.R. Capobianchi, G. Ippolito, and G.   
Rezza. 2009. 'Crimean–Congo hemorrhagic fever, southwestern Bulgaria. ', Emerg. 
Infect. Dis., 15: 3. 
Ciccarelli, F, D.,, T. Doerks, C. Von Mering, C.J. Creevey, B. Snel, and P. Bork. 2006. 
'Toward automatic reconstruction of a highly resolved tree of life', Science, 311 5. 
Clerex-Van, H.C.M., J.P. Clerex, H. Ushijima, H. Akashi, F. Fuller, and D.H. Bishop. 1982. 
'The 30 terinal RNA sequences of bunyaviruses and nairoviruses (Bunyaviridae): 
76 
 
University of Ghana http://ugspace.ug.edu.gh
evidence of end sequence genetic differences within the virus family.', J. Gen. Virol., 
61: 5. 
Coates, D.M., and C. Sweet. 1990. 'Studies on the pathogenicity of a nairovirus, Dugbe virus, 
in normal and immunosuppressed mice', Journal of General Virology, 71: 8. 
Comber, A., P. Fisher, and R. Wadsworth. 2005. 'What is land cover?', Environment and 
Planning B, 32: 11. 
Connolly-Andersen, A. M., I. Douagi, A. A. Kraus, and A.  Mirazimi. 2009. 'Crimean Congo 
hemorrhagic fever virus infects human monocyte-derived dendritic cells.', Virology, 
390: 6. 
Crabtree, M.B., R. Sang, and B.R. Miller. 2009. 'Kupe Virus, a New Virus in the Family 
Bunyaviridae, Genus Nairovirus, Kenya', Emerging Infectious Diseases, 15: 8. 
Crowder, C.D., H.E. Matthews, S. Schutzer, M.A. Rounds, B.J. Luft, O. Nolte, S.R. 
Campbell, C.A. Phillipson, F. Li, R. Sampath, D.J. Ecker, and M.W. Eshoo. 2010. 
'Genotypic variation and mixtures of Lyme Borrelia in Ixodes ticks from North 
America and Europe', PLoS One, 5: e10650. 
Darwish, M.A., I.Z.E. Iman, T. Amer, and H. Hoogstraal. 1976. 'Antibodies to Dugbe virus in 
mammalian sera from Egypt.', J. Egypt. Publ. Health Assoc., 51: 7. 
David-West, T.S., and J.S. Porterfield. 1974. 'Dugbe Virus: a Tick-borne Arbovirus from 
Nigeria', Journal of Gen. Virology, 23: 11. 
de la Fuente, J., S. Antunes, S. Bonnet, A. Cabezas-Cruz, A.G. Domingos, A. Estrada-Pena, 
N. Johnson, K.M. Kocan, K.L. Mansfield, A.M. Nijhof, A. Papa, N. Rudenko, M. 
Villar, P. Alberdi, A. Torina, N. Ayllon, M. Vancova, M. Golovchenko, L. 
Grubhoffer, S. Caracappa, A.R. Fooks, C. Gortazar, and R.O.M. Rego. 2017. 'Tick-
Pathogen Interactions and Vector Competence: Identification of Molecular Drivers 
for Tick-Borne Diseases', Front Cell Infect Microbiol, 7: 114. 
77 
 
