ABSTRACT
Chikungunya virus (CHIKV) is among re-emerging arboviruses that affect human health globally. The spread has been associated with lack of sustainable vector control and viral preventive measures. Studies of the biology and ecology of the key vector, Aedes aegypti can open avenues for control of this virus. Despite the increasing evidence linking plant feeding to the survival and pathogen transmission dynamics as observed in the Anopheles-malaria parasite system, little is known as to whether plant feeding can influence pathogen-Ae. aegypti interaction. This study aimed to determine the effect of Pithecellobium dulce, a host plant for this vector, on the competence of Ae. aegypti to CHIKV. Adult Ae. aegypti females fed orally on dimethyl sulfoxide (DMSO) extracts of P. dulce, reduced female survival in a dose-dependent manner (P<0.0001). Chemical analysis of pools of midgut content after ingestion of the plant extract detected by coupled liquid chromatography triple quadrupole tandem mass spectrometry (LC-QqQ-MS) and coupled gas chromatography-mass spectrometry (GC-MS) identified several plant metabolites namely the amino acid proline, the flavonoid glycoside kaempferol 3-O- rhamnoside, the sterol β sitosterol and the fatty acid linoleic acid. Further, the females were orally exposed to a CHIKV infectious blood using a membrane-feeding assay before and after feeding on an optimal survival dose of the plant extract. Virus infection in the mosquito vector was determined by plaque assays. Highly significant infection and dissemination rates and respective mean titers were observed in the control and post- exposed (mosquitoes fed on glucose solution then the plant extract) treatment (P<0.001). No significant effect was observed in mean titers of the control and the pre-exposed (mosquitoes fed on plant extract then glucose solution) cohort (P<0.001). Although there was no observed significant difference while using either frozen or freshly cultured virus, transmission, which is a measure of vector competence, was only observed in the freshly cultured virus type. The pre-exposed, control and pre + post-exposed treatments recorded transmission although with significantly reduced titer in the latter. The post-exposed treatment recorded no transmission further suggesting possibility of P. dulce activity. These results demonstrate that Ae. aegypti feeding on this plant i) influences its survival, ii) leads to ingestion of secondary metabolites and iii) modulates infection success to chikungunya virus. The known anti-pathogenic effect of the identified metabolites suggests the potential impact on virus transmission occurs through reduced virus titers, thus these findings open a novel avenue towards the development of antiviral strategies targeting vector plant feeding behavior.
TABLE OF CONTENTS
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENT iv
TABLE OF CONTENTS v
LIST OF TABLES ix
LIST OF FIGURES x
ABBREVIATIONS AND ACRONYMS xi
ABSTRACT xii
CHAPTER 1.0: INTRODUCTION
1.1 Background 1
1.2 Statement of problem 4
1.3 Justification and significance of the study 5
1.4 Research objectives 6
1.4.1 Main objective 6
1.4.2 Specific objectives 6
1.5 Research hypothesis 6
CHAPTER 2.0: LITERATURE REVIEW
2.1 Epidemiology, burden and transmission of chikungunya virus 7
2.1.1 Chikungunya virus discovery and genetic diversity 7
2.1.2 Global spread of chikungunya virus 8
2.1.3 Spread of chikungunya virus in Kenya 10
2.1.3 Burden of chikungunya disease 11
2.1.4 Chikungunya virus infection, clinical presentation and diagnosis 12
2.1.5 Chikungunya virus vectors and transmission cycles 13
2.2 Control of chikungunya disease 15
2.3 Vector competence 17
2.3.1 Influence of genetic factors on vector competence 17
2.3.2 Influence of environmental factors on vector competence 18
2.4 Plant feeding and influence on vector competence 19
2.4.1 Pithecellobium dulce and Aedes aegypti interaction 20
CHAPTER 3.0: MATERIALS AND METHODS
3.1 Plant collection and preparation of extracts 22
3.2 Mosquitoes collection and rearing 22
3.3 Determination of optimal concentration of dimethyl sulfoxide (DMSO) for use in survival assays 23
3.4 Survival assays 24
3:5 Chemical analysis of Pithecellobium duce extract and mosquito midguts 25
3.6 Effect of Pithecellobium dulce extract on chikungunya virus infection, dissemination and transmission success in Aedes aegypti 27
3.6.1 Mosquito rearing and identification 27
3.6. 2 Virus amplification and quantification 28
3.6.3 Oral infection of the mosquitoes 29
3.7.2 Virus screening for infection and dissemination 30
3.7.3 : Virus screening for transmission potential 31
3.8. Ethical statement 32
3.9. Statistical analysis 32
CHAPTER 4.0: RESULTS
4.1 Survival analysis 33
4.1.1 Optimal DMSO dose 33
4.1.2 Survival analysis using Pithecellobium dulce extract 35
4.1.3 Dose-response analysis for Pithecellobium dulce extract. 37
4.2 Chemical analysis of the Pithecellobium dulce extract and the mosquito midgut.38
4.2.1 GC-MS analysis of the Pithecellobium dulce extract and the mosquito midgut extract 41
4.3 Effect of Pithecellobium dulce extract on infection success. 43
4.3.1 Proportion rate of infection by Ae. aegypti post-infection with CHIKV before and after feeding on P. dulce extract. 43
