PROGRAM

December 2013

• Message from Scott O’Neill: Our challenge - exciting opportunities PAGE 1

• Indonesia: Communities and regulators show support PAGE 2

• Vietnam: Results on the way as fi rst fi eld trial wraps up PAGE 2

• Next steps: Developing the Wolbachia method PAGE 2

• Australia: Plans underway for further trial PAGE 3

• Brazil: Support growing for Wolbachia release in 2014 PAGE 3

• Colombia: Strong start in newest research site PAGE 4

• China: Positive feedback PAGE 4

Each year on 7 April the World Health Organization (WHO) celebrates World Health Day to mark the founding of the organisation in 1948. A theme is chosen to highlight a priority of public health at that time. In 2014 that theme is vector-borne diseases.

This public awareness event is promoted as ‘providing an opportunity for individuals in

every community to get involved in activities that can lead to better health’.

I encourage everyone to get involved in this important initiative – but I would like to take this opportunity to thank those who are already very involved in reducing the global burden of dengue. Those I would like to thank include the residents of Cairns, Australia and Tri Nguyen Island, Vietnam where we have been undertaking further fi eld trials of our Wolbachia dengue control method this year; residents in Yogyakarta, Indonesia who have registered their support for a trial to begin there in early 2014; our scientifi c partners in Brazil, China, Colombia and Singapore who are engaging with communities and regulatory bodies to progress trials in those countries; and our growing groups of funders, supporters and research project team members.

As a global team we share the WHO’s goal for World Health Day 2014 – better protection from vector-borne diseases – and look forward to working with the WHO and ministries of health not only in countries where dengue has shown to be a major burden in 2013, but also in countries where only a decade ago the disease was unknown and is now an emerging threat.

We will report our research progress in 2014 to you through the media, this newsletter and our website. If you have any questions about our work please email contact@eliminatedengue.com

I wish you all a safe and happy holiday season and a year of exciting opportunities in 2014.

Professor Scott O’Neill Program Leader.

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Our challenge – exciting opportunitiesIn this edition

www.eliminatedengue.com

‘As vector-borne diseases begin to spread beyond their traditional boundaries, action needs to be expanded beyond the countries where these diseases currently thrive.

‘The world’s fastest growing vector-borne disease is dengue’ WHO

Read more about World Health Day

Sukma Aprillya (Lya), from our team in Indonesia, uses puppets to share a story with her son Wisang.

In Yogyakarta, Indonesia, traditional arts such as puppets can be a messenger for communication between parents and children. This is a timely reminder of the need to adapt the way we deliver information to local communities as our research continues to expand across cultures. Photo credit: Puspita Kusuma.

The Eliminate Dengue offi ce at Monash University in Melbourne, Australia will close for the holidays from Friday 20 December and reopen on Thursday 2 January 2014.

Sharing stories

www.eliminatedengue.com

Tri Nguyen Island, on the south central coast of Vietnam, hosted the nation’s fi rst fi eld trial of our research method this year. While the last release of Wolbachia mosquitoes took place in early September, the team continues to visit the island to monitor mosquitoes and meet with community members to discuss the progress of the trial.

While the Wolbachia rate in the mosquito population initially

reached a high level, recent results show there has been a decline in the number of mosquitoes carrying Wolbachia. If this decline continues – as it did in previous fi eld trials in Australia with this strain of Wolbachia – the Eliminate Dengue team will return to Tri Nguyen Island with a Wolbachia strain that has successfully established in wild mosquito populations in Australia.

In the meantime, we expect the existing Wolbachia mosquitoes have been reducing the risk of dengue transmission on the island, where there has not been a dengue case since the trial began.

Program Newsletter December 2013

Indonesia’s fi rst fi eld trials are due to begin early in 2014 with approval from the Provincial Government and ethical clearance from the IRB at Universitas Gadjah Mada (UGM), one of four internationally accredited IRB’s in Indonesia.

The approval follows more than two years of preparation and stakeholder and community engagement by the many teams involved in the research project, led by the Faculty of Medicine at UGM.

As we prepare to release Wolbachia mosquitoes in two Yogyakarta communities, Nogotirto and Kronggahan, in the Sleman district, the project team has sought to meet with all adult residents within these sites to explain our research and give them the opportunity to make an informed decision about supporting the fi eld trial. That support has been overwhelming.

We have also been engaging with the Ministry of Health to keep them informed of our research and will continue to work with residents and all stakeholders as the fi eld trials proceed, to report the fi ndings and respond to any questions.

