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.

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.

4. Diverse mosquito vectors in East Africa
Ajamma et al. (2016) J. Med. Entomol.
63 mosquito species
morphologically and molecularly
identified (CO1 & ITS2 markers)
For most, their role in arbovirus
transmission is poorly understood 0.2
Ma. africana KU056557 LV A10S Chamaunga.Is
Ma. africana KU056527 LB M10YA Logumgum
Ma. africana KU056539 LB M22YA Kokwa.Is
Ma. uniformis KU056584 LB 1G03 Logumgum
M
a. africana
KU056531
LB
b2
Logum
gum
An.gambiaeKU056612LB2A01Sirata
Ma. africana KU056561 LB
M20YA
Molo River
Ae.aegyptiKU056491LV1D07Mfangano.Is
Ma. africana KU056544 LV A7S Mfangano.Is
Ma.uniformisKU056578LBM14YASirata
Ma. africana KU056525 LB M53YA Logumgum
Ma. uniformis KU056582 LB 1F12 Logumgum
M
a.africana
KU056535
LB
1H04
Logum
gum
M
a.uniform
is
KU
056574
LB
U
7B
Logum
gum
Ma.uniformisKU056575LB
M18YAMoloRiver
Ma. uniformis KU056581 LB 1F10 Logumgum
Ma. uniformis KU056597 LB 1G06 Logumgum
Ma. africana KU056545 LV M3YA Mbita
An.arabiensisgenbankDQ287768.1ITS
Ma.uniformisKU056566LVU1SMbita
Ma.uniformisKU056564LBA3BK.Samaki
Ma. africana KU056529 LB M48YA Kokwa.IsMa. africana KU056522 LB M8YA Salabani
Ae.metallicusKU056496LV1F08Mbita
Culex sp. GPA KU056506 LV 1A07 Mbita
M
a.uniform
is
KU056577
LB
M
50YA
Nosuguro
M
a. africana
KU056550
LV
A6S
Cham
aunga.Is
Cx. Lut. tigripes KU056505 LV 1F03 Mbita
Ma. uniformis KU056592 LV U6S Chamaunga.Is
Ma.africanaKU056532LB
M40YA
Salabani
Ad.africanaKU056486LB2B01Logumgum
Cx. tigripes genbank U33033.1 ITS
M
a.africana
KU056553
LV
A1S
M
bita
M
a. uniform
is KU056603
LB
M
17YA
M
olo
River
Ma.africanaKU056538LV
M1YA
Mbita
Ma.uniformisKU056580LBM12YALogumgum
An.gambiaeKU056611LB2A03K.Samaki
Ae.aegyptiKU056492LV1D06Mfangano.Is
Ma. africana KU056524 LB M19YA Molo River
Ma.uniformisKU056565LVafsChamaunga.Is
An.gambiaeKU056614LV
1A03Rusinga.Is
Ma.uniformisKU056601LBM23YANosuguro
Ad.africanaKU056485LB2A11Logumgum
Ma.uniformisKU056587LV1A09L.Nyamasare
Ma. africana KU056530 LB A10B Logumgum
Ma. uniformis KU056583 LB 1G02 Logumgum
Ma. africana KU056541 LV M34YA Chamaunga.Is
Ma. africana KU056548 LV
1C06 Mbita
M
a.africana
KU056555
LV
M
2YA
M
bita
Ae.hirsutusKU056500LV2C02Chamaunga.Is
Ma. africana KU056526 LB M27YA Nosuguro
Ma. africana KU056551 LV
M35YA Chamaunga.Is
Ma.uniformisKU056576LBM24YANosuguro
Ma. africana KU056556 LV M37YA Mfangano.Is
Ma.uniformisKU056596LVunsChamaunga.Is
M
a. africana
KU056546
LV
A2S
Ungoye
Ma.uniformisKU056593LBM28YAK.SamakiM
a. africana
KU056534
LB
M
41YA
Logum
gum
Cx. quinquefasciatus genbank EU359697.1 ITS
Ma. africana KU056520 LB M21YA Kokwa.Is
M
a.africana
KU056536
LB
A9B
Logum
gum
Ma. africana KU056547 LV A5S Ungoye
An.gambiaeKU056615LV1A04Rusinga.Is
Ma.uniformisKU056569LVU7SMfangano.Is
An.gambiaeKU056616LV1D11Mbita
Cx. pipiens KU056509
LV
1F01
Rusinga.Is
Ma.uniformisKU056608LB1G04Logumgum
Ma.uniformisKU056602LBM43YALogumgum
Ae.hirsutusKU056498LV
2D03Rusinga.Is