4.3.2 : Replication dynamics of chikungunya virus in Aedes aegypti among the different treatments days post infection. 44
CHAPTER 5.0: DISCUSSION, CONCLUSION AND RECOMMENDATIONS
5.1 Discussion 47
5.2 Conclusion 50
5.3 Recommendations 50
REFERENCES 51
APPENDICES 67
Appendix1.0: Estimated median survival time for each dimethyl sulfoxide dose 67
Appendix 2.0: Forest plot of hazard ratios for dimethyl sulfoxide 68
Appendix3.0: Schoen field assessment for proportional hazards assumption illustrating that the model used for survival analysis fitted well 69
Appendix 4.0: LC-QqQ MS fragments of identified compounds in the plant extract .70
Appendix 5.0 Compounds identified in Pithecellobium dulce plant extract 72
Appendix 6.0 Compounds identified in mosquito midgut after ingestion of
Pithecellobium dulce extract 73
Appendix 7.0: Turnitin plagiarism certificate 74
Appendix 8.0 NACOSTI Research permit 75
LIST OF TABLES
Table 3.1: Different doses of dimethyl sulfoxide in 6% glucose solution and controls tested against survival of Aedes aegypti 36
Table 3.2: Different concentrations of Pithecellobium dulce extract in 6% glucose solution and 0.103mL of dimethyl sulfoxide and controls tested against survival of Aedes aegypti 36
Table 4.1: Median survival times of female Aedes aegypti fed on Pithecelobium dulce extracts and associated 95% confidence limits (CL). 36
Table 4.2: LC-QqQ-MS fragments of identified compounds in the plant and the midgut extract (pool of 400 midguts). 39
LIST OF FIGURES
Figure 2.1: Global distribution of chikungunya virus infections. 9
Figure 2.2: Distribution of chikungunya virus outbreaks in Kenya 10
Figure 2.3: Typical rashes of chikungunya virus infection 12
Figure 2.4: Transmission of chikungunya virus in both the sylvatic and urban cycle. 14
Figure 2.5: Pithecellobium dulce tree (a) and its leaves and fruits (b) 21
Figure 4.1 Kaplan-Meier survival curves for Aedes aegypti orally fed on different concentrations of dimethyl sulfoxide… 34
Figure 4.2: Estimated dose-response curves showing the probability of Aedes aegypti
dying against dose level for a period of 21 days 34
Figure 4.3 Kaplan-Meier survival curves for Aedes aegypti orally fed on different concentrations of Pithecellobium dulce extracts 35
Figure 4.4: Hazard ratios for survival analysis of female Aedes aegypti on Pithecellobium dulce extract. 36
Figure 4.5: Estimated dose-response curves showing the probability of Aedes aegypti
dying against dose level for a period of 21 days. 37
Figure 4.6: LC-QqQ-MS profile of the Pithecellobium dulce and the mosquito midgut extract. 40
Figure 4.7: GC-MS of identified metabolites in the plant extract and the mosquito midgut. .42
Figure 4.8: Proportion of infection and dissemination by Aedes aegypti post-infection with either freshly cultured CHIKV (A and B) or frozen virus (C and D) before and after feeding on Pithecellobium dulce extract. 44
Figure 4.9: Chikungunya virus replication dynamics in Aedes aegypti before and after feeding on Pithecellobium dulce extract. Infection using both freshly cultured and frozen virus (A), dissemination in freshly cultured virus (B). 46
ABBREVIATIONS AND ACRONYMS
ATSBs Attractive targeted sugar baits
CHIKV Chikungunya virus
CHIK Chikungunya disease
CDC Center for Disease Control
CI Confidence interval
CPE Cytopathic effects
DALYs Disability adjusted life years
DENV Dengue virus
DDT Dichlorodiphenyltrichloroethane
DNA Deoxyribonucleic acid
DMSO Dimethyl sulfoxide
ECSA East Central South Africa lineage
ECSA-IOL East, Central and South Africa-Indian Ocean Lineage
EIP Extrinsic incubation period
GIS Geographic Information System
GC-MS Gas chromatography-mass spectrometry
GC-EAD Gas chromatographic-electroantennographic detection
HEPA High efficient particulate air
LC-QqQ-MS Liquid chromatography triple quadrupole tandem mass spectrometry
ICIPE International Centre of Insect Physiology and Ecology
IOL Indian Ocean Lineage
KEMRI Kenya Medical Research Institute
JAK-STAT Janus kinase signal transducer and activator of transcription
LD Light and Day
LC-MS Liquid chromatography-mass spectrometry
MEB Midgut escape barrier
MIB Midgut infection barrier
NACOSTI National commission for science, technology and innovation
NIST National Institute of Standards and Technology
NHPs Non-human primates
NSAIDs Non-steroid anti-inflammatory drugs
PAHO Pan-American Health organization
QTI Quantitative trait loci
SGIB Salivary gland infection barrier
SIT Sterile insect technique
VC Vectorial capacity
WHO World Health Organization
CHAPTER 1.0
INTRODUCTION
1.1 Background
Chikungunya (CHIK) is a mosquito-borne viral disease first identified during the 1952-53 outbreak in Tanzania (Robinson, 1955). The name chikungunya which means “that which bends up” describes the stooped posture of infected patients suffering from severe arthralgia besides the abrupt onset of fever and rash (Thiberville et al., 2013). Even though infection in humans is self-limiting and acute symptoms resolve within 5-7 days, chikungunya virus (CHIKV) is recurrent in 30-40% of infected patients and may persist for years, impacting productivity (Owino, 2018; Schwartz and Albert, 2010a). Since its discovery, sporadic outbreaks of CHIK have been reported in Africa and Asia (Powers and Logue, 2007).