These small-scale fi eld trials are an exciting step in testing the Wolbachia method to reduce dengue transmission in Indonesia. If these are successful we would then hope to carry out larger citywide trials in Yogyakarta, where there is a high prevalence of dengue. Our goal is a long-term one, which would only proceed with ongoing community support and regulatory approval. Eliminate Dengue Indonesia is funded by Yayasan Tahija (The Tahija Foundation) Indonesia.

Communities and regulators show support

Results on the way as first field trial wraps up

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VIETNAM

Vietnam field trial update

Developing the Wolbachia method

Follow our progresswww.eliminatedengue.com/project/vietnam/progress

INDONESIA

We are now working to optimise the Wolbachia approach so it can be implemented broadly in dengue-endemic countries. To do this we are testing various Wolbachia strains to select those with the strongest dengue transmission blocking potential and also evaluate how these diff erent Wolbachia strains establish and spread under diff erent ecological conditions.

In addition, we need to understand how these diff erent Wolbachia strains can be most eff ectively deployed to achieve optimum coverage, with respect to both effi cacy and public acceptability. Finally, we need to ascertain the likely costs of deploying Wolbachia under diff erent dengue transmission conditions.

After fi eld-testing two Wolbachia strains, results suggest that one strain – wMelPop – may not be as eff ective in the fi eld as we had hoped. With a strong dengue-blocking eff ect, it is proving to be diffi cult to establish in wild mosquito populations.

The fi rst strain we fi eld-tested – wMel – is establishing more easily and once established is maintaining close to 100%. We will continue to deploy this strain in some of our future trial sites while also assessing a third combination strain, which we hope to begin fi eld-testing in 2014, pending regulatory approval.

How we monitor Wolbachia carrying mosquitoes on Tri Nguyen Island

Monitoring for WolbachiaAt the beginning of the year, so that we could see how many mosquitoes there are on the island, we set a number of mosquito traps in homes (pictured). We collected samples from these traps every week.

We always found more mosquitoes two weeks after heavy rainfall; this is because the rain has hatched new eggs that have been laid by adult mosquitoes in containers around the home. Once we began releasing

Wolbachia mosquitoes we continued to collect the samples from the traps and sent them to the laboratory to see if they had Wolbachia.

2. A microscope is used to separate Aedes aegypti from other mosquitoes caught in the bag. The Aedes aegypti are then sent to Hanoi.

NHA TRANG Institut Pasteur HANOI National Institute of Hygiene and Epidemiology

3. In the laboratory all the mosquitoes are put through a machine that shows us how many have Wolbachia.

Sharing the results

We have now completed 23 weeks of releasing Wolbachia mosquitoes

and we would like to say a big thank you to all local households for

your support and involvement in our project’s activities.

Our latest monitoring tells us that on 29 August 80% of Aedes aegypti mosquitoes

had Wolbachia.

Over the coming months we will continue to collect adult mosquitoes to see if

Wolbachia stays in the local Aedes aegypti population.

We hope this will be the case, however there is also the possibility that the

number of Wolbachia mosquitoes in our traps could decrease. This is because the

Wolbachia mosquitoes have a shorter life span and they lay fewer eggs than the

wild mosquitoes without Wolbachia.

Talking to the community on TNI

www.eliminatedengue.com

Eliminate Dengue project progress

4/9/2013

We will keep updating you on our project progress through the

monitoring phase and will talk to the TNI community about the next

steps for our project on the island.

Please contact us if you have any questions.

As always, we encourage all residents on the island to always

protect themselves from being bitten by mosquitoes. If you

think you have dengue, please seek medical advice through

the local health clinic or the Vinh Nguyen health station.

Percentage of mosquitoes with Wolbachia Weeks mosquitoes released Wolbachia mosquitoes released

Our goal

29/8/13 - 80%

1. Every Thursday the catching bag (circled in photo) is collected from the trap and taken to IPNT in Nha Trang.

TRI NGUYEN ISLAND

www.eliminatedengue.com

www.eliminatedengue.com

Community engagement and fi eld entomology activities have intensifi ed over the past months in Brazil’s four study sites of Jurujuba, Tubiacanga, Urca and Vila Valqueire where community support continues to be strong for a release of Wolbachia mosquitoes in these areas.