Ma. uniformis KU056605 LB M25YA Nosuguro
Cx. pipiens KU056510
LV
1E.03
Ungoye
Culex sp. GPB KU056517 LV 2E.01 Ngodhe.Is
Cx. univittatus KU056518 LB 2A06 K.Samaki
Cx. annulioris KU056516 LV 1C11 Mfangano.Is
Ae.aegyptiKU056487LV1B06Mfangano.Is
Mi. splendens KU056513 LB 2B06 K.Samaki
Ma. uniformis KU056595 LB U3B Logumgum
Ma.uniformisKU056590LB1F09K.Samaki
Cx. pipiens
KU056508
LV
1C01
Rusinga.Is
Ma. africana KU056558 LV M36YA Mfangano.Is
Ma.uniformisKU056567LBU1BK.Samaki
Cx. univittatus KU056519 LB 2A09 K.Samaki
Ma. uniformis KU056591 LB M16YA Sirata
Ma. uniformis KU056589 LB
unb Logumgum
Ae.hirsutusKU056501
LV
2B12
Cham
aunga.Is
Cx. annulioris KU056515 LV 1C10 Mfangano.Is
Culex sp. GPA KU056507 LV
1A08 Mbita
Ma. uniformis KU056598 LB U2B Logumgum
Ma.uniformisKU056609LBM15YASirata
M
a. africana
KU056543
LV
A3S
M
bita
Ma. africana KU056554 LV M4YA Mbita
Ma. africana KU056542 LV A4S L.Nyamasare
Ma. africana KU056540 LB M45YA Molo River
Ma. africana KU056533 LB A7B Logumgum
Ma.uniformisKU056604LBM13YALogumgum
Ma. africana KU056528 LB M9YA Logumgum
Ae.aegyptiKU056489LVM31Mbita
Ma.uniformisKU056579LVU8SMfangano.Is
Ae.metallicusKU056493LV1F04Mbita
C
x.pipiens
KU
056511
LV
1E.09
R
usinga.Is
Ma. africana KU056549 LV M32YA Mbita
Ma.africanaKU056563LB
M38YASirata
Ma. uniformis KU056586 LB 1G01 Logumgum
Ma. africana KU056562 LV M33YA Chamaunga.Is
An.gambiaeKU056613LV1A01Rusinga.Is
Ma.uniformisKU056606LBU5BLogumgum
M
a.africana
KU
056552
LV
A9S
M
fangano.Is
M
a.uniform
isKU056573
LB
U10B
Logum
gum
Ae.luteocephalusKU056503LV1D02Mfangano.Is
Ma. africana KU056521 LB A6B Logumgum
Ma. uniformis KU056588 LB
U6B
Logumgum
Ae.luteocephalusKU056502LV1C12Mfangano.Is
Cx. duttoni KU056610 LB 1H11 Ruko
Ae.hirsutusKU056499LV
2B11Chamaunga.Is
Ma.uniformisgenbankJN981958.1ITS
Ae.metallicusKU056494LV1F05Mbita
Ma.uniformisKU056599LBU9BLogumgum
Ae.aegyptiKU056490LV1B07Mfangano.Is
Ma. africana KU056559 LB
M26YA Nosuguro
Ma. uniformis KU056594 LB M42YA Logumgum
M
a. africana
KU056523
LB
A1B
K.Sam
aki
M
a. uniform
is
KU056572
LB
M
11YA
Logum
gum
Ma.uniformisKU056600LBU8BLogumgum
Cx. Lut. tigripes KU056504 LB 2B04 Ruko
Ma.uniformisKU056571LB
M30YA
K.Samaki
Ma.uniformisKU056568LBM55YALogumgum
AeaegyptigenbankM95126.1ITS
Mi. splendens KU056514 LB 2B07 Sirata
Ma.uniformisKU056607LB1F11Logumgum
Cx.pipiens
KU056512
LV
1E.10
Rusinga.Is
An.gambiaeKU056617LB2A02Sirata
Ae.aegyptiKU056488LV1D08Mfangano.Is
Ma. africana KU056560 LB A5B Logumgum
Ae.metallicusKU056495LV1F07Mbita
M
a.africana
KU
056537
LB
1H
02
Logum
gum
Ae.metallicusKU056497LV1D09Mfangano.Is
Ma. uniformis KU056585 LB u2 Logumgum
M
a. uniform
is
KU056570
LB
M
29YA
K.Sam
aki
50
49
64
1
16
8
63
59
16
35
0
3
100
2
84
80
47
33
0
93
10
4
6
23
6
90
0
4
50
0
61
99
0
0
13
23
82
100
100
29
52
1
98
1
69
67
9
76
68
11
61
36
0
99
76
81
10
0
0
0
31
10047
23
99
100
59
1
45
2
67
86
28
66
74
4
42
31
1
62
0
67
43
100
19
1
100
0
46
99
5
21
59
0
34
4
98
1
31
25
3
3
2
70
87
14
48
72
1
73
3
4
0
0
99
99
3
100
0
7
98
4
39
17
49
18
1
73
18
32
100
66
13
94
2
100