University of Ghana http://ugspace.ug.edu.gh
Deyde, V.M., M.L. Khristova, P.E. Rollin, T.G. Ksiazek, and S. T. Nichol. 2006. 'Crimean-
Congo hemorrhagic fever virus genomics and global diversity', J Virol, 80: 8834-42. 
Djikeng, A., R. Kuzmickas, N.G. Anderson, and D.J. Spiro. 2009. 'Metagenomic analysis of 
RNA viruses in a fresh water lake.', PLoS One, 4. 
Drosten, C., B.M. Kummerer, H. Schmitz, and S. Gunther. 2003. 'Molecular diagnostics of 
viral haemorrhagic fevers.', Antiviral Res 57: 61–87. 
Edwards, R.A., and F. Rohwer. 2005. 'Viral metagenomics.', Nat. Rev. Microbiol., 3: 6. 
Ergönül, Ö. 2006. 'Crimean-Congo haemorrhagic fever', The Lancet Infectious Diseases, 6: 
203-14. 
Ergönül, Ö.  2008. 'Treatment of Crimean-Congo hemorrhagic fever', Antiviral Res, 78: 125-
31. 
Estrada-Pena, A. 1999. 'Geostatistics and remote sensing using NOAA–AVHRR satellite 
imagery as predictive tools in tick distribution and habitat suitability estimations for 
Boophilus microplus (Acari: Ixodidae) in South America. ', Veterinary Parasitology,, 
81: 10. 
Estrada-Peña, A., and M. Salman. 2013. 'Current Limitations in the Control and Spread of 
Ticks that Affect Livestock: A Review', Agriculture 2013, 3, 3: 15. 
Estrada-Pena, A., and M.J. Venzal. 2007. 'Climate Niches of Tick Species in the 
Mediterranean Region: Modeling of Occurrence Data, Distributional Constraints, and 
Impact of Climate Change', J. Med. Entomol., 44: 9. 
Fajs, L., I. Humolli, A. Saksida, N. Knap, M. Jelovsek, M. Korva, I. Dedushaj, and T. Avsic-
Zupanc. 2014. 'Prevalence of Crimean-Congo hemorrhagic fever virus in healthy 
population, livestock and ticks in Kosovo', PLoS One, 9: e110982. 
Ferraris, O., M. Moroso, O. Pernet, S. Emonet, R.A. Ferrier, G. Paranhos-Baccala, and C.N. 
Peyrefitte. 2015. 'Evaluation of Crimean-Congo hemorrhagic fever virus in vitro 
78 
 
University of Ghana http://ugspace.ug.edu.gh
inhibition by chloroquine and chlorpromazine, two FDA approved molecules', 
Antiviral Res, 118: 75-81. 
Fisher-Hoch, S.P., J.B. McCormick, R. Swanepoel, A. Van Middlekoop, S. Harvey, and H.G. 
Kustner. 1992. 'Risk of human infections with Crimean–Congo hemorrhagic fever 
virus in a South African rural community.', Am. J. Trop. Med. Hyg, 47: 9. 
Francischetti, M.B.I, A. Sá-Nunes, J.B. Mans, I.M Santos, and J.M.C. Ribeiro. 2010. 'The 
role of saliva during tick feeding', Front Bioscience, 14: 53. 
Garrison, A.R., S. Alakbarova, D.A. Kulesh, D. Shezmukhamedova, S. Khodjaev, T.P. Endy, 
and Paragas J. 2007. 'Development of a TaqMan–Minor Groove Binding Protein 
Assay for the Detection and Quantification of Crimean-Congo Hemorrhagic Fever 
Virus', American Journal of Tropical Medicine, 77: 7. 
Garrison, A.R., S.R. Radoshitzky, K.P. Kota, G. Pegoraro, G. Ruthel, J.H. Kuhn, L.A. 
Altamura, S.A. Kwilas, S. Bavari, V. Haucke, and C.S. Schmaljohn. 2013. 'Crimean-
Congo hemorrhagic fever virus utilizes a clathrin- and early endosome-dependent 
entry pathway', Virology, 444: 45-54. 
Gerrard, S.R., L. Li, A.D. Barrett, and S.T.  Nichol. 2004. 'Ngari virus is a Bunyamwera virus 
reassortant that can be associated with large outbreaks of hemorrhagic fever in 
Africa', J Virol., 78: 5. 
Ginsberg, H., and C. Ewing. 1989. 'Comparison of Flagging, Walking, Trapping, and 
Collecting from Hosts as Sampling Methods for Northern Deer Ticks, Ixodes 
dammini, and Lone-Star ticks, Amblyomma americanum', Experimental and Applied 
Acarology, 7: 10. 
Giorgi, F.M., C. Del Fabbro, and F. Licausi. 2013. 'Comparative study of RNA-seq- and 
microarray-derived coexpression networks in Arabidopsis thaliana', Bioinformatics, 
29: 8. 
79 
 