Members of the Eliminate Dengue Brazil project are working closely with the Ministry of Health and Ministry of Environment to fi nalise regulatory approval for these proposed releases, which it is hoped will begin mid 2014.

Various trials in Cairns in 2013 have been testing the best way to apply the Wolbachia method in diff erent settings, including our fi rst ‘dry season’ trial. We have seen many interesting and promising results and – with the approaching wet season bringing an increase in mosquito numbers – we expect to see strong data on the establishment of Wolbachia.

We are continuing to monitor mosquitoes in all release sites including Yorkeys Knob and Gordonvale, where close to 100% of mosquitoes still carry Wolbachia. We are also monitoring areas adjacent to our central Cairns fi eld sites to see if Wolbachia is spreading into these areas. In 2014 we hope to conduct a further trial in Cairns, subject to government approval and community support, in the suburbs of Stratford and Freshwater. This trial will be the fi rst to test a new combination strain of Wolbachia.

In 2014 Eliminate Dengue Australia will also explore the option of expanding the research trials to the city of Townsville, north Queensland (south of Cairns). The initial stages of this work will involve talking to the community about our research and monitoring to gain an understanding of the existing mosquito population.

Plans underway for further trials

Support growing for Wolbachia release in 2014

AUSTRALIA

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BRAZIL

Program Newsletter December 2013

Cairns field trial update

Follow our progresswww.eliminatedengue.com/project/australia/progress

Researchers awarded Eureka prize

In September, a team of six long-time collaborators from the Eliminate Dengue program received the 2013 Australian Infectious Diseases Research Centre Eureka Prize for Infectious Diseases Research. The Eureka Prizes are presented annually by the Australian Museum.

“This work is a potential game-changer in the battle against dengue and other insect-borne diseases,” Australian Museum director Frank Howarth said at the awards gala dinner.

The award recognised some of the early scientifi c studies that underpin the current Eliminate Dengue program.

Read more about the Eureka prizes at www.australianmuseum.net.au/eureka.

www.eliminatedengue.com

In November, members of the Eliminate Dengue China team again met with the Ministry of Agriculture to further discuss the regulation of the Wolbachia method in China. The meeting involved relevant national experts from a range of scientifi c fi elds and provided an opportunity to discuss our application for approval to undertake fi eld trials in China. Overall feedback from the meeting was positive, particularly around the environmental safety of the Wolbachia method.

These trials would be the fi rst to expand the Wolbachia method into the Aedes albopictus mosquito, the secondary dengue vector. Three proposed fi eld sites have been selected with diff erent environmental conditions, to enable us to test the eff ectiveness of the Wolbachia method in a range of settings. We are continuing to meet with community members and monitor mosquitoes in these sites in preparation for future fi eld trials, which would only go ahead with regulatory approval.

Contact us

Phone: +61 3 9905 0243Email: contact@eliminatedengue.com

The Eliminate Dengue research program is a not-for-profi t,

international collaboration led from Monash University, Melbourne.

Our research is funded by:

• The Foundation for the National Institutes of Health through the Grand Challenges in Global Health Initiative

of the Bill & Melinda Gates Foundation• Yayasan Tahija, Indonesia

• The Australian and Queensland Governments

• The Brazilian Government

As the newest addition to the research program, Eliminate Dengue Colombia has begun preliminary research and community engagement activities in the communities of Niquia and Paris, in the municipality of Bello.

The fi eld team has installed a network of mosquito monitoring traps throughout these communities, which will enable us to gather a baseline understanding of the Aedes aegypti population in the area.

We have also spent time talking with community members – from school children to senior citizens – about our research and holding educational talks to raise community awareness of dengue and how to reduce the risk of dengue transmission.Pictured above: School children in Paris, Bello, have been learning about mosquitoes and dengue transmission from the local Eliminate Dengue team.

Strong start in newest research site

Positive feedback

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In the media

Follow international media coverage about our work www.eliminatedengue.com/progress/index/type/news

Publications

Read about our work www.eliminatedengue.com/publications CHINA

COLOMBIA

Program Newsletter December 2013

Wolbachia-Associated Bacterial Protection in theMosquito Aedes aegyptiYixin H. Ye, Megan Woolfit, Edwige Rances, Scott L. O’Neill, Elizabeth A. McGraw*

School of Biological Sciences, Monash University, Clayton, Victoria, Australia

Abstract

Background: Wolbachia infections confer protection for their insect hosts against a range of pathogens including bacteria,viruses, nematodes and the malaria parasite. A single mechanism that might explain this broad-based pathogen protectionis immune priming, in which the presence of the symbiont upregulates the basal immune response, preparing the insect todefend against subsequent pathogen infection. A study that compared natural Wolbachia infections in Drosophilamelanogaster with the mosquito vector Aedes aegypti artificially transinfected with the same strains has suggested thatinnate immune priming may only occur in recent host-Wolbachia associations. This same study also revealed that whileimmune priming may play a role in viral protection it cannot explain the entirety of the effect.