5. High Resolution Melting (HRM) Analysis
Multiplex of degenerate universal primers
For alpha-, flavi-, nairo-, phlebo-,
orthobunya-, and thogoto-viruses
C°

6. Arbovirus differentiation by HRM
Multiplex of degenerate
universal primers for:
orthobunyaviruses (O)
alphaviruses (A)
flaviviruses (F)
nairoviruses (N)
phleboviruses (P)
thogotoviruses (T)
Villinger et al. (2016) Mol Ecol Res.
20
10
Temperature (º C)
77 78 79 80 81 82 83 84 85 86 87 88
Meltrate(dF/dT)
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.1
0.0
0.2
B
Negative control
Dugbe (N)
Hazara (N)
Dhori (T)
Bunyamwera (O)
Dengue (F)
Sindbis (A)
Middelburg (A)
Ndumu (A)
Semliki Forest (A)
Usutu (F)
Chikungunya (A)
Yellow fever (F)
WN (F)
Thogoto (T)
RVF (P)

7. Arbovirus differentiation by HRM
Villinger et al. (2016) Mol Ecol Res.
Nor
20
10
Temperature (ºC)
77 78 79 80 81 82 83 84 85 86 87 88
Meltrate(dF/dT)
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.1
0.0
Negative control (MH)
Dugbe (N) cDNA
Batai (O) sDNA
Bunyamwera (O) cDNA
Sindbis (A) cDNA
O’nyong-nyong (A) sDNA
Zika (F) sDNA
Wesselsbron (F) sDNA
*Wesselsbron (F) cDNA
Chikungunya (A) sDNA
Chikungunya (A) cDNA
CCHF (N) sDNA
NSD (N) sDNA
Punta Toro (P) sDNA
WN2 (F) sDNA
WN1 (F) sDNA
WN (F) cDNA
RVF1 (P) sDNA
RVF 2 (P) sDNA
RVF (P) cDNA
0.2
0.3

8. ‘Mixed’ arbovirus infections
Villinger et al. (2016) Mol Ecol Res.

9. Bunyamwera Sindbis Dengue Thogoto
Virus
Dilution
CPE
Multiplex
HRM
CPE
Multiplex
HRM
CPE
Multiplex
HRM
CPE
Multiplex
HRM
10
-3 6/6
(20,000)
4/4
(20,000)
6/6
(200,000)
4/4
(200,000)
6/6
(20,000)
3/3
(20,000)
2/2
(200) 4/4 (200)
10-4 6/6
(2,000)
4/4
(2,000)
6/6
(20,000)
4/4
(20,000)
6/6
(2,000)
2/3
(2,000)
1/2
(20)
2/4 (20)
10
-5 4/6
(200)
4/4
(200)
6/6
(2,000)
4/4
(2,000)
2/6
(200)
3/3
(200)
0/2
(2)
0/4 (2)
10
-6 1/6
(20)
3/4
(20)
4/6
(200)
4/4
(200)
1/6
(20)
2/3
(20)
0/2
(<1) 0/4 (<1)
10-7 0/6
(2)
1/4
(2)
2/6
(20)
4/4
(20)
0/6
(2)
0/3
(2)
0/2
(<1)
0/4 (<1)
10
-8 0/6
(<1)
0/4
(<1)
0/6
(2)
2/4
(2)
0/6
(<1)
0/3
(<1)
0/2
(<1)
0/4 (<1)
10
-9 0/6
(<1)
0/4
(<1)
0/6
(<1)
0/4
(<1)
0/6
(<1)
0/3
(<1)
0/2
(<1) 0/4 (<1)
Validation against CPE in Vero cells
Villinger et al. (2016) Mol Ecol Res.
Virus titer detection limits (PFU/1mL) are highlighted.