University of Ghana http://ugspace.ug.edu.gh
Graci, J.D., and C.E. Cameron. 2006. 'Mechanisms of action of ribavirin against distinct 
viruses', Reviews in medical virology, 16: 12. 
Grashchenkov, N.I. 1945. "Investigation of Etiology, Pathogenesis, and Clinical 
Symptomatology of Crimean Hemorrhagic Fever, Reports on the 1944 Scientific 
Investigation of the Institute of Neurology." In, 100–07. Moscow: Akad. Med. Nauk 
SSR. 
Gunes, T., A. Engin, O. Poyraz, N. Elaldi, S. Kaya, I. Dokmetas, M. Bakir, and Z. Cinar. 
2009. 'Crimean-Congo hemorrhagic fever virus in high-risk population, Turkey', 
Emerg Infect Dis, 15: 461-4. 
Health, The Center for Food Security & Public. 2016. "Nairobi Sheep Disease." In Iowa State 
University edited by The Center for Food Security & Public Health and Iowa State 
University, 4. Ames, Iowa  
Hewson, R., A. Gmyl, L. Gmyl, S.E. Smirnova, G. Karganova, B. Jamil, R. Hasan, J. 
Chamberlain, and C. Clegg. 2004. ' Evidence of segment reassortment in Crimean-
Congo haemorrhagic fever virus', J Gen Virol, 85: 3059–70. 
Honig, E.J., J.C. Osborne, and S.T. Nichol. 2004. 'The high genetic variation of viruses of the 
genus Nairovirus reflects the diversity of their predominant tick hosts', Virology, 318: 
7. 
Hoogstraal, H. 1979. 'Review Article: The epidemiology of tick-borne Crimean-Congo 
hemorrhagic fever in Asia, Europe, and Africa.', J. Med. Entmol., 15: 111. 
Hotez, P.J., and A. Kamath. 2009. 'Neglected Tropical Diseases in Sub-Saharan Africa: 
Review of Their Prevalence, Distribution, and Disease Burden', PLOS Neglected 
Tropical Diseases, 3: e412. 
Illumina. 2017. "TruSeq RNA Access Library Prep." In, 36. 
80 
 
University of Ghana http://ugspace.ug.edu.gh
Jabbari, A., S. Besharat, A. Abbasi, A. Moradi, and K. Kalavi. 2006. 'Crimean–Congo 
hemorrhagic fever: case series from a medical center in Golestan province, Northeast 
of Iran (2004).', Indian J. Med. Sci., 60,: 3. 
Kalvatchev, N., and I. Christova. 2008. 'One Step RT-PCR for Rapid Detection of Crimean-
Congo Haemorrhagic Fever Virus, ' Biotechnology & Biotechnological Equipment, 
22: 3. 
Katholi, C.R., and T.R. Unnasch. 2006. 'Important experimental parameters for determining 
infection rates in arthropod vectors using pool screening approaches.', The American 
Journal of Tropical Medicine and Hygiene,, 74: 7. 
Keshtkar-Jahromi, M., M.M. Sajadi, H. Ansari, M. Mardani, and K. Holakouie-Naieni. 2013. 
'Crimean-Congo hemorrhagic fever in Iran', Antiviral Res, 100: 20-8. 
Khurshida, A., M. Hassana, M. M. Alamb, U. B. Aamirb, L. Rehmanb, S. Sharifb, S. 
Shaukatb, M. S. Ranab, M. Angezb, and S. S. Z. Zaidib. 2015. 'CCHF virus variants 
in Pakistan and Afghanistan: Emerging diversityand epidemiology', Journal of 
Clinical Virology, 67: 6. 
Kinsella, E., S.G. Martin, A. Grolla, M. Czub, H. Feldmann, and R. Flick. 2004. 'Sequence 
determination of the Crimean-Congo hemorrhagic fever virus L segment', Virology, 
321: 6. 
Kobayashi, D., M. Ohashi, J.H.N. Osei, E. Agbosu, M. Opoku, A. Agbekudzi, J. Joannides, 
R. Fujita, T. Sasaki, J.H.K. Bonney, S. Dadzie, H. Isawa, K. Sawabe, and N. Ohta. 
2017. 'Detection of a novel putative phlebovirus and first isolation of Dugbe virus 
from ticks in Accra, Ghana', Ticks and Tick-borne Diseases, 8: 6. 
Koehler, J. W., K.L. Delp, A.T. Hall, S.P. Olschner, B.J. Kearney, A.R. Garrison, L.A. 
Altamura, C. A. Rossi, and T.D. Minogue. 2017. 'Sequence optimized realtime 
RTPCR assay for CCHFV ', TR-17-094. 
81 
 