In Kenya, chikungunya virus emerged in Lamu Island in 2004 before spreading to Comoros and La Reunion Islands, India and South East Asia infecting millions of people and causing severe cases of the disease and deaths (Sergon et al., 2008, 2007; Renault et al., 2007). These outbreaks resulted in importation of the virus in Europe and America in 2007 and 2013 respectively (Watson, 2007;Yactayo et al., 2016). Currently, cases of CHIKV have been reported in over 60 countries globally with Asia and America being the most affected (WHO, 2020). The virus re-emerged in 2016 in Mandera County with reports of over 1792 cases (Konongoi et al., 2018), followed by an outbreak in Mombasa County in 2017-2018 involving a novel CHIKV strain (Eyase et al., 2020). The most recent outbreak was reported in Hagadera, Garissa County where 109 cases were recorded (WHO, 2020). In addition to the aforementioned outbreaks, sero-prevalence studies have shown evidence of CHIKV transmission in western Kenya among asymptomatic children (Nyamwaya et al., 2021; Grossi-Soyster et al., 2017; Mease et al., 2011).
Global expansion of CHIKV is instigated by various factors including absence of licensed vaccines and antiviral drugs (Gorcha et al., 2014) and extensive geographic spread of the principal vectors Aedes aegypti and Aedes albopictus. These factors are fueled by globalization of trade and travel and pronounced competence of these vectors in transmitting the virus (Tatem et al., 2006). The risk of transmission of CHIKV however, varies at both local and global scales (Moore et al., 2018; Staples et al., 2009). For instance, in Kenya, while human infections and resultant outbreaks are endemic at the Coastal and Northeastern region, not every region is equally affected. This underscores the need for vector competence studies as an important epidemiological risk factor for spread and establishment of CHIKV.
Vector competence is a complex phenotypic trait determined by both biotic and abiotic factors (Lefèvre et al., 2013b). An example of a biotic factor is plant nutrition, which is an understudied in regards to the biology of Ae. aegypti. As such, studies in this area may open avenues for control of this vector. While mosquitoes primarily depend on plants for sugars, this essential behavior exposes mosquitoes to a range of plant-produced substances which may potentially influence vector survival, and pathogen transmission dynamics (Cory and Hoover, 2006). For instance, whereas feeding on Parthenium hysterophorus, a preferred host plant for the malaria vector Anopheles gambiae enhances survival of the vector, its key secondary metabolite parthenin, a sesquiterpene lactone blocks transmission of the malaria parasite Plasmodium falciparum (Balaich et al.,2016; Manda et al.,2007). On the contrary, plant feeding on the invasive shrub Prosopis juliflora enhances the malaria transmission potential of Anopheles mosquitoes (Muller et al., 2017). Likewise, plant feeding influences the level and transmission of Leishmania parasites by sand flies (Schlein and Muller, 2004). However, beyond a few studies linking plant feeding to its survival and reproductive fitness (Nyasembe et al., 2021), little is known about the influence of plant feeding on pathogen transmission success in the Aedes-virus interactive system.