10. C°
Malaria (Plasmodium) parasite HRM
Detects as few as 236 parasites/mL of blood – very low parasitaemia.
Kipanga et al. (2014) Malaria Journal

11. Mosquito species HRM
Ajamma et al. (2016) F1000 Research
Temperature (ºC)
76 77 78 79 80 81 82 83 84
100
90
80
70
60
50
40
30
20
10
76 77 78 79 80 81 82 83 84
76 77 78 79 80 81 82
30
20
10
100
90
80
70
60
50
40
30
20
10
negative controlMa. africanaMa. uniformis
Norm
B
NormalisedFluorescence(%)
C
NormalisedFluorescence(%)
Temperature (ºC)
76 77 78 79 80 81 82 83 84
30
20
10
76 77 78 79 80 81 82 83 84
100
90
80
70
60
50
40
30
20
10
negative controlMa. africanaMa. uniformis
Normalis
C
NormalisedFluorescence(%)
100
80
70
60
50
40
30
20
10
74 75 76 77 78 79 80 81
Cx. tenagius
Cx. duttoni
A
90
C
Temperature (ºC)
NormalisedFluorescence(%)
B
D
Temperature (ºC)
NormalisedFluorescence(%)NormalisedFluorescence(%)
NormalisedFluorescence(%)
Cx. pipiens
Cx. antennatus
100
80
70
60
50
40
30
20
10
90
100
80
60
40
20
74 75 76 77 78 79 80 81
100
80
60
40
20
40
30
20
10
74 75 76 77 78 79 80 81
Cx. tenagius
Cx. duttoni
C
Temperature (ºC)
NormalisedFluorescence(%)
D
Temperat
Tempera
Temperature (ºC)
NormalisedFluorescence(%)Normalise
Normalis
Cx. pipiens
Cx. antennatus
Cx. neavei
Culex sp. GPA
100
80
70
60
50
40
30
20
10
90
100
80
60
40
20
74 75 76 77 78 79 80 81
74 75 76 77
70 71 72 73
40
20
40
30
20
10
74 75 76 77 78 79 80 81
Cx. tenagius
Cx. duttoni
C
Temperature (ºC)
NormalisedFluorescence(%)
D
Temperature (ºC)
NormalisedFluorescence(%)Normalis
Normalis
Cx. pipiens
Cx. antennatus
Cx. neavei
Culex sp. GP
100
80
70
60
50
40
30
20
10
90
100
80
60
40
20
74 75 76 77 78 79 80 81
74 75
70 71
40
20
40
30
20
10
74 75 76 77 78 79 80 81
Cx. tenagius
Cx. duttoni
C
Temperature (ºC)NormalisedFluorescence(%)
D
Temperature (ºC)
Temperature (ºC)
Temperature (ºC)
NormalisedFluorescence(%)Normalise
Normalis
Cx. pipiens
Cx. antennatus
Cx. neavei
Culex sp. GPA negativ
100
80
70
60
50
40
30
20
10
90
100
80
60
40
20
74 75 76 77 78 79 80 81
74 75 76 77 78 79
70 71 72 73 74 7
40
20
40
30
20
10
74 75 76 77 78 79 80 81
Cx. tenagius
Cx. duttoni
C
Temperature (ºC)
NormalisedFluorescence(%)
D
Temperature (ºC)
Temperature (ºC)
Temperature (ºC)
NormalisedFluorescence(%)Norma
Norm
Cx. pipiens
Cx. antennatus
Cx. neavei
Culex sp. GPA negative control
100
80
70
60
50
40
30
20
10
90
100
80
60
40
20
74 75 76 77 78 79 80 81
74 75 76 77 78 79 80
70 71 72 73 74 75 76
40
20
100
80
70
60
50
40
30
20
10
90
NormalisedFluorescence(%)
Temperature (ºC)
74 75 76 77 78 79 80 8173
negative controlAe. metallicusAe. vittatus
Temperature (ºC)
81 82 83 84 85 86 87 88
NormalisedFluorescence(%)
100
90
80
70
60
50
40
30
20
10
An. arabiensis control
An. arabiensis samples
An. arabiensis control
An. arabiensis samples
negative control
IGS
Adapted from
Zianni et al. 2013
CO1
CO1
CO1

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).