University of Ghana http://ugspace.ug.edu.gh
Koffi, J.K., P.A. Leighton, Y. Pelcat, L. Trudel, LR. Lindsay, F. Milord, and N.H. Ogden. 
2012. 'Passive surveillance for I. scapularis ticks: enhanced analysis for early 
detection of emerging Lyme disease risk', J Med Entomol, 49: 10. 
Kubar, A., M. Haciomeroglu, A. Ozkul, U. Bagriacik, E. Akinci, K. Sener, and H. Bodur. 
2011. 'Prompt administration of Crimean–Congo hemorrhagic fever (CCHF) virus 
hyperimmunoglobulin in patients diagnosed with CCHF and viral load monitorization 
by reverse transcriptase-PCR.', Jpn. J. Infect. Dis., 64: 5. 
Leblebicioglu, H. 2010. 'Crimean-Congo haemorrhagic fever in Eurasia', Int J Antimicrob 
Agents, 36 Suppl 1: S43-6. 
Li, C.X., M. Shi, J.H. Tian, X.D. Lin, Y.J. Kang, L.J. Chen, X.C. Qin, J. Xu, E.C. Holmes, 
and Y.Z. Zhang. 2015. 'Unprecedented genomic diversity of RNA viruses in 
arthropods reveals the ancestry of negative-sense RNA viruses', eLife., 4. 
Madder, M., I. Horak, and H. Stoltsz. 2013a. 'The African Veterinary Information Portal: 
Tick Surveillance'. 
Madder, M., I. Horak, and H.   Stoltsz. 2013b. 'Ticks: Tick identification': 34. 
Mahanty, S., and M. Bray. 2004. 'Pathogenesis of filoviral haemorrhagic fevers', Lancet 
Infect Dis, 4: 487-98. 
Maiga, O., Sas M.A., K. Rosenke, B. Kamissoko, M. Mertens, N. Sogoba, A. Traore, M. 
Sangare, M. Niang, T.G. Schwan, H.M. Maiga, S.F. Traore, H. Feldmann, D. 
Safronetz, and M.H. Groschup. 2017 'Serosurvey of Crimean-Congo Hemorrhagic 
Fever Virus in Cattle, Mali, West Africa.', Am J Trop Med Hyg., 96: 5. 
Mardani, M., M.K. Jahromi, K.H. Naieni, and M. Zeinali. 2003. 'The efficacy of oral 
ribavirin in the treatment of Crimean–Congo hemorrhagic fever in Iran. ', Clin. Infect. 
Dis., 36: 6. 
82 
 
University of Ghana http://ugspace.ug.edu.gh
Marriott, A.C.,, and P.A. Nuttall. 1996. 'Large RNA segment of Dugbe nairovirus encodes 
the putative RNA polymerase', Journal of General Virology, 77: 6. 
Mertens, M., K. Schmidt, A. Ozkul, and M.H. Groschup. 2013 'The impact of Crimean-
Congo hemorrhagic fever virus on public health.', Antiviral Res., 98: 13. 
Messina, J.P., D.M. Pigott, N. Golding, K.A. Duda, J.S. Brownstein, D.J. Weiss, H. Gibson, 
T.P. Robinson, M. Gilbert, G.R. William Wint, P.A. Nuttall, P.W. Gething, M.F. 
Myers, D.B. George, and S.I. Hay. 2015. 'The global distribution of Crimean-Congo 
hemorrhagic fever', Trans R Soc Trop Med Hyg, 109: 503-13. 
Midilli, K., A. Gargili, Ö. Ergönül, G. Sengoz, R. Ozturk, M. Bakar, and F. Jongejan. 2007. 
'Imported Crimean–Congo hemorrhagic fever cases in Istanbul. ', BMC Infect. Dis., 
7,: 1. 
Mild, M., M. Simon, J. Albert, and A. Mirazimi. 2010. 'Towards an understanding of the 
migration of Crimean-Congo hemorrhagic fever virus', J Gen Virol, 91: 199-207. 
Moore, D.L., O.R. Causey, D.E. Carey, S. Reddy, A.R. Cooke, F.M. Akinkugbe, T.S. 
DavidWest, and G.E. Kemp, .   . 1975. 'Arthropod-borne viral infections of man in 
Nigeria, 1964–1970.', Ann. Trop. Med. Parasitol., 69: 16. 
Morikawa, S., M. Saijo, and I. Kurane. 2007. 'Recent progress in molecular biology of 
Crimean-Congo hemorrhagic fever', Comp Immunol Microbiol Infect Dis, 30: 375-89. 
Muianga, A.F., R. Watson, A. Varghese, I.S. Chongo, S. Ali, V. Monteiro, F. Inalda, I. 
Chelene, V. Antonio, R. Hewson, and E.S. Gudo. 2017. 'First serological evidence of 
Crimean-Congo haemorrhagic fever in febrile patients in Mozambique', Int J Infect 
Dis, 62: 119-23. 
Nabeth, P., Cheikh O.D., Lo B., Faye O., Vall M.O.I., Niang M., Wague B., Diop D., Diallo 
M., Diallo B., Diop M. O., and Simon F. 2004. 'Crimean-Congo Hemorrhagic Fever, 
Mauritania', Emerging Infectious Diseases, 10: 7. 
83 
 