Previously, a high degree yet selective plant feeding was observed in nature in both sexes of Ae. aegypti amongst them Pithecellobium dulce (Nyasembe et al., 2018; Olson et al., 2020; Nyasembe et al., 2021). Pithecellobium dulce is a perennial evergreen tree indigenously grown in America and is cultivated in the Coastal region of Kenya (Srinivas et al., 2018). Locally, P. dulce is known by a swahili word “Mkwaju” meaning “Tamarind tree”. Phytochemical analysis of different parts of P. dulce has revealed the presence of various compounds including alkaloids, tannins, flavonoids, glycosides and triterpenoids (Srinivas et al., 2018). For example, the leaves have been reported to possess astringent, emollient, and antidiabetic properties with metabolites such as afzelin, dulcitol and quercetin identified in subsequent studies (Vanitha and Manikandan, 2016). The preference of Ae. aegypti for this plant has largely been attributed to plant sugar content and volatile profile (Nyasembe et al., 2018) and perhaps the presence of plant metabolites whose role in pathogen transmission dynamics is unknown. Thus, in this study we evaluated the effect of P. dulce extract on survival and competence of Ae. aegypti to CHIKV.
1.2 Statement of problem
Chikungunya is a re-emerging mosquito borne viral disease of immense public health importance globally. The disease is characterized by both large and small-scale outbreaks as well as inter-epidemic infections that are of social, health and economic concern. The extensive global expansion of CHIKV is due to absence of licensed vaccines and sustainable vector control measures.
Increasing evidence linking plant-nectar feeding to aspects of mosquito vectorial capacity such as increase in survival and fecundity and reduction of biting frequency provides insights into plant-vector interactions that could open avenues for their control (Gu et al., 2011). For instance, in the Anopheles-Plasmodium and sand fly-Leishmania vectorial systems, plant feeding may expose these vectors to a range of metabolites influencing infection success. (Muller et al., 2017; Balaich et al., 2016; Schlein and Muller, 2004). Recent studies have demonstrated that plant feeding enhances the survival and reproductive success of Ae. aegypti (Nyasembe et al., 2021), despite its known preference to feed on humans (Harrington et al., 2009). However, little is known about the influence of plant feeding on pathogen transmission success in this vector.
Pithecellobium dulce benth is a preferred host plant fed upon by Ae. aegypti in nature (Nyasembe et al., 2018), attributed to sugar and amino acid content which the vector ingests to enhance its survival and reproduction success (Nyasembe et al., 2021). However, it is conceivable that as demonstrated in Anopheles-Plasmodium and sandfly- Leishmania vectorial systems (Muller et al., 2017; Balaich et al., 2016; Schlein and Muller, 1995;), P. dulce could be a source of secondary metabolites whose role in the Aedes-virus interactive system is unknown. Therefore, we proposed to evaluate the effect of P. dulce extract on survival and susceptibility of Ae. aegypti to CHIKV infection.
1.3 Justification and significance of the study
Chikungunya virus is increasingly becoming a global concern. This is due to the numerous outbreaks and the inter-epidemics reported at both global and local scale (WHO 2020). Challenges of current vector control strategies coupled with the lack of CHIKV vaccines as well as variation in the global and local transmission of CHIKV has underscored the need to explore and develop novel strategies that can prevent CHIK infection in the vector. Plant feeding is a neglected aspect in the biology of the key vector, Aedes aegypti that could open avenues for control.
Mosquitoes solely depend on plants for sugars necessary for their survival and reproductive fitness (Nyasembe et al., 2021; Wanjiku et al., 2021; Olson et al., 2020 Nyasembe et al., 2018). Either subsequent studies have also shown that plant nutrition through the effects of ingested secondary metabolites or nutritional content influences the competence of mosquitoes in transmitting pathogens. (Alaux et al., 2010; Lefèvre et al., 2013b). However, the role of secondary metabolites ingested during plant feeding is not known in the Aedes-virus interaction.
Building on earlier reports of plant feeding of Ae. aegypti, we tested whether this behavior could influence its survival and competence to CHIKV by virtue of ingested metabolites acquired during feeding on P. dulce plant. The findings from this study could have epidemiologic importance since identification of metabolites with the potential to regulate mosquito-virus interaction could pave way for transmission blocking.
1.4 Research objectives
1.4.1 Main objective
To determine the effect of Pithecellobium dulce stem, leaf and inflorescence extracts on survival and competence of Ae. aegypti to chikungunya virus.
1.4.2 Specific objectives
i. To screen the effects of P. dulce stem, leaf and inflorescence extracts on survival of Ae. aegypti
ii. To identify plant metabolites ingested by Ae. aegypti after feeding on P. dulce
stem, leaf and inflorescence extracts
iii. To determine the effect of P. dulce stem, leaf and inflorescence extracts on CHIKV infection, dissemination and transmission potential of Ae. aegypti
1.5 Research hypothesis
1. Feeding on P. dulce stem, leaf and inflorescence extracts enhances survival of Ae. aegypti
2. Ae. aegypti ingests secondary metabolites after feeding on P. dulce stem, leaf and inflorescence extracts
3. Feeding on P. dulce stem, leaf and inflorescence extracts reduces the extrinsic development and competence of Ae. aegypti to CHIKV transmission.
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