This document discusses using high-resolution melting analysis (HRM) to conduct integrated surveillance of vectors, pathogens, and host species in East Africa. It describes using HRM to identify over 100 arboviruses transmitted by mosquitoes and ticks in the region, including flaviviruses, alphaviruses, phleboviruses, nairoviruses, orthobunyaviruses, and thogotoviruses. The document also discusses using HRM to identify mosquito vector species and differentiate among arboviruses in a multiplex reaction with degenerate primers. Finally, it presents results demonstrating the ability of HRM to distinguish among several arboviruses.
High-resolution melting analysis approaches to integrated vector surveillance
1. High-resolution melting analysis approaches to integrated
vector surveillance of mosquitoes and ticks, their pathogens
(arboviruses, Plasmodium, bacteria) and bloodmeal hosts
Jandouwe Villinger
Martin Lüscher Emerging Infectious Diseases (ML-EID) Laboratory
icipe Duduville campus, Nairobi, Kenya
International Congress of Entomology (ICE 2016 XXV)
Orlando, FL, September 29th, 2016
2. Vector-borne disease diversity in
East Africa
>100 arboviruses of medical and veterinary importance.
- vectored by mosquitoes, ticks and sand flies.
- Flaviviruses: e.g. Dengue, Zika, yellow fever, West Nile viruses
- Alphaviruses: e.g. Chikungunya, sindbis, Semliki Forest viruses
- Phlebovuruses: e.g. Rift Valley fever virus
- Nairoviruses: e.g. Crimean-Congo hemmorrhagic fever virus
- Orthobunyaviruses: e.g Bunyamwera, Batai viruses
- Thogotovuruses: e.g Thogoto, Dhori viruses
3-5 species of malaria parasites.
- vectored by Anopheles mosquitoes.
- Plasmodium falciparum, P. vivax, P. ovale, P. malariae
Diverse tick-borne diseases.
- Rickettsia, Ehrlichia, Anaplasma, Theileria, Babesia, Coxiella, etc.
Transmission of vector-borne diseases (VBDs) may involve multiple
vertebrate host species.
3. Viral evolution and changing disease risks
Rift Valley fever virus
Full genomes (L, M & S segments) M segment
Baba et al. (2016) Emerg Microbes Infect.
Increased virulence in humans over past century due to genetic mutations and reassortments.
5. High Resolution Melting (HRM) Analysis
Multiplex of degenerate universal primers
For alpha-, flavi-, nairo-, phlebo-,
orthobunya-, and thogoto-viruses
C°
12. C°
Vector blood-meal identification HRM
Vertebrate hosts:
David Omondi et al. 2015 PLoS One
Cyt b blood-meal analysis
16S blood-meal analysis
Temperature (°C)
NormalizedFluourescence(%)
13. Benefits of HRM-based vector surveillance
Low-cost approach for broad-diversity surveillance
of arboviruses, malaria parasites, tick borne-diseases,
vector species and blood-meal host species.
Significantly faster than cell culture, and comparable in
sensitivity.
Can detect mixed pathogen infections and mixed
blood-meal hosts.
Can facilitate pathogen discovery.
14. Arboviruses circulating in Homa Bay County,
The Lake Victoria region of Kenya
David Omondi et al. (PLoS One 2015); In bloodfed
mosquitoes:
Sindbis – Culex pipiens fed on human
Bunyamwera – Aedeomyia africana fed on cattle
– Anopheles coustani fed on sheep
– Mansonia africana fed on man
David Omondi et al. (in prep): 2 sindbis and 4 Bunyamwera
acute infections among 105 febrile illness patients
on Mfangano Island.
Yvonne Ajamma et al. (in prep): Likely transovarial (vertically)
transmitted Bunyamwera isolated from Culex univittatus
mosquitoes (3 pools) reared from field-collected larvae.
15. Discoveries using multiplex PCR-HRM
Novel Wesselsbron virus in Culex and Anopheles
mosquitoes (first time in Kenya) (Villinger et al. 2016 Mol Ecol
Res.).
Bunyamwera, sindbis and chikungunya viruses in
wildlife and sindbis in cattle (Jagero et al. in prep).