University of Ghana http://ugspace.ug.edu.gh
Norval, R.A.I., J.F. Butler, and C.E. Yunker. 1989. 'Use of carbon dioxide and natural or 
synthetic aggregation-attachment pheromone of the bont tick, Amblyomma hebraeum, 
to attract and trap unfed adults in the field.', Experimental & Applied Acarology,, 7: 
11. 
Ntiamoa-Baidu, Y., C. Carr-Saunders, B.E. Matthews, P.M. Preston, and A.R. Walker. 2004. 
'An updated list of the ticks of Ghana and an assessment of the distribution of the 
ticks of Ghanaian wild mammals in different vegetation zones.', Bull. Entomol. Res., 
94,: 16. 
Odhiambo, C., M. Venter, O. Lwande, R. Swanepoel, and R. Sang. 2016. 'Phylogenetic 
analysis of Bunyamwera and Ngari viruses (family Bunyaviridae, genus 
Orthobunyavirus) isolated in Kenya.', Epidemiol Infect, 144: 7. 
Ortega-Prieto, A.M., J. Sheldon, A. Grande-Pérez, H. Tejero, J. Gregori, J. Quer, J.I. Esteban, 
E. Domingo, and C. Perales. 2013. 'Extinction of hepatitis C virus by ribavirin in 
hepatoma cells involves lethal mutagenesis', PLoS One., 8. 
Ozdarendeli, A., N. Canakoglu, E. Berber, K. Aydin, S. Tonbak, M. Ertek, T. Buzgan, Y. 
Bolat, M. Aktas, and A. Kalkan. 2010. 'The complete genome analysis of Crimean-
Congo hemorrhagic fever virus isolated in Turkey', Virus Res, 147: 288-93. 
Palomar, A.M., A. Portillo, P. Santibanez, D. Mazuelas, J. Arizaga, A. Crespo, O. Gutierrez, 
J.F. Cuadrado, and J.A. Oteo. 2013. 'Crimean-Congo hemorrhagic fever virus in ticks 
from migratory birds, Morocco', Emerg Infect Dis, 19: 260-3. 
Papa, A., I. Christova, E. Papadimitriou, and A. Antoniadis. 2004. 'Crimean–Congo 
hemorrhagic fever in Bulgaria.', Emerg. Infect. Dis., 10,: 3. 
Papa, A., B. Ma, S. Kouidou, Q. Tang, C. Hang, and A. Antoniadis. 2002. 'Genetic 
Characterization of the M RNA Segment of Crimean Congo Hemorrhagic Fever 
Virus Strains, China.', Emerg Infect Dis, 8: 4. 
84 
 
University of Ghana http://ugspace.ug.edu.gh
Rajput, Z.I., S.H. Hu, W.J. Chen, A.G. Arijo, and C.W.   Xiao. 2006. ' Importance of ticks 
and their chemical and immunological control in livestock.', J. Zhejiang Univ. Sci. B, 
7: 9. 
Riesenfeld, C.S., P.D. Schloss, and J. Handelsman. 2004. 'Metagenomics: genomic analysis 
of microbial communities', Annu. Rev. Genet., 38: 31. 
Saksida, A., D. Duh, B. Wraber, I. Dedushaj, S. Ahmeti, and T. Avsic-Zupanc. 2010. 
'Interacting roles of immune mechanisms and viral load in the pathogenesis of 
crimean-congo hemorrhagic fever', Clin Vaccine Immunol, 17: 1086-93. 
Sang, R., J. Lutomiah, H. Koka, A. Makio, E. Chepkorir, C. Ochieng, S. Yalwala, J. Mutisya, 
L. Musila, J.H. Richardson, B.R. Miller, and D. Schnabel. 2011. 'Crimean-Congo 
hemorrhagic fever virus in Hyalommid ticks, northeastern Kenya', Emerg Infect Dis, 
17: 1502-5. 
Sas, A.M., L. Comtet, F. Donnet, M. Mertens, Z. Vatansever, N. Tordo, P.P. Philippe, and 
M.H. Groschup. 2018. 'A novel double-antigen sandwich ELISA for the species-
independent detection of Crimean-Congo hemorrhagic fever virus-specific 
antibodies', Antiviral Research, 151: 3. 
Schnittler, H.J., and H.  Feldmann. 2003. 'Viral hemorrhagic fever – a vascular disease? ', 
Thromb. Haemost., 89. 
Sengupta, D., and B.  Cookson. 2010. 'SeqSharp: A general approach for improving cycle-
sequencing that facilitates a robust one-step combined amplification and sequencing 
method', The Journal of molecular diagnostics, 12: 6. 
Shayan, S., M. Bokaean, M.R. Shahrivar, and S. Chinikar. 2015. 'Crimean-Congo 
Hemorrhagic Fever', Lab Med, 46: 180-9. 
Simon, M., K.I. Falk, A. Lundkvist, and A. Mirazimi. 2006. 'Exogenous nitric oxide inhibits 
Crimean Congo hemorrhagic fever virus', Virus Res, 120: 184-90. 
85 
 