16. Insect-specific flaviviruses (ISFVs)
CxFV (GQ165808)-Uganda-Cx. quinquefasciatus
CxFV (JF707815)-Spain-Cx. theileri
Murray Valley encephalitis virus (KF751871)-Australia-Cx. annulirostris
Aedes albopictus (AY223844) INVS
MaFV (KM088040)-Kenya-Ma. africana
AnFV (KM088038)-Kenya-An. squamosus
Nakiwogo virus (GQ165809)-Uganda-Ma. africana
Lammi virus (FJ606789)-Finland-mosquito
OcFV (JF707790)-Spain-Ae. Ochlerotatus caspius
Quang Binh virus (FJ644291)-Vietnam-Culex tritae
CxFV (AB377213)-Japan-Cx. pipiens
CxFV (HQ634597)-USA-Cx. quinquefasciatus
CxFV (EU879060)-Mexico-Cx. quinquefasciatus
AnFV (KM088035)-Kenya-Anopheles squamosus
AnFV (KM088036)-Kenya-An. gambiae
Zika virus (AY632535)-Uganda-monkey
Dengue virus (NC_001477)
West Nile virus (NC_009942)
Usutu virus (NC_006551)-Austria-blackbird
Calbertado virus (EU569288)-Canada-Cx. tarsalis
Barkedji virus (KC496020)-Israel-Cx. perexiguus
AeFV (KM088042)-Kenya-Ae. tricholabis
MaFV (KM088043)-Kenya-Mansonia africana
Kamiti River virus (AY149904)-Kenya-Ae. macintoshi
AnFV (KM088039)-Kenya-An. squamosus
Dongang virus (NC_016997)-China-Aedes sp.
Hanko virus (JQ268258)-Finland-mosquito
Uganda S virus (DQ859065)-Uganda-Ae. longipalpis
Wesselsbron virus (KM088034)-Kenya-Culex sp.
Wesselsbron virus (JX423791)-South Africa-Ae. circumluteolus
Ilomantsi virus (KC692067)-Finland-mosquito
Nanay virus (JX627335)-Peru-mosquito
OcFV (HE997073)-Portugal-Ae. Ochlerotatus caspius
T'Ho virus (EU879061)-Mexico-Cx. quinquefasciatus
AeFV (KM088041)-Kenya-Aedes sp.
AeFV (AB488408)-Japan-Ae. albopictus
AeFV (KF801612)-Italy-Ae. albopictus
West Nile virus lineage 1 (JN819317)
AnFV (KM088037)-Kenya-An. gambiae
Palm Creek virus (KC505248)-Australia-Coquillettidia sp.
Chaoyang virus (FJ883471)-China-mosquito
CFAV (GQ165810)-Puerto Rico-Ae. aegypti
West Nile virus lineage 2 (DQ318019)
Zika virus (KU647676)-Martinique-human
Nounane virus (FJ711167)-Cote d'Ivoire-Uranotaenia sp.
Barjedji virus (EU078325)-Senegal-mosquito
Yellow fever virus vaccine strain (NC_002031)
MBF
AeFV
AnFV
MaFV
CxFV
ISFV
351
608
926
321
433
187
977
999
502
977
999
290
481
466
741
382
355
998
864
570
965
162
997
1000
303
989
799
970
308
464
1000
286
999
930
659
994
944
979
635
997
815
241
997
998
894
Villingeretal.(2016)MolEcolRes.

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)

26. • Shimba photos
Shimba Hills National Reserve
Tick-borne pathogen surveillance

27. OUR CONTACTS
International Centre of Insect
Physiology and Ecology (icipe)
P.O. Box 30772-00100, Nairobi, Kenya
Tel: +254 (20) 8632000
Fax: +254 (20) 8632001/8632002
E-mail: icipe@icipe.org
Website: www.icipe.org
facebook.com/icipe.insects/icipe
twitter.com/icipe
linkedin.com/company/icipe

28. Comparison of assay sensitivity
0
5
10
15
20
25
30
35
40
P. falciparum
Plasmodium spp.
nPCRdPCR-HRMnPCR-HRM
Prevalence(%)
Purity Kipanga et al. 2014 Malaria Journal