High diversity of mosquito-specific flaviviruses (Villinger et
al. 2016; Ajamma et al. in prep).
17. Implications of arbovirus findings
Broad vertebrate host and vector reservoirs, e.g.:
Bunyamwera: humans, 2 wildlife ruminants, 4 mosquito
genera, vertical transmission in mosquitoes
Sindbis: humans, wildlife, livestock, mosquitoes
Insect-specific viruses may be exploited
The Wesselsbron, Chikungunya, Bunyamwera and Sindbis
viruses have been associated with severe human febrile illness,
but are rarely, if ever, identified as a cause of morbidity in
humans or livestock.
This is due to poor diagnostics and surveillance of these
arboviruses.
There is need for differential arbovirus diagnosis and
surveillance to guide appropriate teatment and management,
especially at the interface of humans, livestock and wildlife.
18. From single nucleic acid extractions in the laboratory and field-
based molecular approaches, we can rapidly identify:
Pathogen diversity:
• Arboviruses
• Plasmodium
• Tick-borne pathogens
Vector diversity:
• Mosquitoes
• Ticks
Vertebrate host diversity (blood-meal analysis)
Potential endosymbiotic bacteria and viruses.
Integrated vectored pathogen
transmission surveillance
19. Combined, these assays allow us to integrate surveillance
and discovery of diverse pathogens with information on
diversity of vectors, their blood-meal hosts and potential
vertebrate reservoirs.
We are asking questions regarding:
Pathogen diversity
Transmission and exposure risk factors.
Strategies for blocking pathogen transmission by
investigating co-infection (AnFV, AeFV, MaFV)
Integrated vectored pathogen
transmission surveillance
20. Acknowledgements
• AVID (Arbovirus Incidence and Diversity) Consortium (funded by
google.org)
• THRiVE Consortium (funded by Wellcome Trust)
• Swedish International Development Cooperation Agency (SIDA)
• Internal icipe innovations grant (SIDA, SDC, Kenyan government)
• Sena and Tom Mboya Health Centers staff and participating patients
• Kenya Wildlife Service (KWS), Department of Veterinary Services (DVS),
National Museums of Kenya, FAO, Smithsonian Institution.
• Students and Staff: David Omondi, Yvonne Ajamma, Purity Kipanga,
Thomas Onchuru, Micky Mwamuye, Edwin Ogola, Martin Mbaya, Eunice
Owino, Daniel Ouso, Geoffrey Jagero, James Kabii, Esther Waweru.
• Colleagues: Drs. Daniel Masiga, Rosemary Sang, Baldwyn Torto, Lillian
Wambua, Maamun Jeneby, Damaris Matoke, Marycelin Baba, Jeremy
Herren, Edward Kariuki, Laban Njoroge, Burtram Fielding, Anne Muigai,
Mario Younan.
21. Theme Publications: Arboviruses
Villinger J, Mbaya MK, Ouso DO, Kipanga PN, Lutomiah J & Masiga DK. Arbovirus
and insect-specific virus discovery in Kenya by novel six genera multiplex high-
resolution melting analysis. Molecular Ecology Resources 2016; Epub ahead of
print.
Ajamma YU, Villinger J, Salifu D, Omondi D, Onchuru TO, Njoroge L, Muigai AWT &
Masiga DK. Abundance and species composition of mosquito vectors of
arboviruses in the Lake Victoria and Lake Baringo regions of Kenya. Journal of
Medical Entomology 2016; Epub ahead of print.
Ajamma YU, Mararo E, Omondi D, Onchuru T, Muigai AWT, Masiga D & Villinger J.
Rapid and high throughput molecular identification of diverse mosquito species
by high resolution melting analysis. F1000Research 2016;5:1949.
Baba M, Masiga DK, Sang R & Villinger J. Has Rift Valley fever virus evolved with
increasing severity in human populations in East Africa? Emerging Microbes and
Infections 2016;5:e58.
Baba M, Villinger J & Masiga DK. Repetitive dengue outbreaks in East Africa: A
proposed phased mitigation approach may reduce its impact. Reviews in
Medical Virology 2016;26:183-196.