University of Ghana http://ugspace.ug.edu.gh
Simon, M., C. Johansson, and A. Mirazimi. 2009. 'Crimean-Congo hemorrhagic fever virus 
entry and replication is clathrin-, pH- and cholesteroldependent', Journal of General 
Virology, 90: 6. 
Smego, Jr. R.A., A.R. Sarwari, and A.R. Siddiqui. 2004. 'Crimean–Congo hemorrhagic fever: 
prevention and control limitations in a resource-poor country. ', Clin.Infect. Dis., 38,: 
5. 
Sonenshine, D.E. 2018. 'Range Expansion of Tick Disease Vectors in North America: 
Implications for Spread of Tick-Borne Disease', Int. J. Environ. Res. Public Health, 
15 9. 
Spengler, J. R., A. Estrada-Pena, A. R. Garrison, C. Schmaljohn, C. F. Spiropoulou, E. 
Bergeron, and D. A. Bente. 2016. 'A chronological review of experimental infection 
studies of the role of wild animals and livestock in the maintenance and transmission 
of Crimean-Congo hemorrhagic fever virus', Antiviral Res, 135: 31-47. 
Sprickett, A.M., I.G. Horak, L.E.O. Braack, and H. Van Ark. 1991. 'Drag-sampling of free-
living ixodid ticks in the Kruger National Park.', Onderstepoort Journal of Veterinary 
Research,, 58: 6. 
Suliman, H.M., I.A. Adam, S.I. Saeed, S.A. Abdelaziz, E.M. Haroun, and I.E. Aradaib. 2017. 
'Crimean Congo hemorrhagic fever among the one-humped camel (Camelus 
dromedaries) in Central Sudan', Virol J, 14: 147. 
Tucker, J.B. 2009. 'Six-Legged Soldiers: Using Insects as Weapons of War', BioScience. Am 
Inst of Biol Sci, 59: 3. 
van de Wal, B.W., J.R. Joubert, P.J. van Eeden, and J.B. King. 1985. 'A nosocomial outbreak 
of Crimean–Congo haemorrhagic fever at Tygerberg Hospital. Part IV. Preventive 
and prophylactic measures. ', S. Afr. Med. J., 68: 4. 
86 
 
