High-resolution melting analysis approaches to integrated vector surveillance of mosquitoes and ticks, their pathogens

(arboviruses, Plasmodium, bacteria) and bloodmeal hosts

Jandouwe VillingerMartin Lüscher Emerging Infectious Diseases (ML-EID) Laboratory

icipe Duduville campus, Nairobi, Kenya

International Congress of Entomology (ICE 2016 XXV)Orlando, FL, September 29th, 2016

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.

Viral evolution and changing disease risksRift 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.

Diverse mosquito vectors in East Africa

Ajamma et al. (2016) J. Med. Entomol.

63 mosquito speciesmorphologically and molecularlyidentified (CO1 & ITS2 markers)

For most, their role in arbovirus transmission is poorly understood

High Resolution Melting (HRM) AnalysisMultiplex of degenerate universal primersFor alpha-, flavi-, nairo-, phlebo-, orthobunya-, and thogoto-viruses

C°

Arbovirus differentiation by HRMMultiplex of degenerate universal primers for: orthobunyaviruses (O)

alphaviruses (A)

flaviviruses (F) nairoviruses (N)

phleboviruses (P) thogotoviruses (T)

Villinger et al. (2016) Mol Ecol Res.

Arbovirus differentiation by HRM

Villinger et al. (2016) Mol Ecol Res.

‘Mixed’ arbovirus infections

Villinger et al. (2016) Mol Ecol Res.

Validation against CPE in Vero cells

Villinger et al. (2016) Mol Ecol Res.

Virus titer detection limits (PFU/1mL) are highlighted.

C°

Malaria (Plasmodium) parasite HRM

Detects as few as 236 parasites/mL of blood – very low parasitaemia.Kipanga et al. (2014) Malaria Journal

Mosquito species HRM

Ajamma et al. (2016) F1000 Research

IGSAdapted from Zianni et al. 2013

CO1CO1

CO1

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)

Nor

mal

ized

Fluo

ures

cenc

e (%

)

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.

Arboviruses circulating in Homa Bay County, The Lake Victoria region of KenyaDavid Omondi et al. (PLoS One 2015); In bloodfed

mosquitoes: Sindbis – Culex pipiens fed on humanBunyamwera – 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.

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).

Insect-specific flaviviruses (ISFVs) V

illin

ger e

t al.

(201

6) M

ol E

col R

es.

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.

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 pathogentransmission surveillance

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 pathogentransmission surveillance

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.

Theme Publications: ArbovirusesVillinger 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.

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)

Tick-borne pathogen HRM

Mwamuye et al. 2016 Ticks Tick Borne Dis.

Anaplasma Ehrlichia

Rickettsia Theileria

Tick-borne pathogen HRM

Mwamuye et al. 2016 Ticks Tick Borne Dis.; David Omondi et al. (in review) PLOS NTD

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)

• Shimba photos

Shimba Hills National ReserveTick-borne pathogen surveillance

OUR CONTACTS

International Centre of Insect Physiology and Ecology (icipe)

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Comparison of assay sensitivity

Purity Kipanga et al. 2014 Malaria Journal