ABSTRACT
Phleboviruses transmitted by sand flies are among emerging public health threats. A novel Phlebovirus named Ntepes virus (NPV) was recently described in Kenya and found to infect humans from a wider geographic area. However, the entomologic risk factors such as potential vectors and transmission efficiency remains poorly defined. This study assessed the ability of the sand fly Phlebotomus duboscqi to transmit NPV. Two hundred and five 5-day old laboratory colonized P. duboscqi were exposed to NPV by membrane feeding in a triplicate experiment with a viremic blood meal of a dose of about 106.0pfu/ml. All the 205 NPV-exposed sandflies were randomly picked on the 6th, 10th and 15th days post infection and individually dissected into abdomens, legs and salivary glands to test for mid-gut infection, disseminated infection and transmissible infection, respectively, by cell culture. Of the 205 NPV-exposed sandflies, 40 (19.51%) developed infections which were all limited to the mid gut and that did not disseminate to the legs nor the salivary glands. Mid gut infection rates decreased with increasing extrinsic incubation period (Spearman’s correlation, ρ= -0.7145). These findings signify that P. duboscqi is an incompetent laboratory vector of NPV from ingestion of a viremic blood meal since the mid gut infections did not disseminate to the salivary glands to be transmitted by bites.
TABLE OF CONTENTS
DECLARATION i
TABLE OF CONTENTS ii
List of Figures iv
List of Tables v
List of Appendices vi
List of Abbreviations vii
ABSTRACT viii
DEDICATION ix
ACKNOWLEDGEMENT x
CHAPTER ONE
1.0 INTRODUCTION
1.1 Study Background 1
1.2 Statement of the Problem 4
1.3 Justification 5
1.4 Research Hypothesis 6
1.5 Objectives 6
1.5.1 Main Objective 6
1.5.2 Specific Objectives 6
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 Sandfly borne Phleboviruses Classification 7
2.2 Clinical Presentation of Sandfly Borne Phleboviruses Infections 8
2.3 Maintenance Cycles of Sandfly Borne Phleboviruses 9
2.4 Vectorial Capacity 10
2.5 Vector Competence 11
2.6 The Extrinsic Incubation Period (EIP) of Sandfly Fever Viruses 12
2.7 Vector Profiles of Phleboviruses 13
2.8 Ntepes virus and its Transmission Ecology 15
2.9 Distribution and Vector Biology of Phlebotomine Sandflies 16
CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 Study Samples 18
3.2 Sandfly rearing 18
3.3 Virus Amplification 19
3.4 Virus quantification 19
3.5 Vector Competence Assessment 20
3.5.1 Sandflies infection with NPV 20
3.5.2 Infection and Dissemination Assays 21
3.6 Data Analysis 22
CHAPTER FOUR
4.0 RESULTS
4.1 Virus Amplification and Quantification 23
4.2 Sandflies Infection with NPV 23
CHAPTER FIVE
5.0 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS
5.1 Discussion 27
5.2 Conclusions 30
5.3 Recommendations 31
REFERENCES 32
APPENDICES 44
List of Figures
Figure 1. Box and whiskers plot showing percentage midgut infection rates of NPV in Phlebotomus duboscqi against the extrinsic incubation period in days. 25
Figure 2. Regression line graph showing the correlation between midgut infection rates of NPV in Phlebotomus duboscqi against EIP in days. 26
Figure 3. Regression line graph showing the correlation between mortalities of NPV- exposed sandflies against midgut infection 26
List of Tables
Table 1. Feeding success rates of P. duboscqi on viremic blood meal by membrane feeding on hemotek system during the infection with NPV. 23
Table 2. Titers of NPV in the pre-feeding and post-feeding blood meals during the infections of P. duboscqi in Log10 pfu/ml. 24
Table 3. Infection, dissemination and transmission success of NPV in P. duboscqi across the extrinsic incubation period after oral feeding of the viremic blood meal 25
List of Appendices
Appendix 1. Mortalities of P. duboscqi across the EIP after ingestion of NPV 44
Appendix 2. Regression of mortalities of Phlebotomus duboscqi against EIP in days 44
List of Abbreviations
|
NPV
ZCL
CBRD
KEMRI
ICIPE
EID
WHO
CO1
ICTV
RNA
SFTS
EIP
MEB
MIB
SGIB
RVFV
GFV
PRNT
MEM
FBS
CPE
GIBCO
PFU
RH
HM
IR
USA
VHF
DPI
AFI
|
Ntepes
virus
Zoonotic
Cutaneous Leishmaniasis
Centre for Biotechnology Research and
Development
Kenya Medical Research Institute
International Center for Insect
Physiology and Ecology
Emerging
Infectious Diseases
World Health Organization
Cytochrome Oxidase subunit1
International Committee for Taxonomy of
Viruses
Ribonucleic Acid
Severe fever with
thrombocytopenia syndrome
Extrinsic
Incubation Period
Midgut Escape Barrier
Midgut
Infection Barrier
Salivary
Gland Infection Barrier
Rift
Valley fever virus
Gabek
Forest virus
Plaque
Reduction Neutralization Test
Minimum
Essential Media
Fetal Bovine Serum
Cytopathic Effects
Grand Island Biological Company
Plaque Forming Units
Relative Humidity
Homogenization medium
Infection Rate
United States of America
Viral hemorrhagic fevers
Days Post Infection
Acute febrile illness
|
CHAPTER ONE
1.0 INTRODUCTION
1.1 Study Background
Vector-borne diseases pose a significant burden to human health globally (Peters 2014). Serious arthropod-vector borne infections such as Rift Valley fever virus (RVFV), malaria, dengue and chikungunya are prevalent in the tropics while other new arthropod-vector borne pathogens continue to emerge (Takken and Koenraadt, 2013; Hill et al., 2005). Cases of vector borne viral diseases such as RVFV, chikungunya, and dengue fever continue to be reported in Kenya (Konongoi et al. 2018; Lutomiah et al. 2016; Sang et al. 2010). Of these vector borne diseases, the viruses transmitted by sandflies are inadequately researched on in the Sub-Saharan Africa (Alkan et al. 2013).