University of Ghana http://ugspace.ug.edu.gh
van Eeden, P.J., S.F. van Eeden, J.R. Joubert, J.B. King, B.W. van de Wal, and W.L. Michell. 
1985b. . 'A nosocomial outbreak of Crimean–Congo haemorrhagic fever at Tygerberg 
Hospital. Part II. Management of patients. ', S. Afr. Med. J., 68, : 4. 
Vanhomwegen, J., M.J. Alves, T.A. Zupanc, S. Bino, S. Chinikar, H. Karlberg, G. 
Korukluoglu, M. Korva, M. Mardani, A. Mirazimi, M. Mousavi, A. Papa, A. Saksida, 
B. Sharifi-Mood, P. Sidira, K. Tsergouli, R. Wolfel, H. Zeller, and P. Dubois. 2012. 
'Diagnostic assays for Crimean-Congo hemorrhagic fever', Emerg Infect Dis, 18: 
1958-65. 
Vassilev, T., V. Valchev, G. Kazarov, L. Razsukanova, and T. Vitanov. 1991. 'A reference 
preparation for human immunoglobulin against Crimean/Congo hemorrhagic fever', 
Biologicals, 19: 57. 
Vesco, U., N. Knap, M.B. Labruna, T. Avšič-Županc, A. Estrada-Peña, A.A. Guglielmone, 
G.H. Bechara, A. Gueye, A. Lakos, A. Grindatto, V. Conte, and D. .  De Meneghi. 
2011. ' An integrated database on ticks and tick-borne zoonoses in the tropics and 
subtropics with special reference to developing and emerging countries.', Exp. Appl. 
Acarol., 54: 19. 
Victoria, J.G., A. Kapoor, K. Dupuis, D.P. Schnurr, and E.L. Delwart. 2008. 'Rapid 
identification of known and new RNA viruses from animal tissues.', PLoS Pathog., 4. 
Walker, A.R., A. Bouattour, J.-L. Camicas, A. Estrada-Peña, I.G. Horak, A.A. Latif, R.G. 
Pegram, and P.M.  Preston. 2014. Ticks of Domestic Animals in Africa: a Guide to 
Identification of Species (Bioscience Reports: Edinburgh Scotland, U.K.). 
Whitehouse, C.A. 2004. 'Crimean-Congo hemorrhagic fever', Antiviral Res, 64: 145-60. 
WHO. 2013. 'Crimean-Congo haemorrhagic fever'. 
http://www.who.int/csr/disease/crimean_congoHF/en/. 
87 
 
University of Ghana http://ugspace.ug.edu.gh
Wilhelmsson, P., L. Fryland, S. Bo¨rjesson, J. Nordgren, S. Bergstro¨m, J. Ernerudh, P. 
Forsberg, and P-E. Lindgren. 2010. 'Prevalence and Diversity of Borrelia Species in 
Ticks That Have Bitten Humans in Sweden', JOURNAL OF CLINICAL 
MICROBIOLOGY,, 48: 9. 
Williams, R.W., O.R. Causey, and G.E. Kemp. 1972. 'Ixodid Ticks from Domestic Livestock 
in Ibadan, Nigeria as Carriers of Viral Agents1', Journal of Medical Entomology, 9: 3. 
Willner, D., M. Furlan, M. Haynes, R. Schmieder, F.E. Angly, Silva J., S. Tammadoni, B. 
Nosrat, D. Conrad, and F. Rohwer. 2009. 'Metagenomic analysis of respiratory tract 
DNA viral communities in cystic fibrosis and non-cystic fibrosis individuals.', PLoS 
One., 4. 
Wilson, M.L., J.P. Gonzalez, J.P. Cornet, and J.L. Camicas. 1991. 'Transmission of Crimean–
Congo haemorrhagic fever virus from experimentally infected sheep to Hyalomma 
truncatum ticks.', Res. Virol., 142: 10. 
Wodecka, B., A. Leon´ska, and B. Skotarczak. 2010. 'A comparative analysis of molecular 
markers for the detection and identification of Borrelia spirochaetes in Ixodes ricinus', 
Journal of Medical Microbiology, 59: 6. 
Yilmaz, M., K. Aydin, E. Akdogan, N. Sucu, M. Sonmez, S.B. Omay, and I. Koksal. 2008. 
'Peripheral blood natural killer cells in Crimean–Congo hemorrhagic fever. ', J. Clin. 
Virol., 42, : 3. 
 
 
 
 
 
88 
 
University of Ghana http://ugspace.ug.edu.gh
APPENDICES 
APPENDIX 1 
Global positioning system coordinates for the seven study sites 
  GPS Coordinates 
Regions Study sites Type Latitude Longitude 
 Army Recruit DD 5.87948 0.032874 
Greater Training School, DMS N 5° 52' 46.128'' E 0° 1' 58.346'' 
Accra- Accra Shai Hills 
5BN, Burma Camp DD 5.603544 0.155419 
 DMS N 5°36'12.8" W 0°09'19.5" 
1BN, Michel Camp DD 5.732687 -0.041898 
DMS N 5° 43' 57.673' W 0° 2' 30.832'' 
 Air Borne Force, DD 9.542479 -0.856517 
Northern Tamale DMS N 9° 32' 32.924' W 0° 51' 23.461'' 
Region Air Force Base, DD 9.546874 -0.855683 
(Tamale) Tamale DMS N 9° 32' 48.746'' W 0° 51' 20.458'' 
6BN, Kamina DD 9.46842 -0.85396 
Barracks-Tamale DMS N 9° 28' 6.312'' W 0° 51' 14.256'' 
Upper East Navrongo DD 10.97986559  -0.940994779 
Region DMS N 10° 53' 5" W 1° 05' 25"  
     