Methodology/Findings: Here we assess whether the level of innate immune priming induced by differentWolbachia strainsin A. aegypti is correlated with the degree of protection conferred against bacterial pathogens. We show that Wolbachiastrains wMel and wMelPop, currently being tested for field release for dengue biocontrol, differ in their protective abilities.The wMelPop strain provides stronger, more broad-based protection than wMel, and this is likely explained by both thehigher induction of immune gene expression and the strain-specific activation of particular genes. We also show thatWolbachia densities themselves decline during pathogen infection, likely as a result of the immune induction.

Conclusions/Significance: This work shows a correlation between innate immune priming and bacterial protectionphenotypes. The ability of the Toll pathway, melanisation and antimicrobial peptides to enhance viral protection or toprovide the basis of malaria protection should be further explored in the context of this two-strain comparison. This workraises the questions of whetherWolbachiamay improve the ability of wild mosquitoes to survive pathogen infection or alterthe natural composition of gut flora, and thus have broader consequences for host fitness.

Citation: Ye YH, Woolfit M, Rances E, O’Neill SL, McGraw EA (2013) Wolbachia-Associated Bacterial Protection in the Mosquito Aedes aegypti. PLoS Negl TropDis 7(8): e2362. doi:10.1371/journal.pntd.0002362

Editor: Paulo Filemon Pimenta, Fundacao Oswaldo Cruz, Brazil

Received February 10, 2013; Accepted June 30, 2013; Published August 8, 2013

Copyright: � 2013 Ye et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This research was supported by a grant from the Foundation for the National Institutes of Health through the Grand Challenges in Global HealthInitiative of the Bill and Melinda Gates Foundation and the National Health and Medical Research Council, Australia. The funders had no role in study design, datacollection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: beth.mcgraw@monash.edu

Introduction

Wolbachia pipientis is a maternally inherited intracellular

bacterium that is found in a wide range of arthropod species

and filarial nematodes, with approximately 40% of insect

species infected [1]. Wolbachia spreads rapidly through popula-

tions and to high frequencies by inducing a range of

manipulations of host reproduction that benefit infected

females. In insects, the most common manipulation is cytoplas-

mic incompatibility (CI) [2,3]. Interestingly, some Wolbachia

strains that cannot induce reproductive manipulations still

spread through populations [4]. This would not be predicted

unless there were other positive benefits for Wolbachia-infected

insects. Despite numerous laboratory and semi-field based

experiments examining a range of life history traits, few studies

have identified significant fitness benefits of infection [5,6,7,8].

Most reveal no effect [9] or weak negative effects [10,11,12]. It

is possible, however, that there are benefits to Wolbachia

infections that are only detectable under field conditions or in

circumstances not yet tested in the laboratory.

Recently,Wolbachia was found to either extend the lifespan and/

or increase the survival of Drosophila infected with native viruses,

a trait termed pathogen protection [13,14,15]. Subsequently,

Wolbachia strains native to Drosophila have also been shown to

confer pathogen protection against arboviruses, bacteria, filarial

nematodes and the malaria parasite Plasmodium gallinaceum when

stably transinfected into the mosquito Aedes aegypti [16,17]

[13,17,18,19,20,21,22]. This broad-based pathogen protection

may offer a potential fitness advantage, assisting cytoplasmic

incompatibility in the maintenance and spread ofWolbachia in wild

populations. Understanding the true fitness effects of Wolbachia

infections in mosquitoes is important as these symbiont-infected

mosquitoes are being released into wild populations as part of a

biocontrol strategy for reducing dengue virus transmission to

humans [23].

While the mechanism of pathogen protection is not fully

understood, several recent studies have shed some light on its basis.

It was originally hypothesized that priming of the insect immune

response might provide a single mechanistic explanation for

symbiont-induced protection against viruses, bacteria, nematodes

PLOS Neglected Tropical Diseases | www.plosntds.org 1 August 2013 | Volume 7 | Issue 8 | e2362