Sandfly fever is a group of diseases caused by sandfly borne phleboviruses which manifest as self-limiting febrile illnesses (Burrell, Howard, and Murphy 2017). Sandfly borne phleboviruses also cause severe and sometimes fatal encephalitis and meningitis (Sang and Dunster 2001; Killick-Kendrick 1999). Sandfly fevers often manifest as a “three-day fever” with influenza-like symptoms including fever, myalgia, retro-orbital pain and malaise with patients usually recovering fully within one week (Papa et al. 2015). Toscana virus; one of the sand fly borne phleboviruses, has however been shown to have a strong neurotropism and to sometimes cause meningo-encephalitis (Guler et al. 2012; Alkan et al. 2013).
Sandfly fever viruses occur in the Old World including Mediterranean basin, South and Central Asia, North Africa and middles East where they are transmitted by the genera Phlebotomus and Sergentomyia (Alkan et al. 2013). In the New World including South, North and Central America, they are tranmistted by the genus Lutzomyia (Killick-Kendrick 1999; Alkan et al. 2013). They have been isolated in both vertebrates and sandflies in Europe, Central Asia, America ,North and Central Africa (Guler et al. 2012). Their spread, locally and globally, is conditioned by different factors involving both the virus and the vectors. These factors include vector biology, the geographical distribution of both the virus and the vector as well as climate change. The spread of sandfly-borne viral diseases is restricted to the distribution of their potential vectors and their circulation can spread to a non-endemic areas with competent vectors if a viremic host is introduced (Depaquit et al. 2010).
Phlebotomine sandflies are distributed throughout the tropics and the subtropics including the Sub-Saharan Africa, Mediterranean regions of Europe and the Indian subcontinent of Asia (Alkan et al. 2013). Their distribution is limited to areas that experience temperatures of 15.60C for not less than three months in a year (Lawyer et al. 2017; European Centre for Disease Prevention and Control 2019).
Vaccines and therapeutic drugs have been developed for most of vector borne diseases in attempts to prevent and treat them, however, vector control as an intervention is lagging behind (Takken and Koenraadt 2013). This is despite the fact that vector control has been described as the most effective approach to interrupt disease transmission and can even lead to disease eradication (Takken and Koenraadt, 2013). Understanding the transmission ecology of a vector borne pathogen through assessments of vector competence of among endemic vector species is prerequisite in disease risk assessment and control (Hardy et al. 1983; Agha et al. 2017).
Tchouassi et al., (2019) described a novel Phlebovirus isolated from a pool of sandflies collected from Marigat, Baringo County, Kenya. The virus was isolated in an exploratory study and named Ntepes Virus (NPV) following the name of the village from where it was isolated. Cell lines of selected vertebrates including non-human primates, bats and rodents, livestock and humans, in in-vitro tropism studies, all showed susceptibility to the virus (Tchouassi et al. 2019). Swine and rodents are thought to be the amplificatory hosts of the virus since their cell lines produced higher copy numbers of the virus from the in vitro growth analyses.
Seroprevalence studies by Tchouassi et al., (2019) on human serum samples showed evidence of infections to humans by NPV. Neutralizing antibodies specific to NPV were detected in 13.9% of the tested human serum samples collected from the area where NPV was isolated. Following the CO1 gene sequence analysis of the pool of isolation, NPV is suggested to have been isolated from sandflies from the genus Sergentomyia. The isolation does not, however, guarantee vector status to Sergentomyia species.