 
  
89 
 
University of Ghana http://ugspace.ug.edu.gh
APPENDIX 2 
Total number of tick species and tick sex collected from animals from the three regions 
Tick species Animal Northern region Upper East Greater Accra  
source region region Total 
Male Female Male Female Male Female 
Amblyomma Cattle 677 71 0 1 303 146 1198 
variegatum Dog 0 0 2 0 0 0 2 
Total 677 71 2 1 303 146 1200 
Hyalomma Cattle 8 4 0 0 20 20 52 
rufipes 
Hyalomma Cattle 86 37 0 3 1 0 127 
truncatum 
Rhipicephalus Cattle 0 0 25 29 4 37 95 
sanguineus Dog 0 0 167 145 0 0 312 
Goat 0 0 0 6 0 0 6 
Total 0 0 192 180 4 37 413 
Rhipicephalus Cattle 0 0 2 0 0 7 9 
evertsi Sheep 0 0 8 2 0 0 10 
Goat 0 0 1 0 0 0 1 
Total 0 0 11 2 0 7 20 
Rhipicephalus Cattle 0 0 0 0 1 0 1 
boophilus 
Overall Total 771 112 205 186 329 210 1,813 
 
  
90 
 
University of Ghana http://ugspace.ug.edu.gh
APPENDIX 3 
Total number of ticks collected from animals from the seven study sites in the three 
regions 
  Host  
Region Study site Cattle Dog Goat Sheep Total 
Northern Air borne force 364 0 0 0 364 
region Kamina Barracks 239 0 0 0 239 
Air force base 280 0 0 0 280 
Northern region Total 883 0 0 0 883 
Upper East Navrongo 60 314 7 10 391 
region 
Greater Burma camp 238 0 0 0 238 
Accra region Michel camp 95 0 0 0 95 
Shai hills 206 0 0 0 206 
Greater Accra region Total 539 0 0 0 539 
Over all total 1,482 314 7 10 1,813 
 
 
 
 
 
 
 
91 
 
University of Ghana http://ugspace.ug.edu.gh
APPENDIX 4 
Total number of tick sex collected from the seven study sites in the three regions 
Region Study site Male Female Total 
Northern region Air borne force 301 63 364 
Kamina Barracks 223 16 239 
Air force base 244 36 280 
Northern region Total 768 115 883 
Upper East region Navrongo 208 183 391 
Greater Accra region Burma camp 132 106 238 
Michel camp 60 35 95 
Shai hills 136 70 206 
Greater Accra region Total 328 211 539 
Overall Total 1,304 509 1,813 
 
 
 
 
 
 
 
  
92 
 
University of Ghana http://ugspace.ug.edu.gh
APPENDIX 5.1 
Total number of tick species from seven study sites in the three regions 
Region  Study site Amblyomma Hyalomma Hyalomma Total 
variegatum rufipes truncatum 
 Air borne force 301 9 54 364 
Northern region Kamina 231 0 8 239 
 
Barracks 
Air force base 216 3 61 280 
Northern region Total 748  12  123 883 
Upper East Navrongo 3 0 3 6 
 
region 
Greater Accra Burma camp 214 8 0 222 
region Michel camp 53 24 1 78 
Shai hills 182 8 0 190 
Greater Accra region Total 449 40 1 490 
Overall Total 1,200 52 127 1,379 
 
 
 
 
 
 
 
 
93 
 
University of Ghana http://ugspace.ug.edu.gh
APPENDIX 5.2 
Total number of tick species from seven study sites in the three regions 
Region  Study site Rhipicephalus Rhipicephalus Rhipicephalus Total 
sanguineus evertsi boophilus 
Northern Air borne 0 0 0 0 
region force 
Kamina 0 0 0 0 
Barracks 
Air force base 0 0 0 0 
Northern region Total 0 0 0 0 
Upper East Navrongo 372 13 0 385 
region 
Greater Burma camp 16 0 0 16 
Accra region Michel camp 9 7 1 17 
Shai hills 16 0 0 16 
Greater Accra region Total 41 7 1 49 
Total 413 20 1 434 
 
 
 
94