22. Ticks and Tick-born Diseases
• Anaplasma phagocytophilum in Rhipicephalus maculatus ticks
• Theileria velifera and Rickettsia africae in Amblyomma eburneum ticks
Mwamuye MM, Kariuki E, Omondi D, Kabii J, Odongo D, Masiga D, Villinger
J. Novel Rickettsia and emergent tick-borne pathogens: A molecular survey
of ticks and tick-borne pathogens in Shimba Hills National Reserve, Kenya.
Ticks and Tick-borne Diseases 2016; In Press
Also:
• Anaplasma marginale in Amblyomma ticks (Omondi et al. in review @
PLOS NTD), buffalo and cattle (Jagero et al. in prep)
• Ehrlichia ruminantium in deceased camels and Amblyomma gemma
ticks (OIE immediate notification report, 05/08/2016) and in Amblyomma
latum ticks collected from tortoises (Omondi et al. In review @ PLOS
NTD)
23. Tick-borne pathogen HRM
Mwamuye et al. 2016 Ticks Tick Borne Dis.
Temperature (ºC)
84 85 86 87 88 89 90 91
Meltrate(dF/dT)
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
A. phagocytophilum (ST103)
A. marginale (control)
Temperature (ºC)
79 80 81 82 83 84 85 86 87 88
Meltrate(dF/dT)
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
E. chaffeensis (ST122)
Ehrlichia sp. (ST189)
E. ruminantium (ST130)
Temperature (ºC)
82 83 84 85 86 87 88 89 90 91 92
Meltrate(dF/dT)
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
R. africae (ST138, ST139)
Rickettsia sp. (ST198)
Rickettsia sp. (ST119)
Coxiella sp. (ST269)
Temperature (ºC)
80 81 82 83 84 85 86 87 88
Meltrate(dF/dT)
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
T. parva (control)
T. velifera (ST194, ST260,
ST271, ST272, ST269)
c
a
d
b
Anaplasma Ehrlichia
Rickettsia Theileria
24. Tick-borne pathogen HRM
Mwamuye et al. 2016 Ticks Tick Borne Dis.; David Omondi et al. (in review) PLOS NTD
Temperature (º C)
82 83 84 85 86 87 88 89 90 91 92
3.0
2.5
2.0
1.5
1.0
0.5
0.0
78 79 80 81 82 83 84 85 86 87 88 89
NormalisedFluorescence
110
100
90
80
70
60
50
40
30
20
10
Babesia caballi
Theileria sp.
Hepatozoon fitzsimonsi
E. ruminantium
E. canis
Ehrlichia sp.
Ehlichia sp.
+ Paracoccus sp.
Ehrlichia sp.
Ehrlichia sp.
Paracoccus sp.
E. ruminantium
+ Paracoccus sp.
Anaplasma ovis
Anaplasma platys
Anaplasma bovis
Rickettsia sp.1
R. rhipicephali
R. japonica
R. monasensis
R. montanensis
R. africae
R. aeschlimanni
Rickettsia sp. 2
Rickettsia sp. 3
78 79 80 81 82 83 84 85 86 87 88
78 79 80 81 82 83 84 85 86 87 88 89
3.0
2.5
2.0
1.5
1.0
0.5
0.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Meltrate(dF/dT)
Meltrate(dF/dT)Meltrate(dF/dT)
A
C
B
D
25. Novel tick-pathogen-host associations
• Anaplasma phagocytophilum in Rhipicephalus maculatus
ticks (Mwamuye et al. 2016 Ticks Tick-borne Dis.)
• Theileria velifera and Rickettsia africae in Amblyomma
eburneum ticks (Mwamuye et al. 2016)
• Ehrlichia ruminantium in deceased camels and Amblyomma
gemma ticks (OIE immediate notification report, 05/08/2016) and
in Amblyomma sparsum ticks collected from tortoises and
cattle (Omondi et al. In review @ PLOS NTD)
• Ehrlichia canis in Am. latum from monitor lizards (Omondi et
al. in review @ PLOS NTD)