Phlebotomus duboscqi (Order Diptera, Family Psychodidae) is one of the Phlebotomus species of sandfly in the same ecology from where NPV was isolated (Anjili et al. 2011) . It is also the principal vector of Leishmania major, the causative agent of Zoonotic cutaneous leishmaniasis (ZCL) (Muigai et al. 1987; Beach et al. 1984; Killick-Kendrick 1990) in the same ecology. Involvement of P. duboscqi in the transmission of NPV would be of an epidemiological significance owing to the fact that it has been shown to transmit phleboviruses under laboratory conditions (Hoch, Turell, and Bailey 1984; Turell and Perkins 1990; David et al. 2000). Could P. duboscqi, be responsible for this active circulation of NPV?
There is already evidence of a wider geographical spread of NPV in Kenya (Tchouassi et al. 2019; Marklewitz et al. 2020) but distribution can further be extended to non- endemic areas with its potential competent vectors. According to Depaquit et al., (2010), the distribution of sandfly-borne Phlebovirus diseases may not be confined to just the areas where the viruses have been recorded or isolated but as wide as the non-endemic areas where their potential vectors inhabit. Depaquit et al., (2010), therefore, emphasizes on a need for field work in terms of isolation of the viruses from sandflies as well as their possible vertebrate reservoirs, and lab work to establish the vector competence of colonized sandflies.
1.2 Statement of the Problem
The main promoter of the spread of a sandfly borne virus is the presence of a competent vector of that particular virus (Brett-Major and Claborn 2009). Despite being shown to be an efficient vector of several pathogens of human health importance, phlebotomine sandflies have been significantly neglected in studies to describe their role in the transmission of arboviruses. Most of the studies done on entomological risk assessment of viruses focus on mosquitoes and studies on sandfly as vectors are concentrated on leishmaniasis. This has limited the availability of knowledge necessary in understanding epidemiology and disease dynamics of sandfly-borne viruses.
Vector competence determination is always prerequisite in understanding the disease transmission ecology, epidemic potential and vector control (Hardy et al. 1983). The pioneer study by Tchouassi et al., (2019) highlighted the circulation of a new Phlebovirus tentatively named as Ntepes virus (NPV) isolated from sandflies and with evidence of human infections widely distributed in Northeastern Kenya. The findings of that study suggest that sandflies may have been underestimated and neglected as potential vector for human pathogenic viruses in Kenya and East Africa. The transmission ecology of NPV, however, remains poorly understood. There is therefore, a need to conduct vector competence investigations of suspected arthropod vectors in transmission of NPV.
Tesh, (1988) suggests an involvement of Phlebotomus species in Phlebovirus transmission in the Old World. Phlebotomus duboscqi is an efficient vector of leishmaniasis and is the incriminated vector of Leishmania major in Baringo, where NPV was isolated. Its implication in the transmission of NPV could have ramifications for possible epidemiological links between leishmaniasis and phleboviruses. This study purposes to determine the capacity of colonized P. duboscqi to transmit disseminated infection of NPV in the laboratory.
1.3 Justification
Out of the 10 ICTV recognized arthropod-borne Phlebovirus of human and animal health importance, seven are transmitted by sandflies (Tesh 1988). Like other arboviruses, the risk of spread of sandfly borne viruses are conditioned by the presence of a competent vector (Depaquit et al. 2010; Brett-Major and Claborn 2009). Although Sergentomyia are thought to be the genus from which NPV was isolated, this does not guarantee a vector status. Assessment of the vector status goes beyond merely the isolation of a pathogen from a field collected specimen (Azar and Weaver 2019).
The vector competence of a given arthropod to a particular disease agent is guided by an ecological relevance (Takken and Koenraadt, 2013; Young et al, 2013). According to Tesh (1988), each Phlebovirus tend to have a unique geographical distribution depending on the availability of its vector or the vertebrate host . Phlebotomus duboscqi co-occurs in the same ecology where NPV was isolated in Baringo County. This study focused on determining the vector competence of P. duboscqi to a newly identified Phlebovirus; NPV.
Understanding the vector competence of P. duboscqi to NPV will help understand the transmission ecology of the virus and this knowledge will be useful in managements of epidemics of the virus in terms of vector control.
1.4 Research Hypothesis
H1 Phlebotomus duboscqi is not susceptible to oral infection with NPV.
H2 Phlebotomus duboscqi does not efficiently transmit NPV after oral exposure.
1.5 Objectives
1.5.1 Main Objective
To determine the vector competence of P. duboscqi to Ntepes virus (NPV).
1.5.2 Specific Objectives
i. To assess the oral susceptibility of P. duboscqi to NPV.
ii. To assess the transmission efficiency of NPV by P. duboscqi.
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